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PMID:7370 | Determination of oxalic acid in urine by atomic absorption spectrophotometry. | A method is described for determination of oxalic acid in urine using atomic absorption spectrophotometry. The concentration of urinary oxalic acid is calculated by using the difference between two determinations of calcium: first, the excess of calcium in the supernatant after precipitation as calcium oxalate at pH 5, and second, the total calcium determined at pH less than 1 (endogenous and added). Analytical parameters (pH of precipitation, temperature, calcium and oxalate added, precipitation time, interfering substances) were studied with the aid of [14C]oxalic acid. A constant recovery of 95% of total oxalic acid allows the use of a correction factor. The accuracy and reproducibility of the method make it useful for routine determination of urinary oxalic acid. | Determination of oxalic acid in urine by atomic absorption spectrophotometry. A method is described for determination of oxalic acid in urine using atomic absorption spectrophotometry. The concentration of urinary oxalic acid is calculated by using the difference between two determinations of calcium: first, the excess of calcium in the supernatant after precipitation as calcium oxalate at pH 5, and second, the total calcium determined at pH less than 1 (endogenous and added). Analytical parameters (pH of precipitation, temperature, calcium and oxalate added, precipitation time, interfering substances) were studied with the aid of [14C]oxalic acid. A constant recovery of 95% of total oxalic acid allows the use of a correction factor. The accuracy and reproducibility of the method make it useful for routine determination of urinary oxalic acid. | [
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PMID:7371 | Assessment of the functional activity of human lymphocytes in malignant disease by the local graft-versus-host reaction in rats and the T rosette-forming cell test. | The local graft-versus-host reaction (GVHR) and the spontaneous rosette-forming cell (RFC) test were used to test the functional activity of lymphocytes in blood obtained from twenty normal donors and thirty patients with malignant tumours. The lymphocytes of all twenty control donors gave a positive GVHR and had normal RFC values. In twenty-two of the thirty patients with malignancies the GVHR was negative; of these, only five had low RFC values. In the remaining eight patients the GVHR was positive and RFC values were normal. It is concluded that the local GVHR constitutes a sensitive test for measurement of the functional activity of lymphocytes, while the RFC test can only be used as a quantitative test. | Assessment of the functional activity of human lymphocytes in malignant disease by the local graft-versus-host reaction in rats and the T rosette-forming cell test. The local graft-versus-host reaction (GVHR) and the spontaneous rosette-forming cell (RFC) test were used to test the functional activity of lymphocytes in blood obtained from twenty normal donors and thirty patients with malignant tumours. The lymphocytes of all twenty control donors gave a positive GVHR and had normal RFC values. In twenty-two of the thirty patients with malignancies the GVHR was negative; of these, only five had low RFC values. In the remaining eight patients the GVHR was positive and RFC values were normal. It is concluded that the local GVHR constitutes a sensitive test for measurement of the functional activity of lymphocytes, while the RFC test can only be used as a quantitative test. | [
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PMID:7373 | Plasma levels and effects of metoprolol on blood pressure, adrenergic beta receptor blockade, and plasma renin activity in essential hypertension. | The effects of metoprolol, a selective beta adrenergic receptor antagonist, on blood pressure, beta receptor blockade (antagoinst of isoproterenol and exercise tachycardia), and plasma renin activity (PRA) have been compared with those of placebo in 16 patients with essential hypertension. The dose of metroprolol was 25 mg three times daily for 1 wk and thereafter 100 mg three times daily for 5 wk. The mean decrease in blood pressure during treatment with metoprolol was 24 +/- 3.8 (SEM)/10 +/- 2.1 mm Hg in the lying position and 23 +/- 4.4/9 +/- 3.1 mm Hg after 1 min in the standing position. At a dose of 2.9 to 5.4 mg/kg, steady-state plasma concentrations of metoprolol varied 17-fold (from 20 to 341 ng/ml) between patients and correlated with the interindividual variability in isoproterenol antagonism (r = 0.58, p less than 0.05) and decrease in exercise tachycardia (r = 0.65, p less than 0.01). By contrast, neither of these variables correlated with the dose of metoprolol in mg/kg. Metoprolol decreased PRA by 67 +/- 1.9 and 71 +/- 1.2% in the lying and standing positions, respectively. The decrease in the mean arterial blood pressure in the lying position was significantly correlated to the PRA during the placebo period (r = 0.61, p less than 0.05) but not to the plasma steady-state levels of metoprolol, the degree of beta receptor blockade, and the decrease in PRA. | Plasma levels and effects of metoprolol on blood pressure, adrenergic beta receptor blockade, and plasma renin activity in essential hypertension. The effects of metoprolol, a selective beta adrenergic receptor antagonist, on blood pressure, beta receptor blockade (antagoinst of isoproterenol and exercise tachycardia), and plasma renin activity (PRA) have been compared with those of placebo in 16 patients with essential hypertension. The dose of metroprolol was 25 mg three times daily for 1 wk and thereafter 100 mg three times daily for 5 wk. The mean decrease in blood pressure during treatment with metoprolol was 24 +/- 3.8 (SEM)/10 +/- 2.1 mm Hg in the lying position and 23 +/- 4.4/9 +/- 3.1 mm Hg after 1 min in the standing position. At a dose of 2.9 to 5.4 mg/kg, steady-state plasma concentrations of metoprolol varied 17-fold (from 20 to 341 ng/ml) between patients and correlated with the interindividual variability in isoproterenol antagonism (r = 0.58, p less than 0.05) and decrease in exercise tachycardia (r = 0.65, p less than 0.01). By contrast, neither of these variables correlated with the dose of metoprolol in mg/kg. Metoprolol decreased PRA by 67 +/- 1.9 and 71 +/- 1.2% in the lying and standing positions, respectively. The decrease in the mean arterial blood pressure in the lying position was significantly correlated to the PRA during the placebo period (r = 0.61, p less than 0.05) but not to the plasma steady-state levels of metoprolol, the degree of beta receptor blockade, and the decrease in PRA. | [
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PMID:7374 | Effects of prazosin in patients with hypertension. | A double-blind placebo crossover trail (2 periods, each of 6 wk) was carried out in 12 patients. In the first period the patients were randomly allocated to either their individual established dose of prazosin or to the same number of placebo tablets; treatment was reversed after 6 wk. Blood pressure was higher by 17/8 mm Hgin the lying posture and by 24/14 mm Hg in the standing posture during placebo than during prazosin treatment. Standing pulse rate and body weight were higher and plasma renin activity lower during prazosin treatment. Postural hypotension occurring 1 to 2 hr after the first few doses was noted in one third of the patients when they resumed prazosin treatment after the placebo course. | Effects of prazosin in patients with hypertension. A double-blind placebo crossover trail (2 periods, each of 6 wk) was carried out in 12 patients. In the first period the patients were randomly allocated to either their individual established dose of prazosin or to the same number of placebo tablets; treatment was reversed after 6 wk. Blood pressure was higher by 17/8 mm Hgin the lying posture and by 24/14 mm Hg in the standing posture during placebo than during prazosin treatment. Standing pulse rate and body weight were higher and plasma renin activity lower during prazosin treatment. Postural hypotension occurring 1 to 2 hr after the first few doses was noted in one third of the patients when they resumed prazosin treatment after the placebo course. | [
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PMID:7375 | Blood levels and electroencephalographic effects of diazepam and bromazepam. | Blood levels and electroencephalographic (EEG) data were collected for 2 hr after single oral doses of bromazepam (9 mg), diazepam (10 mg), and placebo in 13 male adult volunteers. Both drugs caused an increase in beta activity (above 13 Hz) and a decrease in alpha activity (9 to 11 Hz) in the EEG. Blood levels of 100 ng/ml of diazepam or 50 ng/ml of bromazepam were associated with significant changes in EEG beta activity. Temporal changes in the EEG after administration of diazepam or bromazepam paralleled development of plasma levels of these drugs. Although a weakly significant correlation was found between measurable diazepam blood levels and amount of increased EEG beta activity, the relationship between measurable bromazepam blood levels and the degree of EEG changes was not significant. Quantitative EEG is a sensitive continuous response measure, useful in defining cerebral activity, response latency, and relative potency of psychoactive benzodiazepines. | Blood levels and electroencephalographic effects of diazepam and bromazepam. Blood levels and electroencephalographic (EEG) data were collected for 2 hr after single oral doses of bromazepam (9 mg), diazepam (10 mg), and placebo in 13 male adult volunteers. Both drugs caused an increase in beta activity (above 13 Hz) and a decrease in alpha activity (9 to 11 Hz) in the EEG. Blood levels of 100 ng/ml of diazepam or 50 ng/ml of bromazepam were associated with significant changes in EEG beta activity. Temporal changes in the EEG after administration of diazepam or bromazepam paralleled development of plasma levels of these drugs. Although a weakly significant correlation was found between measurable diazepam blood levels and amount of increased EEG beta activity, the relationship between measurable bromazepam blood levels and the degree of EEG changes was not significant. Quantitative EEG is a sensitive continuous response measure, useful in defining cerebral activity, response latency, and relative potency of psychoactive benzodiazepines. | [
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PMID:7376 | The influence of food on nitrofurantoin bioavailability. | The effect of food on the absorption of five commercial dosage forms of nitrofurantoin varying widely in drug release and dissolution characteristics was assessed in man after oral administration. Four healthy fasting and nonfasting male subjects received, in a crossover fashion, a single 100-mg dose of microcrystalline nitrofurantoin as an aqueous suspension, three different compressed tablets, and a single 100-mg dose of macrocrystalline nitrofurantoin in a capsule. Both the absorption and the duration of therapeutic urinary concentrations of nitrofurantoin were significantly increased after administration of the five products to nonfasting subjects. The enhancement in the bioavailability of the drug in the presence of food ranged from 20% to 400%, with the greatest absorption-enhancing effect occurring with those dosage forms exhibiting the poorest dissolution characteristics. It is concluded that single-dose comparative bioavailability studies of drug products normally administered with food should be performed in both nonfasting and fasting subjects. | The influence of food on nitrofurantoin bioavailability. The effect of food on the absorption of five commercial dosage forms of nitrofurantoin varying widely in drug release and dissolution characteristics was assessed in man after oral administration. Four healthy fasting and nonfasting male subjects received, in a crossover fashion, a single 100-mg dose of microcrystalline nitrofurantoin as an aqueous suspension, three different compressed tablets, and a single 100-mg dose of macrocrystalline nitrofurantoin in a capsule. Both the absorption and the duration of therapeutic urinary concentrations of nitrofurantoin were significantly increased after administration of the five products to nonfasting subjects. The enhancement in the bioavailability of the drug in the presence of food ranged from 20% to 400%, with the greatest absorption-enhancing effect occurring with those dosage forms exhibiting the poorest dissolution characteristics. It is concluded that single-dose comparative bioavailability studies of drug products normally administered with food should be performed in both nonfasting and fasting subjects. | [
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PMID:7377 | Tricyclic antidepressant overdosage: experimental studies on the management of circulatory complications. | Tricyclic antidepressant overdosage may be complicated by cardiac arrhythmias, which were sometimes difficult to treat prior to the use of sodium bicarbonate. Experiments have been done with several antiarrhythmics in an attempt to define the optimum treatment. Sodium bicarbonate proved the most effective experimentally and this supports our clinical experience. Physostigmine is a useful second drug, having beneficial effects against arrhythmias and central nervous system manifestations of toxicity. Practolol, although reversing the arrhythmias, tends to cause hypotension. Other drugs tried were less effective. | Tricyclic antidepressant overdosage: experimental studies on the management of circulatory complications. Tricyclic antidepressant overdosage may be complicated by cardiac arrhythmias, which were sometimes difficult to treat prior to the use of sodium bicarbonate. Experiments have been done with several antiarrhythmics in an attempt to define the optimum treatment. Sodium bicarbonate proved the most effective experimentally and this supports our clinical experience. Physostigmine is a useful second drug, having beneficial effects against arrhythmias and central nervous system manifestations of toxicity. Practolol, although reversing the arrhythmias, tends to cause hypotension. Other drugs tried were less effective. | [
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PMID:7385 | Synaptic transmission from photoreceptors to bipolar and horizontal cells in the carp retina. | The most important observations in the present study can be summarized as follows: 1. In horizontal and bipolar cells, blocking chemical transmission caused membrane potential changes of the same polarity as the responses to illumination in each cell type. 2. Acceleration of transmitter release from the receptor terminals depolarized the horizontal cells, which is the opposite polarity to the light response. 3. Stimulation by transretinal current flowing from receptor side to vitreous side depolarized the receptor terminals and triggered transmitter release, which in turn evoked transient postsynaptic potentials in horizontal and bipolar cells. The polarity of the postsynaptic responses was the opposite to that of the light-evoked responses in each type of cell. 4. Hyperpolarization of the receptor terminals by the current of relatively long duration flowing from vitreous side to receptor side reduced the transmitter release, as demonstrated by the horizontal cell hyperpolarization. 5. From these observations, it is inferred that the receptors release the transmitter in the dark and that the transmission depolarizes horizontal and off-center bipolar cells by increasing membrane permeability chiefly to Na+ and hyperpolarizes the on-center bipolar cell by decreasing membrane permeability to Na+. | Synaptic transmission from photoreceptors to bipolar and horizontal cells in the carp retina. The most important observations in the present study can be summarized as follows: 1. In horizontal and bipolar cells, blocking chemical transmission caused membrane potential changes of the same polarity as the responses to illumination in each cell type. 2. Acceleration of transmitter release from the receptor terminals depolarized the horizontal cells, which is the opposite polarity to the light response. 3. Stimulation by transretinal current flowing from receptor side to vitreous side depolarized the receptor terminals and triggered transmitter release, which in turn evoked transient postsynaptic potentials in horizontal and bipolar cells. The polarity of the postsynaptic responses was the opposite to that of the light-evoked responses in each type of cell. 4. Hyperpolarization of the receptor terminals by the current of relatively long duration flowing from vitreous side to receptor side reduced the transmitter release, as demonstrated by the horizontal cell hyperpolarization. 5. From these observations, it is inferred that the receptors release the transmitter in the dark and that the transmission depolarizes horizontal and off-center bipolar cells by increasing membrane permeability chiefly to Na+ and hyperpolarizes the on-center bipolar cell by decreasing membrane permeability to Na+. | [
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PMID:7432 | Anxiety therapy in the neoplastic patient. | A clinical study was carried out in 73 neoplastic patients suffering from anxiety and other emotional upsets to assess the effectiveness and tolerance of lorazepam. Patients were given individualised daily doses ranging from 1.5 mg to 3 mg lorazepam for 15 to 60 days. Results, as assessed by the response of anxiety, tension, erethism and insomnia, showed that only 4 (5%) patients failed to obtain some relief. There was complete disappearance of all symptoms in 29 (40%) after 15 days, relief of at least one major symptom and reduction in the others in 27 (37%), and slight reduction in one or more symptoms in 13 (18%) patients. Side-effects were minimal and disappeared within a few days with continued treatment. | Anxiety therapy in the neoplastic patient. A clinical study was carried out in 73 neoplastic patients suffering from anxiety and other emotional upsets to assess the effectiveness and tolerance of lorazepam. Patients were given individualised daily doses ranging from 1.5 mg to 3 mg lorazepam for 15 to 60 days. Results, as assessed by the response of anxiety, tension, erethism and insomnia, showed that only 4 (5%) patients failed to obtain some relief. There was complete disappearance of all symptoms in 29 (40%) after 15 days, relief of at least one major symptom and reduction in the others in 27 (37%), and slight reduction in one or more symptoms in 13 (18%) patients. Side-effects were minimal and disappeared within a few days with continued treatment. | [
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PMID:7438 | Transport of ions and water across the epithelium of fish gills. | The teleostean gill is characterized by an exceptionally low permeability to water. Water moves along the osmotic gradient across the gill, being gained in fresh water and lost in sea water. Coupling of water movement to solute movement has not been reported. In fresh water, the gill is the site of independent active uptake of sodium and chloride. Na+ uptake is coupled to H+ or NH4+ excretion, Cl- uptake to HCO3- excretion. Amiloride blocks sodium transport and thiocyanate inhibits the chloride pump. In sea water, sodium and chloride exchanges across the gill are about 100 times faster than in fresh water, up to 100% of the internal sodium or chloride being exchanged per hour. Chloride is actively excreted, while sodium movement may well be passive. The chloride pump is associated with a mechanism for Na/K exchange; both pump and Na/K exchange are blocked by thiocyanate and possibly by ouabain. Three enzymes are involved in the ionic pumps: carbonate dehydratase (EC 4.2.1.1; carbonic anhydrase), sodium/potassium-stimulated adenosine-triphosphatase (EC 3.6.1.3, ATPase) and anion-stimulated ATPase. Specialized cells ('chloride cells') are presumably the site of the active transport. | Transport of ions and water across the epithelium of fish gills. The teleostean gill is characterized by an exceptionally low permeability to water. Water moves along the osmotic gradient across the gill, being gained in fresh water and lost in sea water. Coupling of water movement to solute movement has not been reported. In fresh water, the gill is the site of independent active uptake of sodium and chloride. Na+ uptake is coupled to H+ or NH4+ excretion, Cl- uptake to HCO3- excretion. Amiloride blocks sodium transport and thiocyanate inhibits the chloride pump. In sea water, sodium and chloride exchanges across the gill are about 100 times faster than in fresh water, up to 100% of the internal sodium or chloride being exchanged per hour. Chloride is actively excreted, while sodium movement may well be passive. The chloride pump is associated with a mechanism for Na/K exchange; both pump and Na/K exchange are blocked by thiocyanate and possibly by ouabain. Three enzymes are involved in the ionic pumps: carbonate dehydratase (EC 4.2.1.1; carbonic anhydrase), sodium/potassium-stimulated adenosine-triphosphatase (EC 3.6.1.3, ATPase) and anion-stimulated ATPase. Specialized cells ('chloride cells') are presumably the site of the active transport. | [
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PMID:7439 | Coupling of water to solute movement in isolated gastric mucosa. | Osmosis is apparently the mechanism responsible for the coupling of water to solute transport in biological membranes. Often a secreted or absorbed fluid is essentially iso-osmotic with the solution of origin, or with plasma, and various models have been constructed by Curran, Diamond and others to account for such observations. More information is needed, however, to test further the predictions of these models and to facilitate correlation with known structural details. This study deals with gastric secretion and the effects of the luminal solution on its composition. Although pure gastric juice collected in vivo is virtually iso-osmotic with plasma, Teorell, Obrink and others found that instillation of a buffer solution (glycine) in the lumen led to a twofold increase in the concentration of gastric acid. This effect is not restricted to buffer solutions: the normality of H+ secreted into an isotonic (120 mM) NaCl solution bathing the isolated bullfrog gastric mucosa was 276 +/- 19 mmol/1 (13 experiments). Clearly the luminal solution affects the concentration of gastric secretion, probably by reducing an endogenous osmotic gradient. Thus the sites responsible for transport of H+ must be accessible from the luminal solution. | Coupling of water to solute movement in isolated gastric mucosa. Osmosis is apparently the mechanism responsible for the coupling of water to solute transport in biological membranes. Often a secreted or absorbed fluid is essentially iso-osmotic with the solution of origin, or with plasma, and various models have been constructed by Curran, Diamond and others to account for such observations. More information is needed, however, to test further the predictions of these models and to facilitate correlation with known structural details. This study deals with gastric secretion and the effects of the luminal solution on its composition. Although pure gastric juice collected in vivo is virtually iso-osmotic with plasma, Teorell, Obrink and others found that instillation of a buffer solution (glycine) in the lumen led to a twofold increase in the concentration of gastric acid. This effect is not restricted to buffer solutions: the normality of H+ secreted into an isotonic (120 mM) NaCl solution bathing the isolated bullfrog gastric mucosa was 276 +/- 19 mmol/1 (13 experiments). Clearly the luminal solution affects the concentration of gastric secretion, probably by reducing an endogenous osmotic gradient. Thus the sites responsible for transport of H+ must be accessible from the luminal solution. | [
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PMID:7440 | Ion transport across amphibian lung. | The simple architecture of the amphibian lung makes it possible to study the movement of substances across a barrier with permeability and bioelectric properties that are dominated by the alveolar epithelium. When mounted as a planar sheet between identical Ringer solutions the excised lung of the bullfrog exhibited a transmural electrical potential difference of nearly 20 mV (pleural surface positive) and a resistance of about 700 omega cm2. Unidirectional fluxes of 36Cl, Br-, I-, and SCN- across the short-circuited lung were asymmetrical. The net 36Cl- flow from pleura to lumen matched the short-circuit current after 1.5 h of voltage clamping, followed the kinetics of a saturable process, and was reduced by inhibitors of oxidative metabolism. These results suggest that halide and certain pseudohalide anions are secreted by the frog alveolar epithelium. Fluxes of Na+, K+, Ca+, HCO3-, TcO4-, SO42-, p-aminohippurate, gluconate, dinitrophenolate and water were compatible with passive diffusion of the probe molecules across the barrier. Measurements of lung oxygen consumption, ion fluxes and bioelectric properties have helped to pinpoint possible sites and modes of action of airborne agents, such as heavy metals, sulphates and nitrates, that may damage the mammalian pulmonary barrier. | Ion transport across amphibian lung. The simple architecture of the amphibian lung makes it possible to study the movement of substances across a barrier with permeability and bioelectric properties that are dominated by the alveolar epithelium. When mounted as a planar sheet between identical Ringer solutions the excised lung of the bullfrog exhibited a transmural electrical potential difference of nearly 20 mV (pleural surface positive) and a resistance of about 700 omega cm2. Unidirectional fluxes of 36Cl, Br-, I-, and SCN- across the short-circuited lung were asymmetrical. The net 36Cl- flow from pleura to lumen matched the short-circuit current after 1.5 h of voltage clamping, followed the kinetics of a saturable process, and was reduced by inhibitors of oxidative metabolism. These results suggest that halide and certain pseudohalide anions are secreted by the frog alveolar epithelium. Fluxes of Na+, K+, Ca+, HCO3-, TcO4-, SO42-, p-aminohippurate, gluconate, dinitrophenolate and water were compatible with passive diffusion of the probe molecules across the barrier. Measurements of lung oxygen consumption, ion fluxes and bioelectric properties have helped to pinpoint possible sites and modes of action of airborne agents, such as heavy metals, sulphates and nitrates, that may damage the mammalian pulmonary barrier. | [
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PMID:7441 | Ion transport and water flow in the mammalian lung. | The coupling of bulk water flow to active ion transport has been described in various epithelia; evidence presented here suggests that this is also a feature of the mammalian lung. Measurements of the ionic composition of lung liquid and its rate of formation in the fetal lamb in vivo have made it possible to estimate the net flux of each ion and, with water tracer measurements of ion one-way fluxes, to calculate flux ratios. When these are compared with the ratios predicted by the Ussing flux ratio equation it is clear that the secretion of lung liquid is linked to active transport of Cl- from plasma; sodium moves passively. In addition there is an apparent uphill transfer of HCO2- out of lung liquid. In an in vitro preparation of adult canine trachea Cl- is actively transported towards the lumen and is associated with a small net flux of Na+ in the opposite direction. Addition of acetylcholine increases the net Cl- flux towards the lumen but reverses the orientation of the net Na+ flux. Changes such as these may be important determinants of bulk liquid flow in vivo as well as in vitro. | Ion transport and water flow in the mammalian lung. The coupling of bulk water flow to active ion transport has been described in various epithelia; evidence presented here suggests that this is also a feature of the mammalian lung. Measurements of the ionic composition of lung liquid and its rate of formation in the fetal lamb in vivo have made it possible to estimate the net flux of each ion and, with water tracer measurements of ion one-way fluxes, to calculate flux ratios. When these are compared with the ratios predicted by the Ussing flux ratio equation it is clear that the secretion of lung liquid is linked to active transport of Cl- from plasma; sodium moves passively. In addition there is an apparent uphill transfer of HCO2- out of lung liquid. In an in vitro preparation of adult canine trachea Cl- is actively transported towards the lumen and is associated with a small net flux of Na+ in the opposite direction. Addition of acetylcholine increases the net Cl- flux towards the lumen but reverses the orientation of the net Na+ flux. Changes such as these may be important determinants of bulk liquid flow in vivo as well as in vitro. | [
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PMID:7442 | Lung carbonate dehydratase (carbonic anhydrase), CO2 stores and CO2 transport. | A study of CO2 storage in excised, exsanguinated lungs revealed that CO2 stores include a compartment which reaches equilibration very rapidly (less than 3s) and a slower compartment which equilibrates with a half-time of approximately 15 s. Inhibition of carbonate dehydratase (carbonic anhydrase, EC 4.2.1.1) does not change the slope of the total CO2 dissociation curve of the lung but does increase the slow compartment at the expense of the fast. CO2 diffusion across the pleura is approximately 20 times faster than that of O2, a relationship that is not affected by inhibition of carbonate dehydratase. The role of tissue CO2 stores in limiting respiratory fluctuations of PCO2 or pH in arterial blood is only minor and may be of significance only in rapid, deep inspiration. CO2 uptake or release by the stores is out of phase with blood CO2 exchange. As a consequence, the time course of CO2 exchange at the mouth during expiration cannot be used to predict alveolar or capillary CO2 exchange. | Lung carbonate dehydratase (carbonic anhydrase), CO2 stores and CO2 transport. A study of CO2 storage in excised, exsanguinated lungs revealed that CO2 stores include a compartment which reaches equilibration very rapidly (less than 3s) and a slower compartment which equilibrates with a half-time of approximately 15 s. Inhibition of carbonate dehydratase (carbonic anhydrase, EC 4.2.1.1) does not change the slope of the total CO2 dissociation curve of the lung but does increase the slow compartment at the expense of the fast. CO2 diffusion across the pleura is approximately 20 times faster than that of O2, a relationship that is not affected by inhibition of carbonate dehydratase. The role of tissue CO2 stores in limiting respiratory fluctuations of PCO2 or pH in arterial blood is only minor and may be of significance only in rapid, deep inspiration. CO2 uptake or release by the stores is out of phase with blood CO2 exchange. As a consequence, the time course of CO2 exchange at the mouth during expiration cannot be used to predict alveolar or capillary CO2 exchange. | [
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PMID:7446 | Prolactin-releasing and release-inhibiting factor activities in the bovine, rat, and human pineal gland: in vitro and in vivo studies. | The effects of crude extracts of bovine, rat, and human pineal glands on prolactin (PRL) release were studied using an in vitro system. In addition, the effects of a known pineal constituent, arginine vasotocin (AVT), and crude bovine pineal extract (bPE) on PRL secretion were studied in vivo. Normal male rat hemipituitaries (HP), incubated with bPE (13 mg tissue/HP)released 200%, 150%, and 285% more PRL into the medium than did their corresponding untreated control halves incubated in either Medium 199 alone, hypothalamic extract, or cerebral cortical extract, respectively. HP incubated with either rat (6 mg of tissue/HP) or human (25 mg of tissue/HP) pineal extract released 110% and 75% more PRL, respectively, than did their corresponding untreated control halves. HP exposed to 10 mg tissue eq of either bovine pineal fraction A1 or bovine pineal fraction A3 released 88% and 63%, respectively, less PRL than did their corresponding untreated control halves incubated in Krebs-Ringer Bicarbonate (KRB) medium. Quantitites of melatonin, thyrotropin-releasing hormone (TRH), or estrogen, comparable to those found in the pineal, had no significant effect on PRL secretion in vitro. The iv injection of either bPE (90 mg tissue/rat) or AVT (10 mug/rat) into estrogen and progesterone-treated male rats resulted in a 40% and 138% increase, respectively, in plasma PRL titers, 10 min after injection, over pre-injection control levels. The per cent of increase in plasma PRL levels in these animals was significantly greater than that observed in control rats receiving either saline or cortical extract. The results suggest that crude extracts of pineal glands of three different species contain prolactin-releasing factor (PRF) activity which is probably not due to any endogenous melatonin, TRH, or estrogen that may be present. Conversely, two bovine pineal fractions, A1 and A3, appeared to exhibit prolactin-inhibiting factor (PIF) activity. We have concluded that the pineal gland may serve as an alternate or supplemental source of PRF and/or PIF. | Prolactin-releasing and release-inhibiting factor activities in the bovine, rat, and human pineal gland: in vitro and in vivo studies. The effects of crude extracts of bovine, rat, and human pineal glands on prolactin (PRL) release were studied using an in vitro system. In addition, the effects of a known pineal constituent, arginine vasotocin (AVT), and crude bovine pineal extract (bPE) on PRL secretion were studied in vivo. Normal male rat hemipituitaries (HP), incubated with bPE (13 mg tissue/HP)released 200%, 150%, and 285% more PRL into the medium than did their corresponding untreated control halves incubated in either Medium 199 alone, hypothalamic extract, or cerebral cortical extract, respectively. HP incubated with either rat (6 mg of tissue/HP) or human (25 mg of tissue/HP) pineal extract released 110% and 75% more PRL, respectively, than did their corresponding untreated control halves. HP exposed to 10 mg tissue eq of either bovine pineal fraction A1 or bovine pineal fraction A3 released 88% and 63%, respectively, less PRL than did their corresponding untreated control halves incubated in Krebs-Ringer Bicarbonate (KRB) medium. Quantitites of melatonin, thyrotropin-releasing hormone (TRH), or estrogen, comparable to those found in the pineal, had no significant effect on PRL secretion in vitro. The iv injection of either bPE (90 mg tissue/rat) or AVT (10 mug/rat) into estrogen and progesterone-treated male rats resulted in a 40% and 138% increase, respectively, in plasma PRL titers, 10 min after injection, over pre-injection control levels. The per cent of increase in plasma PRL levels in these animals was significantly greater than that observed in control rats receiving either saline or cortical extract. The results suggest that crude extracts of pineal glands of three different species contain prolactin-releasing factor (PRF) activity which is probably not due to any endogenous melatonin, TRH, or estrogen that may be present. Conversely, two bovine pineal fractions, A1 and A3, appeared to exhibit prolactin-inhibiting factor (PIF) activity. We have concluded that the pineal gland may serve as an alternate or supplemental source of PRF and/or PIF. | [
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PMID:7447 | Adenylyl cyclase activities in ovarian tissues. I. Homogenization and conditions of assay in graafian follicles and corpora lutea of rabbits, rats, and pigs: regulation by ATP, and some comparative properties. | Responsiveness of ovarian adenylyl cyclases to luteinizing hormone (LH), found to be 5 to 10-fold in cell-free preparations under optimal conditions, required gentle homogenizations and storage in sucrose-containing media. Assay conditions required the use of an ATP-regenerating system consisting of creatine kinase, creatine phosphate, and myokinase for the preservation of ATP levels. LH-stimulated adenylyl cyclase (AC) in rabbit CL showed the following properties: 1) The pH optimum of basal activity was about 8.0; that of LH-stimulated activity was about 7.5. 2) The relative response to LH was low (1.5 to 2-fold) at 0.1 mM ATP and increased with increasing ATP, but not with increasing GTP. At low (0.1 mM) ATP, GTP increased catalytic efficacy of the system, both in the absence and in the presence of LH (no effect on relative stimulation). 3) The optimal relative stimulation by LH was obtained at about 1.0 mM MgCl2 in excess of added magnesium-binding ingredients. 4) The sensitivity to stimulation by LH (about 0.2 mug/ml NIH-LH-B8) was unaffected by either pH, nucleotides (ATP and GTP), or MgCl2 concentration. 5) Under the assay conditions used, activity was stimulated by prostaglandin E1 (PGE1) about 1.5 to 2-fold, and by epinephrine about 3 to 4-fold. In all aspects tested, LH-stimulated AC in rat CL resembled that in rabbit CL, except that about 5-fold higher concentrations of NIH-LH-B8 were needed for half-maximal stimulation. The AC activity in pig Graafian follicles, however, differed from that in rabbit CL in that 1) the ATP concentration needed for optimal stimulation by LH was lower (in the micromolar rather than the millimolar range); 2) catecholamines elicited only a 1.3 to 1.4-fold stimulation; and 3) NIH-LH-B8 elicited half-maximal stimulation at 0.008 to 0.020 mug/ml. We were unable to detect LH-responsive AC activity in either homogenates or washed particles of CL from either cycling or pregnant pigs. LH fractions of three origins (human, bovine, and ovine) and of varying specific activities (from 0.041 to 2.0 NIH-LH-S18 units/mg) were tested and the relative potencies by OAAD assay were found to correlate well with the relative potencies in the adenylyl cyclase assays (rat CL, rabbit CL, and pig follicles), consistent with the possibility that AC receptors are responsible for biologic actions of LH. | Adenylyl cyclase activities in ovarian tissues. I. Homogenization and conditions of assay in graafian follicles and corpora lutea of rabbits, rats, and pigs: regulation by ATP, and some comparative properties. Responsiveness of ovarian adenylyl cyclases to luteinizing hormone (LH), found to be 5 to 10-fold in cell-free preparations under optimal conditions, required gentle homogenizations and storage in sucrose-containing media. Assay conditions required the use of an ATP-regenerating system consisting of creatine kinase, creatine phosphate, and myokinase for the preservation of ATP levels. LH-stimulated adenylyl cyclase (AC) in rabbit CL showed the following properties: 1) The pH optimum of basal activity was about 8.0; that of LH-stimulated activity was about 7.5. 2) The relative response to LH was low (1.5 to 2-fold) at 0.1 mM ATP and increased with increasing ATP, but not with increasing GTP. At low (0.1 mM) ATP, GTP increased catalytic efficacy of the system, both in the absence and in the presence of LH (no effect on relative stimulation). 3) The optimal relative stimulation by LH was obtained at about 1.0 mM MgCl2 in excess of added magnesium-binding ingredients. 4) The sensitivity to stimulation by LH (about 0.2 mug/ml NIH-LH-B8) was unaffected by either pH, nucleotides (ATP and GTP), or MgCl2 concentration. 5) Under the assay conditions used, activity was stimulated by prostaglandin E1 (PGE1) about 1.5 to 2-fold, and by epinephrine about 3 to 4-fold. In all aspects tested, LH-stimulated AC in rat CL resembled that in rabbit CL, except that about 5-fold higher concentrations of NIH-LH-B8 were needed for half-maximal stimulation. The AC activity in pig Graafian follicles, however, differed from that in rabbit CL in that 1) the ATP concentration needed for optimal stimulation by LH was lower (in the micromolar rather than the millimolar range); 2) catecholamines elicited only a 1.3 to 1.4-fold stimulation; and 3) NIH-LH-B8 elicited half-maximal stimulation at 0.008 to 0.020 mug/ml. We were unable to detect LH-responsive AC activity in either homogenates or washed particles of CL from either cycling or pregnant pigs. LH fractions of three origins (human, bovine, and ovine) and of varying specific activities (from 0.041 to 2.0 NIH-LH-S18 units/mg) were tested and the relative potencies by OAAD assay were found to correlate well with the relative potencies in the adenylyl cyclase assays (rat CL, rabbit CL, and pig follicles), consistent with the possibility that AC receptors are responsible for biologic actions of LH. | [
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PMID:7449 | Further observations on the properties of serum and tissue guanase from man and some animal species. Optimum pH and activation energy in the presence of 8-azaguanine. | The activation energy and the optimum pH of guanine deaminase in man, the rat, guinea pig and mouse were studied using 8-azaguanine as a substrate. The serum guanase in man and in all the animal species studied differs in activation energy from the guanase of the liver. In man, moreover, the serum guanase is also different from the brain and kidney enzyme. In the rat and guinea pig the brain enzyme has thermic activation energy different from the liver and kidney enzyme. The guanase of the serum and tissues of the guinea pig differs from the enzyme of the serum and tissues of man, rat and mouse for optimum pH. | Further observations on the properties of serum and tissue guanase from man and some animal species. Optimum pH and activation energy in the presence of 8-azaguanine. The activation energy and the optimum pH of guanine deaminase in man, the rat, guinea pig and mouse were studied using 8-azaguanine as a substrate. The serum guanase in man and in all the animal species studied differs in activation energy from the guanase of the liver. In man, moreover, the serum guanase is also different from the brain and kidney enzyme. In the rat and guinea pig the brain enzyme has thermic activation energy different from the liver and kidney enzyme. The guanase of the serum and tissues of the guinea pig differs from the enzyme of the serum and tissues of man, rat and mouse for optimum pH. | [
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PMID:7450 | Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae. | Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants. | Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae. Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants. | [
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PMID:7451 | Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions. | The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway. | Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions. The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway. | [
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PMID:7452 | Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition. | Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000. | Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition. Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000. | [
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PMID:7453 | A spectroscopic investigation of S-trifluoroethylthiopapain. An investigation of the active site of papain. | The pH dependence of the 19F chemical shift and the fluorescence spectrum of S-2,2,2-trifluoro-1,1-dideuteroethyl-thio-paapain are analysed in terms of dependence on the ionisation of aspartic-acid-158 and histidine-159. The 19F probe causes negative cooperativity between these groups, and does not detected any ionisation at high pH. The intermediate chemical exchange rates for the ionisation of aspartic-acid-158 and histidine-159 allow the approxmate rate constants for proton transfer to be calculated. The rather low rate constants are explained in terms of the hydrophobicity of the active-site region and the net positive charge on the enzyme resulting from its high isoelectric point. | A spectroscopic investigation of S-trifluoroethylthiopapain. An investigation of the active site of papain. The pH dependence of the 19F chemical shift and the fluorescence spectrum of S-2,2,2-trifluoro-1,1-dideuteroethyl-thio-paapain are analysed in terms of dependence on the ionisation of aspartic-acid-158 and histidine-159. The 19F probe causes negative cooperativity between these groups, and does not detected any ionisation at high pH. The intermediate chemical exchange rates for the ionisation of aspartic-acid-158 and histidine-159 allow the approxmate rate constants for proton transfer to be calculated. The rather low rate constants are explained in terms of the hydrophobicity of the active-site region and the net positive charge on the enzyme resulting from its high isoelectric point. | [
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PMID:7454 | Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax). | A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed. | Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax). A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed. | [
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PMID:7455 | Interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol in rat kidney: heterogeneity of 3alpha-hydroxysteroid oxidoreductases. | 3alpha-Hydroxysteroid oxidoreductases catalyzing the interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol (3alpha-androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH-dependent oxidoreductase, a microsomal NADPH-dependent enzyme and a microsomal NADH-linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108 000 X g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and co-factor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH-dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH-dependent oxidoreductase displays favorable characteristics to catalyze the 3alpha-dehydrogenation of 3alpha-androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3alpha-androstanediol as an efficient precursor for the local formation of 5alpha-dihydrotestosterone. | Interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol in rat kidney: heterogeneity of 3alpha-hydroxysteroid oxidoreductases. 3alpha-Hydroxysteroid oxidoreductases catalyzing the interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol (3alpha-androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH-dependent oxidoreductase, a microsomal NADPH-dependent enzyme and a microsomal NADH-linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108 000 X g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and co-factor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH-dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH-dependent oxidoreductase displays favorable characteristics to catalyze the 3alpha-dehydrogenation of 3alpha-androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3alpha-androstanediol as an efficient precursor for the local formation of 5alpha-dihydrotestosterone. | [
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PMID:7456 | Influence of DNA acidification on DNA premelting and template properties. | Acidification of a T7 DNA sample was found to be partly irreversible as ultraviolet difference spectra measured at various sub-melting temperatures were different from those observed for a 'normal' DNA sample. This implies some subtle conformational change which is not reversed by return to neutral pH. In the same conditions, only poly(purine)-poly(pyrimidine) polymers behaved in a different manner, during premelting, according to whether they were previously acidified or not. The properties of acidified and reneutralized T7 DNA were also investigated for Escherichia coli RNA polymerase binding and transcription. An inhibition of RNA synthesis and chain initiation was observed. The results suggest that the binding of the enzyme is affected. RNA synthesized is specific but there is a decrease in the number and in the stability of the RNA-polymerase-DNA complexes. | Influence of DNA acidification on DNA premelting and template properties. Acidification of a T7 DNA sample was found to be partly irreversible as ultraviolet difference spectra measured at various sub-melting temperatures were different from those observed for a 'normal' DNA sample. This implies some subtle conformational change which is not reversed by return to neutral pH. In the same conditions, only poly(purine)-poly(pyrimidine) polymers behaved in a different manner, during premelting, according to whether they were previously acidified or not. The properties of acidified and reneutralized T7 DNA were also investigated for Escherichia coli RNA polymerase binding and transcription. An inhibition of RNA synthesis and chain initiation was observed. The results suggest that the binding of the enzyme is affected. RNA synthesized is specific but there is a decrease in the number and in the stability of the RNA-polymerase-DNA complexes. | [
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PMID:7457 | Reconstitution of chemically synthesized ribooligonucleotides with naturally occurring tRNA fragments. | Chemically synthesized yeast tRNA terminal fragments were reconstituted with natural tRNA fragments which were obtained by partial digestion with RNase T1. The synthetic 3'-nonanucleotide (I) accepted alanine (3% with respect to the intact tRNA) when combined with a 4-fold excess of the natural 5'-quarter and the chemically synthesized hexanucleotide (II) stimulated the aminoacylation of the natural 3'-half molecule. | Reconstitution of chemically synthesized ribooligonucleotides with naturally occurring tRNA fragments. Chemically synthesized yeast tRNA terminal fragments were reconstituted with natural tRNA fragments which were obtained by partial digestion with RNase T1. The synthetic 3'-nonanucleotide (I) accepted alanine (3% with respect to the intact tRNA) when combined with a 4-fold excess of the natural 5'-quarter and the chemically synthesized hexanucleotide (II) stimulated the aminoacylation of the natural 3'-half molecule. | [
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PMID:7458 | Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L. | An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000. | Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L. An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000. | [
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PMID:7459 | Cerebral blood flow, cerebral metabolism and cerebrospinal fluid biochemistry in brain-injured patients after exposure to hyperbaric oxygen. | A series of head-injured patients, in coma, were treated with hyperbaric oxygen (OHP) at 2.5 atm. Cerebral blood flow (CBF), cerebral metabolic rates of oxygen (CMRO2), glucose (CMRGL), and lactate (CMRL act), and various cerebrospinal fluid (CSF) parameters were measured before and 2 h after the treatment. Pre-OHP and post-OHP average values of arterial blood and CSF lactate, and CMRL act were higher than normal, while CBF, CMRO2 and CSF oxygen pressure (PO2) were lower. CBF tended to increase after OHP in some patients and to decrease in others. This discrepancy and the conflicting results of the literature can be tentatively explained in assuming that there is a different effect of OHP on normal brain circulation as compared to impaired brain circulation. Changes of cerebral metabolic rates were inconsistent and did not relate to changes of CBF, except with repeated studies of the same patient. A correlation was found between the variations of CMRGL and those of arterial blood and CSF glucose content. CSF PO2, CSF acid-base balance, and CSF lactate content did not vary, and arterial PO2 showed a consistent fall. In two patients who were neurologically improved after OHP exposure, the CBF and metabolic changes were not the same. | Cerebral blood flow, cerebral metabolism and cerebrospinal fluid biochemistry in brain-injured patients after exposure to hyperbaric oxygen. A series of head-injured patients, in coma, were treated with hyperbaric oxygen (OHP) at 2.5 atm. Cerebral blood flow (CBF), cerebral metabolic rates of oxygen (CMRO2), glucose (CMRGL), and lactate (CMRL act), and various cerebrospinal fluid (CSF) parameters were measured before and 2 h after the treatment. Pre-OHP and post-OHP average values of arterial blood and CSF lactate, and CMRL act were higher than normal, while CBF, CMRO2 and CSF oxygen pressure (PO2) were lower. CBF tended to increase after OHP in some patients and to decrease in others. This discrepancy and the conflicting results of the literature can be tentatively explained in assuming that there is a different effect of OHP on normal brain circulation as compared to impaired brain circulation. Changes of cerebral metabolic rates were inconsistent and did not relate to changes of CBF, except with repeated studies of the same patient. A correlation was found between the variations of CMRGL and those of arterial blood and CSF glucose content. CSF PO2, CSF acid-base balance, and CSF lactate content did not vary, and arterial PO2 showed a consistent fall. In two patients who were neurologically improved after OHP exposure, the CBF and metabolic changes were not the same. | [
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PMID:7460 | A new type of fluorocarbon liquid oxygenator. | A new oxygenator based on gas transfer across liquid-liquid interfaces (liquid oxygenator) is introduced. Perfluorinated chemicals are used as gas carriers. This liquid oxygenator differs from others in its concept basing on the dispersion of the blood droplets in an inert liquid by means of shear forces. This method allows high efficiency both with respect to saturation and to required blood flow. The results of gas transfer experiments in vitro and in vivo are satisfactory. | A new type of fluorocarbon liquid oxygenator. A new oxygenator based on gas transfer across liquid-liquid interfaces (liquid oxygenator) is introduced. Perfluorinated chemicals are used as gas carriers. This liquid oxygenator differs from others in its concept basing on the dispersion of the blood droplets in an inert liquid by means of shear forces. This method allows high efficiency both with respect to saturation and to required blood flow. The results of gas transfer experiments in vitro and in vivo are satisfactory. | [
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PMID:7461 | A simplified bloodless procedure for extensive hepatectomy. Experimental study in the pig. | An original procedure of extensive bloodless hepatectomy is described in the pig. Complete vascular liver isolation with aorta clamping up to 25 min allows 60-75% bloodless hepatectomy without any portal triad dissection. Perfect hemostasis and shortening of liver ischemia is obtained with the use of a gelatin-resorcin-formaldehyde adhesive. Hemodynamic tolerance is good, and liver regeneration appears to be as fast as in minor species. | A simplified bloodless procedure for extensive hepatectomy. Experimental study in the pig. An original procedure of extensive bloodless hepatectomy is described in the pig. Complete vascular liver isolation with aorta clamping up to 25 min allows 60-75% bloodless hepatectomy without any portal triad dissection. Perfect hemostasis and shortening of liver ischemia is obtained with the use of a gelatin-resorcin-formaldehyde adhesive. Hemodynamic tolerance is good, and liver regeneration appears to be as fast as in minor species. | [
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PMID:7466 | Purification and some properties of NADP+ -specific isocitrate dehydrogenase from an extreme thermophile, Thermus flavus AT-62. | Thermostable NADP+ -specific isocitrate dehydrogenase (EC 1.1.1.42) was purified from crude extract of an extremely thermophilic bacterium Thermus flavus AT-62 through DEAE-cellulose column, acetone fractionation, DEAE-Sephadex A-50 column and isoelectric focussing. The enzyme was purified about 500-folds in its specific activity and purity was found to be about 96%. The enzyme was not inactivated after 60 min at 70 degrees C, but 20 and 80% of the activity were lost after 60 min at 80 degrees and 90 degrees C, respectively. Oxalacetate plus glyoxylate (each 1 nM) demonstrated 75% inhibition of the activity in concerted manner. The degree of the inhibition and the affinity of the enzyme for isocitrate and NADP+ decreased with the rise of temperature, especially above 60 degrees C. The activation energy below and above 60 degrees C were 14,500 and 8,000 cal per mole respectively. In CD spectra negative bands at 210 and 220nm were observed and alpha-helix content was calculated to be about 26%. In the course of heating up to 60 degrees practically no change in CD bands are observed, but above 60 degrees the depth of CD bands decreased gradually and remarkably above 80 degrees C. The effect of temperature on kinetic parameters and secondary structures of the enzyme was discussed in relation to the temperature adaptation of the organism. | Purification and some properties of NADP+ -specific isocitrate dehydrogenase from an extreme thermophile, Thermus flavus AT-62. Thermostable NADP+ -specific isocitrate dehydrogenase (EC 1.1.1.42) was purified from crude extract of an extremely thermophilic bacterium Thermus flavus AT-62 through DEAE-cellulose column, acetone fractionation, DEAE-Sephadex A-50 column and isoelectric focussing. The enzyme was purified about 500-folds in its specific activity and purity was found to be about 96%. The enzyme was not inactivated after 60 min at 70 degrees C, but 20 and 80% of the activity were lost after 60 min at 80 degrees and 90 degrees C, respectively. Oxalacetate plus glyoxylate (each 1 nM) demonstrated 75% inhibition of the activity in concerted manner. The degree of the inhibition and the affinity of the enzyme for isocitrate and NADP+ decreased with the rise of temperature, especially above 60 degrees C. The activation energy below and above 60 degrees C were 14,500 and 8,000 cal per mole respectively. In CD spectra negative bands at 210 and 220nm were observed and alpha-helix content was calculated to be about 26%. In the course of heating up to 60 degrees practically no change in CD bands are observed, but above 60 degrees the depth of CD bands decreased gradually and remarkably above 80 degrees C. The effect of temperature on kinetic parameters and secondary structures of the enzyme was discussed in relation to the temperature adaptation of the organism. | [
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PMID:7467 | Maintainance of specificity, information, and thermostability in thermophilic Bacillus sp. glutamine synthetase. | Glutamine synthetase has been purified to homogeneity from B. subtilis (37 degrees) B. stearothermophilus (55 degrees), and B. caldolyticus (75 degrees). Those characteristics compared include size (6.0 +/- 0.3 X 10(5) daltons), quaternary structure (12 SU) amino acid content, substrate Km's and specificity for structural analogs, metal ion activation, number and kind of separate feedback modifier sites, and the complexity of modifier-substrate and modifier-modifier site interactions. Although the 37 degrees and 55 degrees systems are quite similar, the 75 degrees system shows important alterations in substrate specificity and modes of modifier action. Whereas at 37 degrees and 55 degrees AMP inhibits synergistically with amino acids (glycine, glutamine, histidine), the 75 degrees enzyme is inhibited directly by the products ADP, (which assumes the role of AMP) and glutamine, plus other ligands. Ligand binding domains are compared and found to be very different. Thermostabilization occurs by (a) protection by bound L-glutamate, (b) protein aggregation, (c) trends in the content of total polar residues, total Asx + Flx residues, the average hydrophobicity, and (d) disulfide bond cross-linking. Such studies provide insights to molecular evolution occurring with changes in environmental stress. | Maintainance of specificity, information, and thermostability in thermophilic Bacillus sp. glutamine synthetase. Glutamine synthetase has been purified to homogeneity from B. subtilis (37 degrees) B. stearothermophilus (55 degrees), and B. caldolyticus (75 degrees). Those characteristics compared include size (6.0 +/- 0.3 X 10(5) daltons), quaternary structure (12 SU) amino acid content, substrate Km's and specificity for structural analogs, metal ion activation, number and kind of separate feedback modifier sites, and the complexity of modifier-substrate and modifier-modifier site interactions. Although the 37 degrees and 55 degrees systems are quite similar, the 75 degrees system shows important alterations in substrate specificity and modes of modifier action. Whereas at 37 degrees and 55 degrees AMP inhibits synergistically with amino acids (glycine, glutamine, histidine), the 75 degrees enzyme is inhibited directly by the products ADP, (which assumes the role of AMP) and glutamine, plus other ligands. Ligand binding domains are compared and found to be very different. Thermostabilization occurs by (a) protection by bound L-glutamate, (b) protein aggregation, (c) trends in the content of total polar residues, total Asx + Flx residues, the average hydrophobicity, and (d) disulfide bond cross-linking. Such studies provide insights to molecular evolution occurring with changes in environmental stress. | [
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PMID:7468 | Properties of enzymes from Clostridium thermoaceticum and Clostridium formicoaceticum. | Methylenetetrahydrofolate dehydrogenase from C. thermoaceticum and C. formicoaceticum have been purified to homogeneity and compared. The two enzymes are very similar physically, chemically, and kinetically, but he C. thermoaceticum enzyme has a higher thermostablility, which is an intrinsic property of the protein. Formate dehydrogenase enzymes from both bacteria require selenite and tungstate for formation and these enzymes also appear to have similar properties, although the C. thermoaceticum is stable at 70 degrees C for more than one hour. Acetate kinase from C. thermoaceticum appears to be under metabolic control. It can be concluded that enzymes from C. thermoaceticum, although they are more thermostable, are very similar to corresponding enzymes from mesophilic organisms. | Properties of enzymes from Clostridium thermoaceticum and Clostridium formicoaceticum. Methylenetetrahydrofolate dehydrogenase from C. thermoaceticum and C. formicoaceticum have been purified to homogeneity and compared. The two enzymes are very similar physically, chemically, and kinetically, but he C. thermoaceticum enzyme has a higher thermostablility, which is an intrinsic property of the protein. Formate dehydrogenase enzymes from both bacteria require selenite and tungstate for formation and these enzymes also appear to have similar properties, although the C. thermoaceticum is stable at 70 degrees C for more than one hour. Acetate kinase from C. thermoaceticum appears to be under metabolic control. It can be concluded that enzymes from C. thermoaceticum, although they are more thermostable, are very similar to corresponding enzymes from mesophilic organisms. | [
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PMID:7470 | Studies on the inhibition of thermolysin. | Thei nhibition of the thermolysin catalyzed hydrolysis of FA-Gly-Leu-NH2 and FA-Gly-Phe-NH2 has been reported. The results suggest a model for substrate and inhibitor binding involving the hydrophobic specificity pocket, Arg-203 and Glu-143. | Studies on the inhibition of thermolysin. Thei nhibition of the thermolysin catalyzed hydrolysis of FA-Gly-Leu-NH2 and FA-Gly-Phe-NH2 has been reported. The results suggest a model for substrate and inhibitor binding involving the hydrophobic specificity pocket, Arg-203 and Glu-143. | [
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PMID:7472 | Muscle metabolism investigated at rest and during exercise and/or pharmacological treatment by vasodilators. | The Ruderman's preparation was utilized to investigate in situ some aspects of the muscular metabolism both at rest and during submaximal exercise upon bilateral sciatic nerve stimulation, and/or during perfusion with some vasodilators: papaverine, caffeine, nicergoline, bamethan. O2 and glucose uptake, the production of lactate and the glycogen level of the muscle were studied. The availability of acetoacetate modifies the muscular metabolism; other fuels (e.g. the ketone body itself) are probably used for energetic purposes both under basal conditions and during exercise, even in the presence of high glycogen concentrations in the tissues. Some vasodilators increase the utilization of other fuels in replacement of glycogen (e.g. nicergoline), some others increase the ulilization of glycogen itself (e.g. bamethan and caffeine), whereas others (e.g. papaverine) do not effect the biochemical parameters studied. The present data confirm the importance both of the substrate availability and of the power of drugs to interfere with some enzymatic systems which modulate the utilization of available substrates, especially during exercise. | Muscle metabolism investigated at rest and during exercise and/or pharmacological treatment by vasodilators. The Ruderman's preparation was utilized to investigate in situ some aspects of the muscular metabolism both at rest and during submaximal exercise upon bilateral sciatic nerve stimulation, and/or during perfusion with some vasodilators: papaverine, caffeine, nicergoline, bamethan. O2 and glucose uptake, the production of lactate and the glycogen level of the muscle were studied. The availability of acetoacetate modifies the muscular metabolism; other fuels (e.g. the ketone body itself) are probably used for energetic purposes both under basal conditions and during exercise, even in the presence of high glycogen concentrations in the tissues. Some vasodilators increase the utilization of other fuels in replacement of glycogen (e.g. nicergoline), some others increase the ulilization of glycogen itself (e.g. bamethan and caffeine), whereas others (e.g. papaverine) do not effect the biochemical parameters studied. The present data confirm the importance both of the substrate availability and of the power of drugs to interfere with some enzymatic systems which modulate the utilization of available substrates, especially during exercise. | [
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PMID:7473 | Factors in the evolution of hemoglobin function. | The packaging of vertebrate blood hemoglobins within cells places subtle constraints on hemoglobin evolution. Since the concentration of hemoglobin is near the solubility limit a selective advantage should exist for a noncomplementary external topology of amino acid residues. Further, any change in charge on the protein should alter ion distribution across the cell membrane and so modify ion-sensitive oxygen transport. An efficient hemoglobin must not only combine readily with oxygen at prevailing environmental oxygen pressures, but must also release it at metabolically appropriate pressures. These adaptations frequently employ different strategies to achieve the same objective in different animals. Some hemoglobins have evolved special properties unrelated to the transport of oxygen to metabolizing tissues. Thus many teleost fish have hemoglobins that discharge much of their oxygen at low pH even at high oxygen pressures. This property appears to aid in filling the swim bladder with oxygen. The hemoglobins of elasmobranchs have evoked a unique resistance to urea as a consequence of the high urea content of their blood. Sometimes the functional adaptations of hemoglobins are achieved by multiple hemoglobins in the same cells. Often, however, different red cell populations with functionally unique hemoglobins arise sequentially during ontogeny. | Factors in the evolution of hemoglobin function. The packaging of vertebrate blood hemoglobins within cells places subtle constraints on hemoglobin evolution. Since the concentration of hemoglobin is near the solubility limit a selective advantage should exist for a noncomplementary external topology of amino acid residues. Further, any change in charge on the protein should alter ion distribution across the cell membrane and so modify ion-sensitive oxygen transport. An efficient hemoglobin must not only combine readily with oxygen at prevailing environmental oxygen pressures, but must also release it at metabolically appropriate pressures. These adaptations frequently employ different strategies to achieve the same objective in different animals. Some hemoglobins have evolved special properties unrelated to the transport of oxygen to metabolizing tissues. Thus many teleost fish have hemoglobins that discharge much of their oxygen at low pH even at high oxygen pressures. This property appears to aid in filling the swim bladder with oxygen. The hemoglobins of elasmobranchs have evoked a unique resistance to urea as a consequence of the high urea content of their blood. Sometimes the functional adaptations of hemoglobins are achieved by multiple hemoglobins in the same cells. Often, however, different red cell populations with functionally unique hemoglobins arise sequentially during ontogeny. | [
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PMID:7476 | Testicular microlithiasis with sterility. | A case report of male infertility associated with testicular microlithiasis is presented. This rare condition has not previously been associated with infertility. A variable maturation arrest was observed in the spermatogenic epithelium. The question of whether the microliths are the cause of the infertility or whether both conditions are the result of some unknown agent must await further elucidation. | Testicular microlithiasis with sterility. A case report of male infertility associated with testicular microlithiasis is presented. This rare condition has not previously been associated with infertility. A variable maturation arrest was observed in the spermatogenic epithelium. The question of whether the microliths are the cause of the infertility or whether both conditions are the result of some unknown agent must await further elucidation. | [
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PMID:7477 | Quantitative parameters for light microscopic assessment of the tubuli seminiferi. | For quantitative, histologic assessment of the testis tubuli there are four suitable parameters: diameter of tubulus, area of tubulus, number of cell nuclei per tubulus, and area of nuclei of the various cells participating in spermatogenesis. For estimating areas, the integrative method of measurement, either with a grid or with the aid of the manual optical picture-analysis system, is proposed. The tubulus diameter can be determined rapidly and easily, although the tubulus size can be assessed more accurately by area estimation. An assessment of reproductive capacity in detail is possible by means of rather time-consuming determination of cell nucleus number and nuclear area, whereby the stages of the cycle of the seminiferous epithelium must be considered. | Quantitative parameters for light microscopic assessment of the tubuli seminiferi. For quantitative, histologic assessment of the testis tubuli there are four suitable parameters: diameter of tubulus, area of tubulus, number of cell nuclei per tubulus, and area of nuclei of the various cells participating in spermatogenesis. For estimating areas, the integrative method of measurement, either with a grid or with the aid of the manual optical picture-analysis system, is proposed. The tubulus diameter can be determined rapidly and easily, although the tubulus size can be assessed more accurately by area estimation. An assessment of reproductive capacity in detail is possible by means of rather time-consuming determination of cell nucleus number and nuclear area, whereby the stages of the cycle of the seminiferous epithelium must be considered. | [
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PMID:7482 | Influence of a meal on the absorption of cimetidine. A new histamine H2-receptor antagonist. | Absorption of an oral dose of 300 mg of Cimetidine was studied after an overnight fast in six healthy male volunteers on three separate occasions, 20 min before, during, and 2 h after a standard breakfast. Compared to the doses taken before and after the meal, the dose taken with the meal showed a significant delay in the time taken to reach therapeutic blood concentrations of the drug with no reduction in the period of time during which this concentration was maintained. We, therefore, suggest that the drug should be taken with a meal to achieve optimal acid inhibition in the interdigestive period. | Influence of a meal on the absorption of cimetidine. A new histamine H2-receptor antagonist. Absorption of an oral dose of 300 mg of Cimetidine was studied after an overnight fast in six healthy male volunteers on three separate occasions, 20 min before, during, and 2 h after a standard breakfast. Compared to the doses taken before and after the meal, the dose taken with the meal showed a significant delay in the time taken to reach therapeutic blood concentrations of the drug with no reduction in the period of time during which this concentration was maintained. We, therefore, suggest that the drug should be taken with a meal to achieve optimal acid inhibition in the interdigestive period. | [
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PMID:7517 | Hemoglobin function in stored blood. XIII. A citrate-adenine preservative with optimal pH to maintain red cell 2,3-DPG (function) and ATP (viability). | Increasing pH by a 0.5 increment over the commonly used preservative, acid-citrate-dextrose with adenine (ACD-Ad), results in a significant improvement in 2,3-DPG, with no significant loss in concentrations of ATP. The intermediate pH preservative, 6.0, also had ATP concentrations which equaled those of the low pH preservatives, 5.0 and 5.5, from the 21st to the 42nd day of storage. A citrate-adenine preservative, with a pH between 5.5 and 6.0, would seem to be optimal for maintenance of hemoglobin function and red cell viability, as determined by measurements of 2,3-DPG and ATP concentrations. | Hemoglobin function in stored blood. XIII. A citrate-adenine preservative with optimal pH to maintain red cell 2,3-DPG (function) and ATP (viability). Increasing pH by a 0.5 increment over the commonly used preservative, acid-citrate-dextrose with adenine (ACD-Ad), results in a significant improvement in 2,3-DPG, with no significant loss in concentrations of ATP. The intermediate pH preservative, 6.0, also had ATP concentrations which equaled those of the low pH preservatives, 5.0 and 5.5, from the 21st to the 42nd day of storage. A citrate-adenine preservative, with a pH between 5.5 and 6.0, would seem to be optimal for maintenance of hemoglobin function and red cell viability, as determined by measurements of 2,3-DPG and ATP concentrations. | [
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PMID:7519 | [Studies on biosynthesis of short-chain fatty acids (author's transl)]. | Studies were reported here on biosynthesis of short-chain fatty acids in lactating rabbit mammary glands. The maximal incorporation from [1-14C] acetate into total fatty acids were observed in microsomes and supernatant fractions of mammary glands but the synthetic rate either in the microsomes alone or in the supernatant alone was rather low. However approximately 80% of the maximal rate was restored in the supernatant for n-butyric acid synthesis. The incorporation from [1-14C] acetate into-n-butyric acid was markedly stimulated in the presence of NADH, compared to NADPH while total fatty acids synthesis was more dependent on NADPH. Long-chain fatty acids synthesis from [1-14C] acetate was decreased markedly by the addition of avidin although n-butyric acid formation was restored to 80%. Then by the addition of malonyl CoA or biotin the avidin system, middle- and long-chain fatty acids were recovered again. [1-14C] propionate was incorporated into even-numbered chain fatty acids as well as odd chain fatty acids. The synthesis of total fatty acids from [1-14C] propionate was more dependent on NADPH-generating system than either NADH or NADPH. [1-14C] bicarbonate was also incorporated slightly into fatty acids, such as decanoic or dodecanoic acids. In the reduction from either acetoacetyl CoA or crotonyl CoA, the tritium of NADP3H was stereospecifically incorporated into the beta-position of n-butyric acid. The reaction of acetoacetyl CoA reduction was much more dependent on NADH while crotonyl CoA was more reduced with NADPH. There was no difference between the dependencies on NADH and NADPH in the reduction of 2-hexenyl CoA. | [Studies on biosynthesis of short-chain fatty acids (author's transl)]. Studies were reported here on biosynthesis of short-chain fatty acids in lactating rabbit mammary glands. The maximal incorporation from [1-14C] acetate into total fatty acids were observed in microsomes and supernatant fractions of mammary glands but the synthetic rate either in the microsomes alone or in the supernatant alone was rather low. However approximately 80% of the maximal rate was restored in the supernatant for n-butyric acid synthesis. The incorporation from [1-14C] acetate into-n-butyric acid was markedly stimulated in the presence of NADH, compared to NADPH while total fatty acids synthesis was more dependent on NADPH. Long-chain fatty acids synthesis from [1-14C] acetate was decreased markedly by the addition of avidin although n-butyric acid formation was restored to 80%. Then by the addition of malonyl CoA or biotin the avidin system, middle- and long-chain fatty acids were recovered again. [1-14C] propionate was incorporated into even-numbered chain fatty acids as well as odd chain fatty acids. The synthesis of total fatty acids from [1-14C] propionate was more dependent on NADPH-generating system than either NADH or NADPH. [1-14C] bicarbonate was also incorporated slightly into fatty acids, such as decanoic or dodecanoic acids. In the reduction from either acetoacetyl CoA or crotonyl CoA, the tritium of NADP3H was stereospecifically incorporated into the beta-position of n-butyric acid. The reaction of acetoacetyl CoA reduction was much more dependent on NADH while crotonyl CoA was more reduced with NADPH. There was no difference between the dependencies on NADH and NADPH in the reduction of 2-hexenyl CoA. | [
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PMID:7520 | An analysis of the relationships among obesity, plasma insulin and hepatic lipogenic enzymes in "viable yellow obese" mice (Avy/a). | The development of obesity, hyperinsulinemia and six hepatic lipogenic enzymes in Avy/a mice were compared to that in a/a mice. Correlation between body weight, liver weight, plasma insulin concentration and activities of hepatic enzymes was analyzed. In the Avy/a mice, body weight, liver weight and plasma insulin level increased steadily as the mice aged. In the a/a mice, the change of these three parameters was much slower. Plasma insulin concentration in a/a mice did not increase until eight months of age. Compared with a/a mice, Avy/a mice had higher 6-phosphogluconate dehydrogenase and fatty acid synthetase activities at two months of age; lower citrate cleavage enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities at three months of age; lower citrate cleavage enzyme and glucose-6-phosphate dehydrogenase and higher acetyl CoA carboxylase activities at five months of age; and higher malic enzyme, citrate cleavage enzyme and 6-phosphogluconate dehydrogenase activities at eight months of age. There were significant correlations between plasma insulin level and body weight and between plasma insulin level and the activities of malic enzyme and citrate cleavage enzyme in Avy/a mice. The correlation between body weight and malic enzyme and citrate cleavage enzyme activities disappeared after the analysis was adjusted for plasma insulin level. | An analysis of the relationships among obesity, plasma insulin and hepatic lipogenic enzymes in "viable yellow obese" mice (Avy/a). The development of obesity, hyperinsulinemia and six hepatic lipogenic enzymes in Avy/a mice were compared to that in a/a mice. Correlation between body weight, liver weight, plasma insulin concentration and activities of hepatic enzymes was analyzed. In the Avy/a mice, body weight, liver weight and plasma insulin level increased steadily as the mice aged. In the a/a mice, the change of these three parameters was much slower. Plasma insulin concentration in a/a mice did not increase until eight months of age. Compared with a/a mice, Avy/a mice had higher 6-phosphogluconate dehydrogenase and fatty acid synthetase activities at two months of age; lower citrate cleavage enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities at three months of age; lower citrate cleavage enzyme and glucose-6-phosphate dehydrogenase and higher acetyl CoA carboxylase activities at five months of age; and higher malic enzyme, citrate cleavage enzyme and 6-phosphogluconate dehydrogenase activities at eight months of age. There were significant correlations between plasma insulin level and body weight and between plasma insulin level and the activities of malic enzyme and citrate cleavage enzyme in Avy/a mice. The correlation between body weight and malic enzyme and citrate cleavage enzyme activities disappeared after the analysis was adjusted for plasma insulin level. | [
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PMID:7532 | Studies on normal human bone marrow cultures in controlled environment thin-film culture systems. | Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first two weeks while an increasing number of monocytes and macrophages appeared in the media of of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO2 concentrations, i.e. 2%, 5%, 10% similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. | Studies on normal human bone marrow cultures in controlled environment thin-film culture systems. Two thin film culture systems, the controlled environment steady state system (SS) and the rocker tube configuration of that system (RT), were used to identify some of the conditions that appear to maintain morphologic and functional characteristics of cells of human bone marrow explants in vitro. The systems configuration assured continual gassing, control and easy monitoring of the cultures. Cytocentrifuge preparations of media of specimens cultured in RT disclosed, though in decreasing numbers, various hematopoietic cells for periods exceeding one month. Hematopoietic cells shed from specimens cultured in the SS system were retained in the culture tubes; cells of the myelocytic series predominated for the first two weeks while an increasing number of monocytes and macrophages appeared in the media of of older cultures. Histologic examination of cultured explants disclosed preservation of the marrow architecture and the persistence of hematopoietic cells. Specimens cultured in RT tubes tended to be less cellular than similar cultures placed in dialysis bags or as cultured in the SS system. Immunoglobulins (Ig) were released into the culture media at a constant rate throughout the period of culture. Specimens that were cultured at a controlled pH of 7.4 released 2 to more than 4 times as much Ig as similar specimens maintained at a pH level of 7.1. There were no definitive differences in Ig levels in the cultures maintained at comparable pH levels and overlaid with various CO2 concentrations, i.e. 2%, 5%, 10% similarly, no differences in Ig levels were found in specimens cultured in media containing fetal bovine sera as opposed to horse sera. | [
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PMID:7533 | Bactericidal mechanisms in rabbit alveolar macrophages: evidence against peroxidase and hydrogen peroxide bactericidal mechanisms. | The role of peroxidase-mediated bacterial killing by rabbit alveolar macrophages was examined. During 3 h of incubation in vitro, alveolar macrophages ingested and killed greater than 88% of the Streptococcus faecalis, Proteus mirabilis, or Streptococcus pneumoniae present in the incubation mixture. Preincubation of alveolar macrophages with inhibitors of catalase, 3-amino-1,2,4-triazole or sodium nitrite, did not alter their bactericidal potential. Iodination of ingested zymosan particles, a peroxidase-dependent and hydrogen peroxide-dependent reaction, was not observed, in spite of vigorous phagocytosis by alveolar macrophages. Furthermore, iodination by alveolar macrophages was not significantly increased when peroxidase-coated zymosan particles were ingested. The results suggest that hydrogen peroxide may not be available to the phagocytic vacuole for microbial killing. Since tetrazolium dye reduction reflects the activity of an oxidase responsible for stimulated oxygen consumption by polymorphonuclear leukocytes, this reaction was also measured. Rabbit alveolar macrophages incubated with latex particles did not exhibit an increased dye reduction compared with resting cells. The absence of significant stimulation of tetrazolium dye reduction indicates that the oxidase reaction does not occur in the proximity of the phagocytic vacuole of alveolar macrophages. | Bactericidal mechanisms in rabbit alveolar macrophages: evidence against peroxidase and hydrogen peroxide bactericidal mechanisms. The role of peroxidase-mediated bacterial killing by rabbit alveolar macrophages was examined. During 3 h of incubation in vitro, alveolar macrophages ingested and killed greater than 88% of the Streptococcus faecalis, Proteus mirabilis, or Streptococcus pneumoniae present in the incubation mixture. Preincubation of alveolar macrophages with inhibitors of catalase, 3-amino-1,2,4-triazole or sodium nitrite, did not alter their bactericidal potential. Iodination of ingested zymosan particles, a peroxidase-dependent and hydrogen peroxide-dependent reaction, was not observed, in spite of vigorous phagocytosis by alveolar macrophages. Furthermore, iodination by alveolar macrophages was not significantly increased when peroxidase-coated zymosan particles were ingested. The results suggest that hydrogen peroxide may not be available to the phagocytic vacuole for microbial killing. Since tetrazolium dye reduction reflects the activity of an oxidase responsible for stimulated oxygen consumption by polymorphonuclear leukocytes, this reaction was also measured. Rabbit alveolar macrophages incubated with latex particles did not exhibit an increased dye reduction compared with resting cells. The absence of significant stimulation of tetrazolium dye reduction indicates that the oxidase reaction does not occur in the proximity of the phagocytic vacuole of alveolar macrophages. | [
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PMID:7534 | Interactions between aminoglycoside antibiotics and carbenicillin or ticarillin. | Carbenicillin or ticarcillin were incubated individually with each of the following antibiotics: gentamicin, tobramycin, sisomicin, amikacin. The residual activity of each aminoglycoside in this mixture was assayed enzymatically. Amikacin was inactivated the least of the aminoglycosides. Both penicillins inactivated each aminoglycoside to a similar extent by a degree which varied according to the medium of incubaiton, the least inactivation being seen in pooled human serum and the most in phosphate buffer at pH 7.4. | Interactions between aminoglycoside antibiotics and carbenicillin or ticarillin. Carbenicillin or ticarcillin were incubated individually with each of the following antibiotics: gentamicin, tobramycin, sisomicin, amikacin. The residual activity of each aminoglycoside in this mixture was assayed enzymatically. Amikacin was inactivated the least of the aminoglycosides. Both penicillins inactivated each aminoglycoside to a similar extent by a degree which varied according to the medium of incubaiton, the least inactivation being seen in pooled human serum and the most in phosphate buffer at pH 7.4. | [
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PMID:7535 | Effects of beta-adrenergic stimulation on bone-marrow function in normal and sublethally irradiated mice. I. The effect of isoproterenol on cAMP content in bone-marrow cells in vivo and in vitro. | The effect of isoproterenol (IPR) on bone-marrow cAMP content was investigated in vivo and in vitro. In unirradiated CFW mice, the bone-marrow cAMP content was found to be elevated by the administration of noradrenaline, adrenaline and isoproterenol. After IPR administration, the increase in cAMP was biphasic with maxima at 1 and 15 min. An increase in cAMP content was also noted in bone-marrow of sublethally-irradiated mice, but no further increase was observed 15 min after the administration of IPR. Elevation of cAMP by either IPR or radiation was prevented by pretreatment with the beta-adrenergic blocking agent--propranolol. IPR was also effective in increasing the cAMP content when added to suspension of bone-marrow cells. This effect was abolished by propranolol. IPR did not increase cAMP levels in bone-marrow cells isolated from irradiated animals. The results suggest that the differentiated bone-marrow cells have beta-adrenergic receptors. | Effects of beta-adrenergic stimulation on bone-marrow function in normal and sublethally irradiated mice. I. The effect of isoproterenol on cAMP content in bone-marrow cells in vivo and in vitro. The effect of isoproterenol (IPR) on bone-marrow cAMP content was investigated in vivo and in vitro. In unirradiated CFW mice, the bone-marrow cAMP content was found to be elevated by the administration of noradrenaline, adrenaline and isoproterenol. After IPR administration, the increase in cAMP was biphasic with maxima at 1 and 15 min. An increase in cAMP content was also noted in bone-marrow of sublethally-irradiated mice, but no further increase was observed 15 min after the administration of IPR. Elevation of cAMP by either IPR or radiation was prevented by pretreatment with the beta-adrenergic blocking agent--propranolol. IPR was also effective in increasing the cAMP content when added to suspension of bone-marrow cells. This effect was abolished by propranolol. IPR did not increase cAMP levels in bone-marrow cells isolated from irradiated animals. The results suggest that the differentiated bone-marrow cells have beta-adrenergic receptors. | [
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PMID:7537 | Lorazepam and diazepam in anxious outpatients. A controlled study. | The response of 134 anxious neurotic outpatients to lorazepam, diazepam, and placebo was assessed in a 4-week double-blind trial. Both active drugs produced significantly more symptom reduction than placebo. Lorazepam, however, proved effective primarily in those patients who did not complain of sedation, and produced greatest improvement in initially sicker patients. Sedation was significantly more disturbing to lorazepam-treated patients than to diazepam-treated patients. Present findings suggested that 3 mg/day of lorazepam may be too high a dosage for mildly anxious patients, while 15 mg/day of diazepam seems an appropriate dosage for mildly anxious patients but may be too low a dosage for highly anxious patients. | Lorazepam and diazepam in anxious outpatients. A controlled study. The response of 134 anxious neurotic outpatients to lorazepam, diazepam, and placebo was assessed in a 4-week double-blind trial. Both active drugs produced significantly more symptom reduction than placebo. Lorazepam, however, proved effective primarily in those patients who did not complain of sedation, and produced greatest improvement in initially sicker patients. Sedation was significantly more disturbing to lorazepam-treated patients than to diazepam-treated patients. Present findings suggested that 3 mg/day of lorazepam may be too high a dosage for mildly anxious patients, while 15 mg/day of diazepam seems an appropriate dosage for mildly anxious patients but may be too low a dosage for highly anxious patients. | [
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PMID:7542 | Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root. | Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into a dimethylsulfoxide (DMSO)-methanol mixture and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with a methanol mixture; a portion of the eluate is treated with nitrous acid and the reserpine-rescinnamine content is determined by measuring the intensity of fluorescence of the oxidation product. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 mg and 50 mg commercial tablets, and a 45 mg synthetic tablet formulation) : 0.174% +/- 0.0112, 0.131% +/- 0.0047, 0.160% +/- 0.0100, and 0.153% +/- 0.0083, respectively. | Collaborative study of a spectrofluorometric assay for Rauwolfia serpentina tablets and powdered root. Reserpine-rescinnamine group alkaloids are extracted from Rauwolfia serpentina preparations into a dimethylsulfoxide (DMSO)-methanol mixture and diluted with 0.5N H2SO4. The chloroform extract of this solution is passed through a 0.1N NaOH-Celite column and then through a silica gel column. The weakly basic alkaloids trapped on the latter column are eluted with a methanol mixture; a portion of the eluate is treated with nitrous acid and the reserpine-rescinnamine content is determined by measuring the intensity of fluorescence of the oxidation product. The following means and standard deviations (11 collaborators) were obtained for the determination of reserpine-rescinnamine group alkaloids in 4 samples of Rauwolfia serpentina (NF reference powder, 100 mg and 50 mg commercial tablets, and a 45 mg synthetic tablet formulation) : 0.174% +/- 0.0112, 0.131% +/- 0.0047, 0.160% +/- 0.0100, and 0.153% +/- 0.0083, respectively. | [
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PMID:7543 | Iron uptake in colicin B-resistant mutants of Escherichia coli K-12. | Four classes of colicin B-resistant mutants of Escherichia coli K-12 were examined for defects in iron uptake. All four mutant classes (cbt, exbC, exbB, and tonB) were defective in the uptake of ferri-ennterochelin. The tonB mutant was also defective in citrate-, ferrichrome-, and rhodoturulic acid-mediated iron uptake. The defects in iron transport were reflected in increased sensitivity to iron chelators and to chromium and aluminium salts, and in hypersecretion of enterochelin. One of the mutants (cbt) was apparently defective in outer membrane ferri-enterochelin receptor activity. aroE derivatives (unable to synthesize enterochelin) of the four mutant classes and the parent strain produced increased amounts of two outer membranes polypeptides when grown under iron stress. These polypeptides are implicated in ferri-enterochelin receptor activity. | Iron uptake in colicin B-resistant mutants of Escherichia coli K-12. Four classes of colicin B-resistant mutants of Escherichia coli K-12 were examined for defects in iron uptake. All four mutant classes (cbt, exbC, exbB, and tonB) were defective in the uptake of ferri-ennterochelin. The tonB mutant was also defective in citrate-, ferrichrome-, and rhodoturulic acid-mediated iron uptake. The defects in iron transport were reflected in increased sensitivity to iron chelators and to chromium and aluminium salts, and in hypersecretion of enterochelin. One of the mutants (cbt) was apparently defective in outer membrane ferri-enterochelin receptor activity. aroE derivatives (unable to synthesize enterochelin) of the four mutant classes and the parent strain produced increased amounts of two outer membranes polypeptides when grown under iron stress. These polypeptides are implicated in ferri-enterochelin receptor activity. | [
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PMID:7544 | Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of competent pneumococci. | The conversion of surface-adsorbed deoxyribonucleic acid (DNA) molecules to a state in which they are inaccessible to exogenous deoxyribonuclease requires specifically calcium ions; magnesium ions cannot replace calcium ions. Virtually maximal levels of nuclease-resistant DNA binding and genetic transformation can be obtained in media free from magnesium and containing only calcium ions. It is suggested that the calcium-requiring process is the transport of DNA molecules across the plasma membrane. Magnesium ions stimulate both the loss of surface-adsorbed DNA to the medium and the extracellular degradation of DNA. | Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of competent pneumococci. The conversion of surface-adsorbed deoxyribonucleic acid (DNA) molecules to a state in which they are inaccessible to exogenous deoxyribonuclease requires specifically calcium ions; magnesium ions cannot replace calcium ions. Virtually maximal levels of nuclease-resistant DNA binding and genetic transformation can be obtained in media free from magnesium and containing only calcium ions. It is suggested that the calcium-requiring process is the transport of DNA molecules across the plasma membrane. Magnesium ions stimulate both the loss of surface-adsorbed DNA to the medium and the extracellular degradation of DNA. | [
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PMID:7545 | Mechanism of autolysis of Neisseria gonorrhoeae. | The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid). | Mechanism of autolysis of Neisseria gonorrhoeae. The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid). | [
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PMID:7546 | Magnitude of the protonmotive force in respiring Staphylococcus aureus and Escherichia coli. | The membrane potential and pH gradient developed across the plasma membranes of whole cells of Staphylococcus aureus and spheroplasts of Escherichia coli were estimated. The distributions of potassium ions in the presence of valinomycin and the pH gradient across the membrane were determined from the changes in pK and pH observed in the external medium during transition from the energized respiring state to the de-engerized resting condition. The protonmotive force in respiring cells was estimated at 211 mV for S. aureus and 230 mV for E. coli at external pH values of approximately 6.5. The adequacy of these protonmotive forces as a driving force for substrate accumulation or adenosine 5'-triphosphate synthesis is discussed. | Magnitude of the protonmotive force in respiring Staphylococcus aureus and Escherichia coli. The membrane potential and pH gradient developed across the plasma membranes of whole cells of Staphylococcus aureus and spheroplasts of Escherichia coli were estimated. The distributions of potassium ions in the presence of valinomycin and the pH gradient across the membrane were determined from the changes in pK and pH observed in the external medium during transition from the energized respiring state to the de-engerized resting condition. The protonmotive force in respiring cells was estimated at 211 mV for S. aureus and 230 mV for E. coli at external pH values of approximately 6.5. The adequacy of these protonmotive forces as a driving force for substrate accumulation or adenosine 5'-triphosphate synthesis is discussed. | [
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PMID:7547 | Proton movements coupled to lactate and alanine transport in Escherichia coli: isolation of mutants with altered stoichiometry in alanine transport. | The addition of lactate to lightly buffered suspensions of resting cells of Escherichia coli caused an increase in the pH of the extracellular phase as lactate and protons entered the cell together. From the magnitude of the pH change and the non-electrogenic character of lactate uptake, we concluded that the stoichiometry of the process was 1 proton/lactate anion. The addition of alanine caused a slow increase in pH, also apparently due to the transport of the amino acid by a symport mechanism with 1 proton/alanine stoichiometry. When cells were grown in the chemostat with alanine as sole carbon source and as limiting nutrient, this stoichiometry was found to alter to 2 protons/alanine, and then to 4 protons/alanine. These increases stoichiometries were due to the selection of mutants. The consequences of these changes on the potential uptake capacity of the cells are discussed. | Proton movements coupled to lactate and alanine transport in Escherichia coli: isolation of mutants with altered stoichiometry in alanine transport. The addition of lactate to lightly buffered suspensions of resting cells of Escherichia coli caused an increase in the pH of the extracellular phase as lactate and protons entered the cell together. From the magnitude of the pH change and the non-electrogenic character of lactate uptake, we concluded that the stoichiometry of the process was 1 proton/lactate anion. The addition of alanine caused a slow increase in pH, also apparently due to the transport of the amino acid by a symport mechanism with 1 proton/alanine stoichiometry. When cells were grown in the chemostat with alanine as sole carbon source and as limiting nutrient, this stoichiometry was found to alter to 2 protons/alanine, and then to 4 protons/alanine. These increases stoichiometries were due to the selection of mutants. The consequences of these changes on the potential uptake capacity of the cells are discussed. | [
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PMID:7548 | Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli. | The enzyme L-threonine dehydrogenase was demonstrated in extracts of Escherichia coli K-12, and was shown to be the first enzyme of the pathway converting threonine to glycine. The enzyme was induced by L-leucine, but not by its substrate, L-threonine. The metabolic significance of leucine as a catabolic signal for amino acid degradation is considered. | Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli. The enzyme L-threonine dehydrogenase was demonstrated in extracts of Escherichia coli K-12, and was shown to be the first enzyme of the pathway converting threonine to glycine. The enzyme was induced by L-leucine, but not by its substrate, L-threonine. The metabolic significance of leucine as a catabolic signal for amino acid degradation is considered. | [
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PMID:7549 | Partial characterization of a temperature-sensitive mutation affecting acetyl coenzyme A carboxylase in Escherichia coli K-12. | A temperature-sensitive mutation in Escherichia coli K-12 was shown to affect acetyl coenzyme A carboxylase and to map at min 63. This site is designated fabE. | Partial characterization of a temperature-sensitive mutation affecting acetyl coenzyme A carboxylase in Escherichia coli K-12. A temperature-sensitive mutation in Escherichia coli K-12 was shown to affect acetyl coenzyme A carboxylase and to map at min 63. This site is designated fabE. | [
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PMID:7550 | Potentiometric studies on benzeneboronic acid-alpha-chymotrypsin interactions. | When the competitive inhibitor benzeneboronic acid (BBA) forms a complex with alpha-chymotrypsin [EC 3.4.21.1] protons are released in the acidic pH region. The proton release can be measured by a difference potentiometric technique. The proton release is also observed in chymotrypsinogen A but not in TRCK-, DIP-, and anhydrochymotrypsins. Based on these observations, a simple procedure to estimate the equilibrium constants of the trigonal-tetrahedral interconversion of BBA is proposed. Thermodynamic parameters of the ionization of His 57 and of each step involved in BBA binding can be estimated from the temperature dependence of the proton release. Those of His 57 are essentially the same as those of imidazole in water. Regarding the interconversion of BBA on the enzyme, the value of delta S is similar to delta S not equal to of the deacylation step of nonspecific substrates, and delta H is remarkably reduced from that for the ionization of BBA in water. The enthalpic gain of enzymic process is suggested to be due to the change of the proton acceptor, which is water in the case of the ionization of BBA in water, to imidazole on the enzyme. | Potentiometric studies on benzeneboronic acid-alpha-chymotrypsin interactions. When the competitive inhibitor benzeneboronic acid (BBA) forms a complex with alpha-chymotrypsin [EC 3.4.21.1] protons are released in the acidic pH region. The proton release can be measured by a difference potentiometric technique. The proton release is also observed in chymotrypsinogen A but not in TRCK-, DIP-, and anhydrochymotrypsins. Based on these observations, a simple procedure to estimate the equilibrium constants of the trigonal-tetrahedral interconversion of BBA is proposed. Thermodynamic parameters of the ionization of His 57 and of each step involved in BBA binding can be estimated from the temperature dependence of the proton release. Those of His 57 are essentially the same as those of imidazole in water. Regarding the interconversion of BBA on the enzyme, the value of delta S is similar to delta S not equal to of the deacylation step of nonspecific substrates, and delta H is remarkably reduced from that for the ionization of BBA in water. The enthalpic gain of enzymic process is suggested to be due to the change of the proton acceptor, which is water in the case of the ionization of BBA in water, to imidazole on the enzyme. | [
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PMID:7551 | The removal of non-collagen components from newborn calf dermis with magnesium chloride solution. | 1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis. | The removal of non-collagen components from newborn calf dermis with magnesium chloride solution. 1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis. | [
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PMID:7552 | Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum. | The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed. | Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum. The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed. | [
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PMID:7553 | Two glycosulfatases from the liver of a marine gastropod, Charonia lampas. Partial purification and properties. | Two glycosulfatases [EC 3.1.6.3], I and II, were purified 31.3- and 33.9-fold respectively, from a crude extract of the liver of Charonia lampas. The purification was carried out by the following chromatographic procedures; phosphocellulose, Sephadex G-150, Concanavalin A-Sepharose and isoelectric focussing. The enzyme preparations obtained were practically free from arylsulfatase [EC 3.1.6.1] contamination. Both glycosulfatases are probably glycoproteins differing in their carbohydrate moieties. The molecular weights of glycosulfatase I and II were estimated to be about 112,000 and 79,000 respectively. They had the same optimum pH of 5.5, and the same Km value of 25.0 mM for glucose 6-sulfate. | Two glycosulfatases from the liver of a marine gastropod, Charonia lampas. Partial purification and properties. Two glycosulfatases [EC 3.1.6.3], I and II, were purified 31.3- and 33.9-fold respectively, from a crude extract of the liver of Charonia lampas. The purification was carried out by the following chromatographic procedures; phosphocellulose, Sephadex G-150, Concanavalin A-Sepharose and isoelectric focussing. The enzyme preparations obtained were practically free from arylsulfatase [EC 3.1.6.1] contamination. Both glycosulfatases are probably glycoproteins differing in their carbohydrate moieties. The molecular weights of glycosulfatase I and II were estimated to be about 112,000 and 79,000 respectively. They had the same optimum pH of 5.5, and the same Km value of 25.0 mM for glucose 6-sulfate. | [
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PMID:7554 | Comparative studies on polyguanylate polymerase and polyadenylate polymerase activities in the DNA-dependent RNA polymerase I fraction from cauliflower. | The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium. | Comparative studies on polyguanylate polymerase and polyadenylate polymerase activities in the DNA-dependent RNA polymerase I fraction from cauliflower. The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium. | [
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PMID:7555 | Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells. | DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment. | Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells. DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment. | [
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PMID:7556 | Formation of interchain disulfide bonds in Bence Jones proteins and immunoglobulins. | The formation of the interchain disulfide bonds in partially reduced Bence Jones proteins and immunoglobulins was studied in the presence of glutathione. It was found that only oxidized glutathione (GSSG) was effective for the formation of the interchain disulfide bonds in type gamma Bence Jones proteins and IgG. In type kappa Bence Jones proteins, on the other hand, no formation of the inter L-L disulfide bond was observed in the presence of GSSG at above pH 6. The kinetic pattern of disulfide bond formation of Bence Jones proteins was well interpreted by assuming that two monomers of a type gamma protein dimer are discriminated (monomers 1 and 2) and that only an intermediate in which the SH group on monomer 1 is blocked with GSSG can form a disulfide-bonded dimer and the intermediate in which the SH group on monomer 2 is blocked with GSSG can not. Comparison of the kinetic data for the formation of the interchain disulfide bonds of IgG with those for Bence Jones proteins suggested that H chain-GSSG mixed disulfide is a principal intermediate for the formation of the inter H-L disulfide bond. | Formation of interchain disulfide bonds in Bence Jones proteins and immunoglobulins. The formation of the interchain disulfide bonds in partially reduced Bence Jones proteins and immunoglobulins was studied in the presence of glutathione. It was found that only oxidized glutathione (GSSG) was effective for the formation of the interchain disulfide bonds in type gamma Bence Jones proteins and IgG. In type kappa Bence Jones proteins, on the other hand, no formation of the inter L-L disulfide bond was observed in the presence of GSSG at above pH 6. The kinetic pattern of disulfide bond formation of Bence Jones proteins was well interpreted by assuming that two monomers of a type gamma protein dimer are discriminated (monomers 1 and 2) and that only an intermediate in which the SH group on monomer 1 is blocked with GSSG can form a disulfide-bonded dimer and the intermediate in which the SH group on monomer 2 is blocked with GSSG can not. Comparison of the kinetic data for the formation of the interchain disulfide bonds of IgG with those for Bence Jones proteins suggested that H chain-GSSG mixed disulfide is a principal intermediate for the formation of the inter H-L disulfide bond. | [
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PMID:7557 | Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase. | The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy. | Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase. The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy. | [
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PMID:7558 | Activation process of pepsinogen. | The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding. | Activation process of pepsinogen. The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding. | [
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PMID:7559 | An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes. | Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5. | An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes. Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5. | [
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PMID:7560 | Purification and properties of cathepsin D from porcine spleen. | Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species. | Purification and properties of cathepsin D from porcine spleen. Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species. | [
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PMID:7561 | Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH. | The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo. | Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH. The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo. | [
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PMID:7562 | Studies on the subcellular localization and properties of bis(monoacylglyceryl)phosphate biosynthesis in rat liver. | Phosphatidylglycerol conversion to bis(monoacylglyceryl)phosphate by rat liver homogenate was studied and maximum rates of synthesis were observed at pH 4.4. The distribution of bis(monoacylglyceryl)P synthetase in rat liver subcellular fractions was determined, and evidence is presented establishing the lysosomes as the site of bis(monoacylglyceryl)P synthesis. In addition to phosphatidylglycerol, 1-acyl- and 2-acyllysophosphatidylglycerol also served as precursors for bis(monoacylglyceryl)P with lysosomes as an enzyme source. Bis(monoacylglyceryl)P synthesis did not require high energy intermediates or cofactors. The possibility that a lysosomal phospholipase A with acyl transferase activity catalyzes the formation of bis(monoacylglyceryl)P was investigated. Heat stability and inhibitor studies suggested that this is probably not the case. Lysosomes were shown to be unable to synthesize phosphatidylglycerol, and lipid analyses showed that lysosomes do not contain phosphatidylglycerol or lysophosphatidylglycerol. Bis(monoacylglyceryl)P synthesis in the cell may require the interaction of lysosomes with a phosphatidylglycerol-containing membrane. | Studies on the subcellular localization and properties of bis(monoacylglyceryl)phosphate biosynthesis in rat liver. Phosphatidylglycerol conversion to bis(monoacylglyceryl)phosphate by rat liver homogenate was studied and maximum rates of synthesis were observed at pH 4.4. The distribution of bis(monoacylglyceryl)P synthetase in rat liver subcellular fractions was determined, and evidence is presented establishing the lysosomes as the site of bis(monoacylglyceryl)P synthesis. In addition to phosphatidylglycerol, 1-acyl- and 2-acyllysophosphatidylglycerol also served as precursors for bis(monoacylglyceryl)P with lysosomes as an enzyme source. Bis(monoacylglyceryl)P synthesis did not require high energy intermediates or cofactors. The possibility that a lysosomal phospholipase A with acyl transferase activity catalyzes the formation of bis(monoacylglyceryl)P was investigated. Heat stability and inhibitor studies suggested that this is probably not the case. Lysosomes were shown to be unable to synthesize phosphatidylglycerol, and lipid analyses showed that lysosomes do not contain phosphatidylglycerol or lysophosphatidylglycerol. Bis(monoacylglyceryl)P synthesis in the cell may require the interaction of lysosomes with a phosphatidylglycerol-containing membrane. | [
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PMID:7563 | Properties of the guanylate cyclase-guanosine 3':5'-monophosphate system of rat renal cortex. Activation of guanylate cyclase and calcium-independent modulation of tissue guanosine 3':5'-monophosphate by sodium azide. | The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism. | Properties of the guanylate cyclase-guanosine 3':5'-monophosphate system of rat renal cortex. Activation of guanylate cyclase and calcium-independent modulation of tissue guanosine 3':5'-monophosphate by sodium azide. The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism. | [
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PMID:7564 | Requirement of an essential thiol group and ferric iron for the activity of the progesterone-induced porcine uterine purple phosphatase. | The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L-cysteine, sulfite, and cyanide. It is inhibited by various mercurials, iodoacetamide, O-iodosobenzoate, and hydrogen peroxide. Thiols increase the specific phosphatase activity from 25 to about 300 units per mg of enzyme. This activation is accompanied by a shift in the extinction maximum to higher energy to yield a protein with a pink coloration. Following maximum activation there is a gradual decrease in enzyme activity and protein color which is accompanied by loss of ferrous iron from the protein. Sodium dithionite at 10 mM or higher causes an immediate inhibition of phosphatase activity and bleaching of color, and can be used to prepare the iron-free apoprotein. The latter can be partially reactivated by Fe3+ salts but not by Fe2+. The Fe3+ restores the pink form of the enzyme with a specific activity of about 200 units/mg of protein. Cu2+ also causes some reactivation, but other metal ions were ineffective. ESR studies showed that the pink form of phosphatase contains approximately 1 atom of high spin ferric iron per molecule. It is concluded that the phosphatase requires a free thiol and Fe3+ for activity. Reduction of the iron leads to complete loss of both color and enzyme activity. The color change from purple to pink represents disulfide reduction and is not due to reduction of iron. | Requirement of an essential thiol group and ferric iron for the activity of the progesterone-induced porcine uterine purple phosphatase. The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L-cysteine, sulfite, and cyanide. It is inhibited by various mercurials, iodoacetamide, O-iodosobenzoate, and hydrogen peroxide. Thiols increase the specific phosphatase activity from 25 to about 300 units per mg of enzyme. This activation is accompanied by a shift in the extinction maximum to higher energy to yield a protein with a pink coloration. Following maximum activation there is a gradual decrease in enzyme activity and protein color which is accompanied by loss of ferrous iron from the protein. Sodium dithionite at 10 mM or higher causes an immediate inhibition of phosphatase activity and bleaching of color, and can be used to prepare the iron-free apoprotein. The latter can be partially reactivated by Fe3+ salts but not by Fe2+. The Fe3+ restores the pink form of the enzyme with a specific activity of about 200 units/mg of protein. Cu2+ also causes some reactivation, but other metal ions were ineffective. ESR studies showed that the pink form of phosphatase contains approximately 1 atom of high spin ferric iron per molecule. It is concluded that the phosphatase requires a free thiol and Fe3+ for activity. Reduction of the iron leads to complete loss of both color and enzyme activity. The color change from purple to pink represents disulfide reduction and is not due to reduction of iron. | [
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PMID:7565 | Distribution of enzyme activities in subcellular fractions of bovine retina. | Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations. | Distribution of enzyme activities in subcellular fractions of bovine retina. Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations. | [
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PMID:7566 | Kinetic studies of sheep kidney gamma-glutamyl transpeptidase. | The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions. | Kinetic studies of sheep kidney gamma-glutamyl transpeptidase. The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions. | [
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PMID:7567 | Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure. | Neurospora crassa glutamine synthetase was purified to homogeneity by a procedure based on affinity chromatography. The enzyme is adsorbed to a matrix of anthranilic acid bound to Sepharose and eluted with AMP. Different experimental approaches indicate that the enzyme has an octameric structure formed by subunits of identical molecular weight. | Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure. Neurospora crassa glutamine synthetase was purified to homogeneity by a procedure based on affinity chromatography. The enzyme is adsorbed to a matrix of anthranilic acid bound to Sepharose and eluted with AMP. Different experimental approaches indicate that the enzyme has an octameric structure formed by subunits of identical molecular weight. | [
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PMID:7570 | Chromatographic behaviour of alkaloids on thin layers of cation exchangers. II. Alginic acid, Rexyn 102, Dowex 50-X4 and CMCNa. | The chromatographic behaviour of 48 alkaloids on cation exchangers with cellulose, paraffin and polystyrene matrices in both the acid and sodium salt forms has been investigated. Water-organic solvent mixtures, aqueous buffer solutions and organic and mineral acid solutions in both water and in aqueous-organic solvents have been used as eluents. The retention mechanisms of these compounds on alginic acid, Rexyn 102 (Hplus) and Dowex 50-X4 (Hplus) thin layers are discussed. Interesting separations of the alkaloids were carried out on alginic acid and Rexyn 102 (Hplus). | Chromatographic behaviour of alkaloids on thin layers of cation exchangers. II. Alginic acid, Rexyn 102, Dowex 50-X4 and CMCNa. The chromatographic behaviour of 48 alkaloids on cation exchangers with cellulose, paraffin and polystyrene matrices in both the acid and sodium salt forms has been investigated. Water-organic solvent mixtures, aqueous buffer solutions and organic and mineral acid solutions in both water and in aqueous-organic solvents have been used as eluents. The retention mechanisms of these compounds on alginic acid, Rexyn 102 (Hplus) and Dowex 50-X4 (Hplus) thin layers are discussed. Interesting separations of the alkaloids were carried out on alginic acid and Rexyn 102 (Hplus). | [
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PMID:7569 | Membrane receptors for hormones and neurotransmitters. | Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function. | Membrane receptors for hormones and neurotransmitters. Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function. | [
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PMID:7571 | Prolactin secretion by metoclopramide in man. | Six men and nine women were given intravenous injections of 2.5 mg of metoclopramide to assess its potential as a stimulus to prolactin release. Following the administration of metoclopramide, there was prompt increase in serum prolactin to a peak response of 38.2 +/- 3.9 ng/ml in men and 103 +/- 10.2 ng/ml in women. The prolactin response to metoclopramide in men was compared with the response to 400 mug of TRH in 10 men. The peak response after TRH was 22.4 +/- 2.2 ng/ml, which was significantly less than that observed after metoclopramide. Pretreatment with 500 mg of L-dopa suppressed the prolactin response to metoclopramide in 6 men to a mean response of 16.3 +/- 4.3 ng/ml. We have concluded that metoclopramide is a safe, reliable, and potent stimulus of prolactin secretion and exerts this effect by blocking dopamine receptors in the hypothalamus and decreasing prolactin inhibiting factor. It is free of side effects and is a useful alternative to chlorpromazine. | Prolactin secretion by metoclopramide in man. Six men and nine women were given intravenous injections of 2.5 mg of metoclopramide to assess its potential as a stimulus to prolactin release. Following the administration of metoclopramide, there was prompt increase in serum prolactin to a peak response of 38.2 +/- 3.9 ng/ml in men and 103 +/- 10.2 ng/ml in women. The prolactin response to metoclopramide in men was compared with the response to 400 mug of TRH in 10 men. The peak response after TRH was 22.4 +/- 2.2 ng/ml, which was significantly less than that observed after metoclopramide. Pretreatment with 500 mg of L-dopa suppressed the prolactin response to metoclopramide in 6 men to a mean response of 16.3 +/- 4.3 ng/ml. We have concluded that metoclopramide is a safe, reliable, and potent stimulus of prolactin secretion and exerts this effect by blocking dopamine receptors in the hypothalamus and decreasing prolactin inhibiting factor. It is free of side effects and is a useful alternative to chlorpromazine. | [
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PMID:7572 | Deficient 17 beta-hydroxysteroid oxidoreductase activity in testes from a male pseudohermaphrodite. | 17beta-Hydroxysteroid oxidoreductase (17 beta-HOR) activity in testicular tissue from a male pseudohermaphrodite (MP) who had elevated plasma LH and androstenedione (A) levels, and normal dehydroepiandrosterone (DHA) levels was compared by in vitro studies to that of testicular tissue from a normal man. The 17 beta-HOR activity from the MP was localized predominantly in the microsomal fraction, as it is in normal testicular tissue. With DHA, A, and estrone as substrates, the 17 beta-HOR activity of the MP was decreased in the presence of NADPH, but not NADH, in comparison with the normal. NADPH was the preferred co-factor for 17 beta-HOR from the normal testes, while 17 beta-HOR activity from the MP testes was less with NADPH than with NADH as cofactor. These results indicate that the inefficient testosterone production in the MP testes may be accounted for by a deficiency of NADPH-dependent 17 beta-HOR activity. Further studies suggested that 3 beta-hydroxysteroid oxidoreductase-isomerase (3 beta-HOR) was increased in the MP testes and was much greater than 17 beta-HOR activity with DHA as substrate. These findings largely explain the elevated plasma A levels with normal DHA levels, and suggest that DHA leads to A leads to testosterone was the major route of testosterone biosynthesis in the MP testes. | Deficient 17 beta-hydroxysteroid oxidoreductase activity in testes from a male pseudohermaphrodite. 17beta-Hydroxysteroid oxidoreductase (17 beta-HOR) activity in testicular tissue from a male pseudohermaphrodite (MP) who had elevated plasma LH and androstenedione (A) levels, and normal dehydroepiandrosterone (DHA) levels was compared by in vitro studies to that of testicular tissue from a normal man. The 17 beta-HOR activity from the MP was localized predominantly in the microsomal fraction, as it is in normal testicular tissue. With DHA, A, and estrone as substrates, the 17 beta-HOR activity of the MP was decreased in the presence of NADPH, but not NADH, in comparison with the normal. NADPH was the preferred co-factor for 17 beta-HOR from the normal testes, while 17 beta-HOR activity from the MP testes was less with NADPH than with NADH as cofactor. These results indicate that the inefficient testosterone production in the MP testes may be accounted for by a deficiency of NADPH-dependent 17 beta-HOR activity. Further studies suggested that 3 beta-hydroxysteroid oxidoreductase-isomerase (3 beta-HOR) was increased in the MP testes and was much greater than 17 beta-HOR activity with DHA as substrate. These findings largely explain the elevated plasma A levels with normal DHA levels, and suggest that DHA leads to A leads to testosterone was the major route of testosterone biosynthesis in the MP testes. | [
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PMID:7573 | Counterimmunoelectrophoresis of staphylococcal antibody. | Modifications of the staphylococcal counterimmunoelectrophoresis technique were evaluated to determine how variations in the procedure affected results. Neither a buffer pH range of 7.8 to 9.0 nor buffer molarity of 0.015 or 0.025 when tested in combinations caused appreciable differences. However, use of different agar preparations or delay in addition of antigen to the test slide altered the location of the precipitin band. Agarose was found to be more sensitive in determining the serum precipitin titer and provided a better photographic record than either Ionagar or Noble agar. | Counterimmunoelectrophoresis of staphylococcal antibody. Modifications of the staphylococcal counterimmunoelectrophoresis technique were evaluated to determine how variations in the procedure affected results. Neither a buffer pH range of 7.8 to 9.0 nor buffer molarity of 0.015 or 0.025 when tested in combinations caused appreciable differences. However, use of different agar preparations or delay in addition of antigen to the test slide altered the location of the precipitin band. Agarose was found to be more sensitive in determining the serum precipitin titer and provided a better photographic record than either Ionagar or Noble agar. | [
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PMID:7574 | Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement. | Recent work has indicated that superoxide is involved in the manganese-stimulated oxidation of NADPH by crude granule preparations of guinea pig neutrophils. The characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from these results that it is unnecessary to invoke a manganese-requiring enzyme as a component of the phagocytically stimulated respiratory system of the neutrophil. | Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement. Recent work has indicated that superoxide is involved in the manganese-stimulated oxidation of NADPH by crude granule preparations of guinea pig neutrophils. The characteristics of a model manganese-requiring NADPH-oxidizing system that employs a defined O2-generator have now been compared to the original neutrophil-granule system. With respect to pH dependence, cyanide sensitivity, and reduced pyridine nucleotide specificity, the properties of the two systems are very similar. Additional information has been obtained concerning cation specificity and the kinetics of the metal-catalyzed NADPH oxidation. From the similarities between the properties of the model and neutrophil particle systems, we postulate that the manganese-dependent NADPH oxidation observed in the presence of neutrophil granules represents in large part of nonenzymatic free radical chain involving the oxidation of NADPH to NADP, with O2- as both the chain initiator and one of the propagating species. In this reaction, the neutrophil particles serve only as a source of O2-. Further, the same changes in kinetics (decrease in apparent Km for NADPH) observed previously when granules from phagocytizing rather than resting cells were employed could be mimicked by varying the rate of O2-generation by the model system. We conclude from these results that it is unnecessary to invoke a manganese-requiring enzyme as a component of the phagocytically stimulated respiratory system of the neutrophil. | [
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PMID:7575 | Determinants of lung bacterial clearance in normal mice. | The determinants of the lung clearance of Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus were studied in normal mice after exposure to an aerosol of viable bacteria and 99mTc-labeled dead bacteria. The fraction of bacteria in lungs that remained viable 4 h after exposure were: S. pneumoniae, 7.3%; K. pneumoniae, 121%; E. coli, 88.5%; S. aureus, 27.6%. The rate of physical removal of bacterial particles (Kmc) was determined from the change in lung 99mTc counts with time: Kmc ranged between 7 and 12%/h and and was similar in all species. The rate of mucociliary clearance and of intrapulmonary bacterial killing (Kk + Kmc) was calculated from the change in bacterial counts with time in animals that had received tetracycline to inhibit bacterial multiplication. Kk, the rate of intrapulmonary killing, was obtained by subtraction of Kmc from (Kk + Kmc). The calculated values for Kk were: S. pneumoniae, - 87%/h; K. pneumoniae, - 17%/h; E. coli, - 18%/h; S. aureus, - 22%/h. The rate of intrapulmonary bacterial multiplication (Kg) was estimated from the relationship of bacterial counts in tetracycline and nontetracycline-treated animals, assuming that tetracycline altered only Kg. Kg, expressed as the doubling time, was: S. pneumoniae, 310 min; K. pneumoniae, 217 min; E.coli, 212 min; S. aureus, infinity (no multiplication). The data indicate that the marked differences in the clearance of these species from the normal mouse lung result from the interaction of differing rates of in vivo bacterial multiplication and killing. | Determinants of lung bacterial clearance in normal mice. The determinants of the lung clearance of Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus were studied in normal mice after exposure to an aerosol of viable bacteria and 99mTc-labeled dead bacteria. The fraction of bacteria in lungs that remained viable 4 h after exposure were: S. pneumoniae, 7.3%; K. pneumoniae, 121%; E. coli, 88.5%; S. aureus, 27.6%. The rate of physical removal of bacterial particles (Kmc) was determined from the change in lung 99mTc counts with time: Kmc ranged between 7 and 12%/h and and was similar in all species. The rate of mucociliary clearance and of intrapulmonary bacterial killing (Kk + Kmc) was calculated from the change in bacterial counts with time in animals that had received tetracycline to inhibit bacterial multiplication. Kk, the rate of intrapulmonary killing, was obtained by subtraction of Kmc from (Kk + Kmc). The calculated values for Kk were: S. pneumoniae, - 87%/h; K. pneumoniae, - 17%/h; E. coli, - 18%/h; S. aureus, - 22%/h. The rate of intrapulmonary bacterial multiplication (Kg) was estimated from the relationship of bacterial counts in tetracycline and nontetracycline-treated animals, assuming that tetracycline altered only Kg. Kg, expressed as the doubling time, was: S. pneumoniae, 310 min; K. pneumoniae, 217 min; E.coli, 212 min; S. aureus, infinity (no multiplication). The data indicate that the marked differences in the clearance of these species from the normal mouse lung result from the interaction of differing rates of in vivo bacterial multiplication and killing. | [
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PMID:7576 | Alpha- and beta-adrenergic receptor blocking agents combined with a diuretic in the treatment of essential hypertension. | The antihypertensive efficacy and side effects of the combined therapy with propranolol, phenoxybenzamine, and hydrochlorothiazide were examined in 17 patients with moderate and moderately severe hypertension. Following a control period two to three weeks' duration, propranolol was started in nine patients as the sole antihypertensive agent and together with phenoxybenzamine, in eight. By titrating the dosage against pulse rate and blood pressure response, propranolol was given, in divided doses, from 80 to 160 mg and phenoxybenzamine, from 20 to 50 mg per day. When propranolol was given alone in nine patients, for four to 12 weeks, normal blood pressure was not attained in any patients. During three to ten weeks of combined propranolol and phenoxybenzamine therapy in 17 patients, normal blood pressure (150/90 mm Hg or less) or near-normal pressure (150/100 or less) was attained in 12 patients in the recumbent position and in 15 patients in the upright position, while orthostatic hypotension was not observed. Except for a reduction of ejaculation in three out of six male patients, no other side effects were encountered. The addition of hydrochlorothiazide diuretic in all of the above 17 patients, at a dose of 50-100 mg per day with a concomitant decrease in the dose of phenoxybenzamine, produced a further reduction in blood pressure, and normal or near-normal blood pressure was attained in all subjects. Symptomatic orthostatic hypotension, observed in two patients, was treated by a further readjustment of the dose of phenoxybenzamine, while inhibition of ejaculation was persistent in only one patient. It is concluded that the combined administration of propranolol, phenoxybenzamine, and hydrochlorothiazide in individualized doses is very effective in lowering the blood pressure with minimal side effects. | Alpha- and beta-adrenergic receptor blocking agents combined with a diuretic in the treatment of essential hypertension. The antihypertensive efficacy and side effects of the combined therapy with propranolol, phenoxybenzamine, and hydrochlorothiazide were examined in 17 patients with moderate and moderately severe hypertension. Following a control period two to three weeks' duration, propranolol was started in nine patients as the sole antihypertensive agent and together with phenoxybenzamine, in eight. By titrating the dosage against pulse rate and blood pressure response, propranolol was given, in divided doses, from 80 to 160 mg and phenoxybenzamine, from 20 to 50 mg per day. When propranolol was given alone in nine patients, for four to 12 weeks, normal blood pressure was not attained in any patients. During three to ten weeks of combined propranolol and phenoxybenzamine therapy in 17 patients, normal blood pressure (150/90 mm Hg or less) or near-normal pressure (150/100 or less) was attained in 12 patients in the recumbent position and in 15 patients in the upright position, while orthostatic hypotension was not observed. Except for a reduction of ejaculation in three out of six male patients, no other side effects were encountered. The addition of hydrochlorothiazide diuretic in all of the above 17 patients, at a dose of 50-100 mg per day with a concomitant decrease in the dose of phenoxybenzamine, produced a further reduction in blood pressure, and normal or near-normal blood pressure was attained in all subjects. Symptomatic orthostatic hypotension, observed in two patients, was treated by a further readjustment of the dose of phenoxybenzamine, while inhibition of ejaculation was persistent in only one patient. It is concluded that the combined administration of propranolol, phenoxybenzamine, and hydrochlorothiazide in individualized doses is very effective in lowering the blood pressure with minimal side effects. | [
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PMID:7579 | Milk lipoprotein lipases: a review. | Lipoprotein lipase activity has been found in the milks from severals species where it is assumed to result from leakage from the mammary gland into milk. The function of the enzyme in the gland is apparently to assist in the transfer of blood lipoprotein triacylglycerol fatty acids into milk triacylglycerols. Bovine skim milk is one of the richest sources of lipoprotein lipase and this enzyme has been purified extensively (7000 fold) by affinity chromatography. The lipase has a molecular weight of about 62000, is inhibited by protamine sulfate, 1.0 M sodium chloride, apolipoprotein C-I (apolipoprotein-serine), and apolipoprotein C-III (apolipoprotein-alanine). The enzyme is activated by apolipoprotein C-II (apolipoprotein-glutamic acid), serum, and by heparin to which it also binds. The lipase is highly specific for the primary esters of acylglycerols and exhibits a slight stereospecificity for the sn-1 ester in preference to the sn-3-ester. Bovine milk also has separate activity toward 1-monoacylglycerols. Human milk contains a serum stimulated lipoprotein lipase with many of the characteristics of the enzyme in bovine milk, as well as an enzyme stimulated by bile salts which resembles the sterol ester hydrolase of rat pancreatic juice. The assay, function, purification, characteristics, and substrate specificities of these enzyme are discussed. | Milk lipoprotein lipases: a review. Lipoprotein lipase activity has been found in the milks from severals species where it is assumed to result from leakage from the mammary gland into milk. The function of the enzyme in the gland is apparently to assist in the transfer of blood lipoprotein triacylglycerol fatty acids into milk triacylglycerols. Bovine skim milk is one of the richest sources of lipoprotein lipase and this enzyme has been purified extensively (7000 fold) by affinity chromatography. The lipase has a molecular weight of about 62000, is inhibited by protamine sulfate, 1.0 M sodium chloride, apolipoprotein C-I (apolipoprotein-serine), and apolipoprotein C-III (apolipoprotein-alanine). The enzyme is activated by apolipoprotein C-II (apolipoprotein-glutamic acid), serum, and by heparin to which it also binds. The lipase is highly specific for the primary esters of acylglycerols and exhibits a slight stereospecificity for the sn-1 ester in preference to the sn-3-ester. Bovine milk also has separate activity toward 1-monoacylglycerols. Human milk contains a serum stimulated lipoprotein lipase with many of the characteristics of the enzyme in bovine milk, as well as an enzyme stimulated by bile salts which resembles the sterol ester hydrolase of rat pancreatic juice. The assay, function, purification, characteristics, and substrate specificities of these enzyme are discussed. | [
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PMID:7580 | Synthetic peptides for chymosin and pepsin assays: pH effect and pepsin independent-determination in mixtures. | Peptide I [H-Phe-Gly-His-Phe(NO2)-Phe-Ala-Phe-OMe] hydrolyzed by chymosin with kcat=.3+/-.3 s-1 and KM=7+/-3 mM (pH 4.7) inhibited competitively peptide II [H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe] hydrolysis by chymosin with KI=.23 +/- .12 mM at pH 4.7. In reference conditions (.4 mM peptide, .01 M acetate buffer pH 4.7), the specific activities of porcine pepsin and chymosin on peptide I were 470 +/- 70 nM S-1 and .8 nM S-1 per mg of enzyme. This difference in specific activity for peptide I allowed development of a chymosin-independent pepsin assay for mixtures of these enzymes. In addition, peptide II with a specific activity of 2400 +/- 300 nM S-1 and 154 +/- 20 nM S-1 per mg of porcine pepsin and chymosin provides an alternative to measurement of milk clotting for measurement of chymosin- and pepsin-like activities in commercial rennets. Hydrolysis products of peptide II by chymosin exhibited one ionized group of apparent pK of 3.5 +/- .2 and a molar absorption coefficient change of 1000 +/- 100 at pH 4.7 and at 310 nm. From measurements of the kinetic constants, kcat and KM, from pH 2.5 to 7 with peptide II, chymosin activity depends on the protonation of one group of apparent pK 5.3 +/- .2 in the free enzyme. Rennet powder proved to be fairly stable after a 17-month storage at 4 C. Within the same period, a crystalline chymosin solution kept at --18 C lost 30 to 50% of its activity. | Synthetic peptides for chymosin and pepsin assays: pH effect and pepsin independent-determination in mixtures. Peptide I [H-Phe-Gly-His-Phe(NO2)-Phe-Ala-Phe-OMe] hydrolyzed by chymosin with kcat=.3+/-.3 s-1 and KM=7+/-3 mM (pH 4.7) inhibited competitively peptide II [H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe] hydrolysis by chymosin with KI=.23 +/- .12 mM at pH 4.7. In reference conditions (.4 mM peptide, .01 M acetate buffer pH 4.7), the specific activities of porcine pepsin and chymosin on peptide I were 470 +/- 70 nM S-1 and .8 nM S-1 per mg of enzyme. This difference in specific activity for peptide I allowed development of a chymosin-independent pepsin assay for mixtures of these enzymes. In addition, peptide II with a specific activity of 2400 +/- 300 nM S-1 and 154 +/- 20 nM S-1 per mg of porcine pepsin and chymosin provides an alternative to measurement of milk clotting for measurement of chymosin- and pepsin-like activities in commercial rennets. Hydrolysis products of peptide II by chymosin exhibited one ionized group of apparent pK of 3.5 +/- .2 and a molar absorption coefficient change of 1000 +/- 100 at pH 4.7 and at 310 nm. From measurements of the kinetic constants, kcat and KM, from pH 2.5 to 7 with peptide II, chymosin activity depends on the protonation of one group of apparent pK 5.3 +/- .2 in the free enzyme. Rennet powder proved to be fairly stable after a 17-month storage at 4 C. Within the same period, a crystalline chymosin solution kept at --18 C lost 30 to 50% of its activity. | [
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PMID:7581 | Diet calcium and pH versus mineral balance in Holstein cows 84 days pre- to 2 days postpartum. | Sixteen dry Holstein cows were assigned four groups combining either .2 or 2.1% calcium and pH of either 4.5 or 6.1 in a 2 X 2 factorial arrangement of treatments to study the effect of diet calcium and acidity (pH) on voluntary intake of dry matter, mineral metabolism, and blood constituents. The 86-day experiment was started 84 days prepartum and completed 2 days postpartum. Voluntary dry matter ingestion tended to be higher with higher calcium and acidity (lower pH) diets. Calcium nutriture was improved by more acid diets. More total calcium was apparently absorbed (26 and 7 g) when the diet contained 2.1% calcium, but the .2% calcium diet resulted in a higher apparent percent absorption (25 and 17%) during the dry period. An increased apparent absorption (70 and 5 g) and utilization of diet calcium occurred with the higher calcium treatment at 2 days postpartum. Magnesium and phosphorus utilizations were improved with lower diet calcium. Although experimental treatments had no significant effect on blood minerals, serum calcium decreased 12% 2 days postpartum. A better calcium nutriture was provided by the higher calcium diets when the diets maintained a positive balance for magnesium and phosphorus. | Diet calcium and pH versus mineral balance in Holstein cows 84 days pre- to 2 days postpartum. Sixteen dry Holstein cows were assigned four groups combining either .2 or 2.1% calcium and pH of either 4.5 or 6.1 in a 2 X 2 factorial arrangement of treatments to study the effect of diet calcium and acidity (pH) on voluntary intake of dry matter, mineral metabolism, and blood constituents. The 86-day experiment was started 84 days prepartum and completed 2 days postpartum. Voluntary dry matter ingestion tended to be higher with higher calcium and acidity (lower pH) diets. Calcium nutriture was improved by more acid diets. More total calcium was apparently absorbed (26 and 7 g) when the diet contained 2.1% calcium, but the .2% calcium diet resulted in a higher apparent percent absorption (25 and 17%) during the dry period. An increased apparent absorption (70 and 5 g) and utilization of diet calcium occurred with the higher calcium treatment at 2 days postpartum. Magnesium and phosphorus utilizations were improved with lower diet calcium. Although experimental treatments had no significant effect on blood minerals, serum calcium decreased 12% 2 days postpartum. A better calcium nutriture was provided by the higher calcium diets when the diets maintained a positive balance for magnesium and phosphorus. | [
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PMID:7583 | The role of precipitins and complement activation in the etiology of allergic lung disease. | An experimental model of allergic lung disease has been described that is monitored by analysis of arterial oxygen tension following aerosol challenge with antigen. Rabbits immunized to a classical soluble antigen, human serum albumin (HSA), to the point where severe Arthus skin reactivity was demonstrable, were aerosol-challenged with antigen. Arterial oxygen tension measurements made on pre- and post-challenge samples yielded early, late, and continuous response patterns, reminiscent of those obtained in humans following provocation testing. Aerosol challenge of unimmunized animals with HSA resulted in no change from baseline conditions. Unimmunized rabbits exposed to small and massive (10X) aerosols of Aspergillus spores also demonstrated various postchallenge depressions in arterial oxygen tension as well as decreased levels in hemolytic complement activity, depending on the species of fungus and dose of spores used. Unimmunized animals pretreated with cobra venom factor in a manner known to achieve complement depletion failed to respond with altered arterial oxygen tensions following similar aerosol challenge. It is postulated that although precipitins may play a role in artificial disease initiated by soluble antigens, nonspecific complement activation may be more important in understanding the etiology of spontaneous disease in humans brought about by inhalation of moldy particulate matter. | The role of precipitins and complement activation in the etiology of allergic lung disease. An experimental model of allergic lung disease has been described that is monitored by analysis of arterial oxygen tension following aerosol challenge with antigen. Rabbits immunized to a classical soluble antigen, human serum albumin (HSA), to the point where severe Arthus skin reactivity was demonstrable, were aerosol-challenged with antigen. Arterial oxygen tension measurements made on pre- and post-challenge samples yielded early, late, and continuous response patterns, reminiscent of those obtained in humans following provocation testing. Aerosol challenge of unimmunized animals with HSA resulted in no change from baseline conditions. Unimmunized rabbits exposed to small and massive (10X) aerosols of Aspergillus spores also demonstrated various postchallenge depressions in arterial oxygen tension as well as decreased levels in hemolytic complement activity, depending on the species of fungus and dose of spores used. Unimmunized animals pretreated with cobra venom factor in a manner known to achieve complement depletion failed to respond with altered arterial oxygen tensions following similar aerosol challenge. It is postulated that although precipitins may play a role in artificial disease initiated by soluble antigens, nonspecific complement activation may be more important in understanding the etiology of spontaneous disease in humans brought about by inhalation of moldy particulate matter. | [
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PMID:7584 | The effect of pertussis and beta adrenergic-blocking agents on mast cells. | Pertussis vaccine injected ip in doses known to cause hypersensitization resulted in a marked decrease in the number of mast cells from the peritoneal washings of rats and mice. A significant reduction was obtained as early as one day after pertussis injection of ten billion cells in rats and was marked after 5 to 7 days. A maximum reduction in the number of mast cells was obtained by a dose of 20 billion cells. There was no detectable histamine biological activity in the supernatant from peritoneal washings obtained after 10 min, 60 min, and 24 hr from control and pertussis-treated rats, indicating that pertussis did not cause degranulation of mast cells in vivo. The histamine content in the precipitated mast cell pellets from control rats was much higher than the corresponding histamine content from pertussis-treated rats. In rats and mice, propranolol and other beta adrenergic-blocking agents caused degranulation of mast cells in the peritoneal washings in vitro. Practolol was the least effective beta adrenergic-blocking agent in degranulating mast cells. Catecholamines, histamine, 6-hydroxydopamine, methacholine, and pertussis failed to cause any degranulation. Isoproterenol protected the mast cell against the degranulation induced by propranolol. Propranolol caused bluing in rat and mice skin when injected id. Mast cells from control and pertussis-injected rats were equally sensitive to propranolol in vitro. The low recovery of mast cells from the peritoneal washings of rats and mice is thought to be due to mobilization of mast cells away from the peritoneum. | The effect of pertussis and beta adrenergic-blocking agents on mast cells. Pertussis vaccine injected ip in doses known to cause hypersensitization resulted in a marked decrease in the number of mast cells from the peritoneal washings of rats and mice. A significant reduction was obtained as early as one day after pertussis injection of ten billion cells in rats and was marked after 5 to 7 days. A maximum reduction in the number of mast cells was obtained by a dose of 20 billion cells. There was no detectable histamine biological activity in the supernatant from peritoneal washings obtained after 10 min, 60 min, and 24 hr from control and pertussis-treated rats, indicating that pertussis did not cause degranulation of mast cells in vivo. The histamine content in the precipitated mast cell pellets from control rats was much higher than the corresponding histamine content from pertussis-treated rats. In rats and mice, propranolol and other beta adrenergic-blocking agents caused degranulation of mast cells in the peritoneal washings in vitro. Practolol was the least effective beta adrenergic-blocking agent in degranulating mast cells. Catecholamines, histamine, 6-hydroxydopamine, methacholine, and pertussis failed to cause any degranulation. Isoproterenol protected the mast cell against the degranulation induced by propranolol. Propranolol caused bluing in rat and mice skin when injected id. Mast cells from control and pertussis-injected rats were equally sensitive to propranolol in vitro. The low recovery of mast cells from the peritoneal washings of rats and mice is thought to be due to mobilization of mast cells away from the peritoneum. | [
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PMID:7585 | Disodium cromoglycate in ragweed-allergic rhinitis. | This study was designed to test the effectiveness of disodium cromoglycate when compared to placebo in a double-blind study in patients with ragweed allergic rhinitis. Patients were selected on the basis of a clinical history and a 4+ reaction to the intradermal injection of water-soluble ragweed, 0.02 c.c. of 500 PNU/c.c. Active agent/placebo groups were selected at random and were on the drug for approximately 8 wk, commencing 1 wk prior to the onset of the ragweed pollen season. Patient response was evaluated using patient diary cards, number of antihistamine tablets taken, and patient interviews. In the Toronto study, of 17 patients on the active drug, 15 were graded as improved, compared to only 6 of 21 placebo-treated patients who were improved. However, in the Hamilton study, results were less impressive. Nonetheless, it appears that intranasal insufflation of disodium cromoglycate was more effective in reducing ragweed hay fever symptoms than placebo. | Disodium cromoglycate in ragweed-allergic rhinitis. This study was designed to test the effectiveness of disodium cromoglycate when compared to placebo in a double-blind study in patients with ragweed allergic rhinitis. Patients were selected on the basis of a clinical history and a 4+ reaction to the intradermal injection of water-soluble ragweed, 0.02 c.c. of 500 PNU/c.c. Active agent/placebo groups were selected at random and were on the drug for approximately 8 wk, commencing 1 wk prior to the onset of the ragweed pollen season. Patient response was evaluated using patient diary cards, number of antihistamine tablets taken, and patient interviews. In the Toronto study, of 17 patients on the active drug, 15 were graded as improved, compared to only 6 of 21 placebo-treated patients who were improved. However, in the Hamilton study, results were less impressive. Nonetheless, it appears that intranasal insufflation of disodium cromoglycate was more effective in reducing ragweed hay fever symptoms than placebo. | [
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PMID:7588 | Piperacetazine versus thioridazine in the treatment of organic brain disease: a controlled double-blind study. | In a double-blind cross-over study, 50 geriatric patients with organic brain disease were divided into two groups. One group first received piperacetazine for 15 days and then thioridazine for 15 days. For the other group the sequence was reversed. Piperacetazine proved to be at least as effective as thioridazine and seemed to be more effective against certain target symptoms; side effects were less common and less severe. | Piperacetazine versus thioridazine in the treatment of organic brain disease: a controlled double-blind study. In a double-blind cross-over study, 50 geriatric patients with organic brain disease were divided into two groups. One group first received piperacetazine for 15 days and then thioridazine for 15 days. For the other group the sequence was reversed. Piperacetazine proved to be at least as effective as thioridazine and seemed to be more effective against certain target symptoms; side effects were less common and less severe. | [
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PMID:7629 | Duodenal immunoglobulin deficiency in graft versus host disease (GVHD) mice. | The small intestine is a well documented target organ in mouse and human GVHD, and diarrhea is a prominent part of the clinical GVHD syndrome. Although a plethora of systemic immune deficits has been documented in GVHD, the integrity of the small intestinal immune system has not been investigated. A correlation has not been demonstrated between systemic immune dysfuction and the incidence of lymphomas in mouse GVHD survivors. If gastrointestinal immune deficiency exists in mouse GVHD, its possible relationship to GVHD lymphomas, frequently abdominal. should be investigated. GVHD was produced in newborn BLA (C57 BL/Ka females x BALB-C males) mice house in a specific pathogen-free environment by the i.p. inoculation of 10(7) male BALB-C spleen cells. Control mice received syngeneic spleen cells. Twenty GVHD and 16 control mice were sacrificed at 3 weeks and specimens of duodenum were removed for routine histologic and immunofluorescent examination. All but one GVHD mouse (95%) had virtually absent duodenal IgA and IgM. Duodenal cellular fluorescence was demonstrated in all controls. A significant duodenal immunoglobulin deficit has been demonstrated in 3-week-old GVHD mice. The relationship of this finding to GVHD diarrhea, wasting, and neoplasia remains to be determined. | Duodenal immunoglobulin deficiency in graft versus host disease (GVHD) mice. The small intestine is a well documented target organ in mouse and human GVHD, and diarrhea is a prominent part of the clinical GVHD syndrome. Although a plethora of systemic immune deficits has been documented in GVHD, the integrity of the small intestinal immune system has not been investigated. A correlation has not been demonstrated between systemic immune dysfuction and the incidence of lymphomas in mouse GVHD survivors. If gastrointestinal immune deficiency exists in mouse GVHD, its possible relationship to GVHD lymphomas, frequently abdominal. should be investigated. GVHD was produced in newborn BLA (C57 BL/Ka females x BALB-C males) mice house in a specific pathogen-free environment by the i.p. inoculation of 10(7) male BALB-C spleen cells. Control mice received syngeneic spleen cells. Twenty GVHD and 16 control mice were sacrificed at 3 weeks and specimens of duodenum were removed for routine histologic and immunofluorescent examination. All but one GVHD mouse (95%) had virtually absent duodenal IgA and IgM. Duodenal cellular fluorescence was demonstrated in all controls. A significant duodenal immunoglobulin deficit has been demonstrated in 3-week-old GVHD mice. The relationship of this finding to GVHD diarrhea, wasting, and neoplasia remains to be determined. | [
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PMID:7630 | Partial regain of activity in heterologous recombinants of phosphorylcholine-binding M-components from different species. | Recombination experiments were performed with heavy and light chains derived from a Waldenström's IgM with specificity against phosphorylcholine. The recombinant molecules had an association constant for phosphorylcholine in the same order of magnitude as the native IgM; the number of binding sites at saturation was only slightly decreased in the reconstituted molecules, indicating regain of binding activity after recombination of IgM heavy and light chains. Heterologous recombinants obtained with polypeptide chains of another monoclonal IgM without demonstrable binding activity recovered only 5 to 10% of the binding activity of homologous recombinants. Hybrid molecules prepared with heavy and light chains from the phosphorylcholine-binding mouse IgA myeloma protein TEPC-15, however, regained as much as 41% of the binding activity of the homologous recombinants; these data suggest a considerable degree of structural homology shared by the human IgM and the murine IgA proteins with phosphorylcholine-binding specifity. | Partial regain of activity in heterologous recombinants of phosphorylcholine-binding M-components from different species. Recombination experiments were performed with heavy and light chains derived from a Waldenström's IgM with specificity against phosphorylcholine. The recombinant molecules had an association constant for phosphorylcholine in the same order of magnitude as the native IgM; the number of binding sites at saturation was only slightly decreased in the reconstituted molecules, indicating regain of binding activity after recombination of IgM heavy and light chains. Heterologous recombinants obtained with polypeptide chains of another monoclonal IgM without demonstrable binding activity recovered only 5 to 10% of the binding activity of homologous recombinants. Hybrid molecules prepared with heavy and light chains from the phosphorylcholine-binding mouse IgA myeloma protein TEPC-15, however, regained as much as 41% of the binding activity of the homologous recombinants; these data suggest a considerable degree of structural homology shared by the human IgM and the murine IgA proteins with phosphorylcholine-binding specifity. | [
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PMID:7631 | Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction. | Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node. | Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction. Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node. | [
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PMID:7589 | [Voluntary choice of sex. The present state of our knowledge]. | Whether we like it or not, the next demand that the modern couple will make after birth control will be voluntary selection of the sex of their babies. Furthermore, this selection does have an undoubted medical value in preventing certain sex-linked congenital or hereditary diseases. Many research workers have turned their attention to these problems over the past years, especially in the U.S.A., in England and in Germany. After a historical review of the progress that has taken place in this area a study has been undertaken into the actual state of research in the U.S.A. and in Germany, in which the country the Federal Government has created a special department for scientific research. Our present knowledge into the methods that are adopted to obtain a foetus of male of female sex up to the present is then briefly explained. The results obtained by the principle research workers have been summarised. These are between 80 and 90 per cent success. The steps that have marked these discoveries in this field that is ill understood by the majority of doctors and the French public are outlined. | [Voluntary choice of sex. The present state of our knowledge]. Whether we like it or not, the next demand that the modern couple will make after birth control will be voluntary selection of the sex of their babies. Furthermore, this selection does have an undoubted medical value in preventing certain sex-linked congenital or hereditary diseases. Many research workers have turned their attention to these problems over the past years, especially in the U.S.A., in England and in Germany. After a historical review of the progress that has taken place in this area a study has been undertaken into the actual state of research in the U.S.A. and in Germany, in which the country the Federal Government has created a special department for scientific research. Our present knowledge into the methods that are adopted to obtain a foetus of male of female sex up to the present is then briefly explained. The results obtained by the principle research workers have been summarised. These are between 80 and 90 per cent success. The steps that have marked these discoveries in this field that is ill understood by the majority of doctors and the French public are outlined. | [
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PMID:7632 | Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose. | The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase. | Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose. The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase. | [
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PMID:7633 | Urticaria. | Urticaria is a problem often as vexing to the physician as to the patient. The approach to the patient with hives first demands a search for the etiology, whether endogenous and triggered by emotions or occult systemic disease, exogenous and triggered by allergy to inhaled or ingested antigens, or physical and due to abnormal sensitivity to heat, cold, light, or pressure. Often a fruitless search, the diagnostic evaluation must be accompanied by appropriate symptomatic therapy requiring familiarity with the antihistamines and their relative advantages in the various forms of urticaria. Elimination diets are of diagnostic as well as therapeutic value: pencillin-free, yeast-free, and salicylate-free diets are particularly useful. Therapeutic trials of tetracycline, nystatin and griseofulvin may be helpful, while corticosteroids and specific desensitization are rarely of value. | Urticaria. Urticaria is a problem often as vexing to the physician as to the patient. The approach to the patient with hives first demands a search for the etiology, whether endogenous and triggered by emotions or occult systemic disease, exogenous and triggered by allergy to inhaled or ingested antigens, or physical and due to abnormal sensitivity to heat, cold, light, or pressure. Often a fruitless search, the diagnostic evaluation must be accompanied by appropriate symptomatic therapy requiring familiarity with the antihistamines and their relative advantages in the various forms of urticaria. Elimination diets are of diagnostic as well as therapeutic value: pencillin-free, yeast-free, and salicylate-free diets are particularly useful. Therapeutic trials of tetracycline, nystatin and griseofulvin may be helpful, while corticosteroids and specific desensitization are rarely of value. | [
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PMID:7635 | Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes. | The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems. | Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes. The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems. | [
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PMID:7636 | Studies on the rumen flagellate Sphaeromonas communis. | The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates. | Studies on the rumen flagellate Sphaeromonas communis. The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates. | [
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PMID:7637 | Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture. | A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells. | Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture. A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells. | [
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PMID:7642 | Differences between the anorexic actions of amphetamine and fenfluramine--possible effects on hunger and satiety. | The inhition of feeding in rats brought about by amphetamine and fenfluramine was continuously monitored for periods of up to 24 h using a pellet detecting eatometer. For rats tested under conditions of food deprivation the two drugs gave rise to distinctive anorexic profiles: amphetamine delayed the onset of eating whereas fenfluramine allowed eating to commence normally but brought about an early termination of the initial bout of feeding. When the drugs were administrated to rats with free access to food, analysis of the meal pattern showed that amphetamine gave rise to a small increase in the inter-meal interval while fenfluramine brought about a clear reduction tion in meal size. It is suggested that the contrasting modes of action of these drugs represent an effect of amphetamine upon hunger and an action of fenfluramine on satiety. This suggestion is in keeping with the proposed mechanisms of action of these drugs, amphetamine acting upon a hpothalamic motivational system and fenfluramine acting by means of a postulated serotoninergic satiety system. Use of the continuous monitoring technique has pointed pointed to certain limitations in the assessment of anorexic drug action by means of discrete food sampling periods. | Differences between the anorexic actions of amphetamine and fenfluramine--possible effects on hunger and satiety. The inhition of feeding in rats brought about by amphetamine and fenfluramine was continuously monitored for periods of up to 24 h using a pellet detecting eatometer. For rats tested under conditions of food deprivation the two drugs gave rise to distinctive anorexic profiles: amphetamine delayed the onset of eating whereas fenfluramine allowed eating to commence normally but brought about an early termination of the initial bout of feeding. When the drugs were administrated to rats with free access to food, analysis of the meal pattern showed that amphetamine gave rise to a small increase in the inter-meal interval while fenfluramine brought about a clear reduction tion in meal size. It is suggested that the contrasting modes of action of these drugs represent an effect of amphetamine upon hunger and an action of fenfluramine on satiety. This suggestion is in keeping with the proposed mechanisms of action of these drugs, amphetamine acting upon a hpothalamic motivational system and fenfluramine acting by means of a postulated serotoninergic satiety system. Use of the continuous monitoring technique has pointed pointed to certain limitations in the assessment of anorexic drug action by means of discrete food sampling periods. | [
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PMID:7643 | Alpha-methyl derivatives of biogenic amines as inhibitors of monoamin oxidase. | The values of Km app and Vmax for three natural substrates of monoamine oxidase have been determined at various stages in the isolation of the enzyme from rat liver tissue. The results are consistent with the presence in the enzyme preparation of at least two distinct molecular forms of the enzyme. Using the alpha-methyl derivatives of the natural substrates as inhibitors of the enzyme, the substrate dependence of Ki further substantiates this view. In addition, the kinetics of the inhibition suggest that the value of Km app may not for all substrates, necessarily be a measure of the affinity of the substrate for the enzyme. | Alpha-methyl derivatives of biogenic amines as inhibitors of monoamin oxidase. The values of Km app and Vmax for three natural substrates of monoamine oxidase have been determined at various stages in the isolation of the enzyme from rat liver tissue. The results are consistent with the presence in the enzyme preparation of at least two distinct molecular forms of the enzyme. Using the alpha-methyl derivatives of the natural substrates as inhibitors of the enzyme, the substrate dependence of Ki further substantiates this view. In addition, the kinetics of the inhibition suggest that the value of Km app may not for all substrates, necessarily be a measure of the affinity of the substrate for the enzyme. | [
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PMID:7644 | Comparative study of the effects of mianserin, a tetracyclic antidepressant, and of imipramine on uptake and release of neurotransmitters in synaptosomes. | The effects of mianserin, a tetracyclic antidepressant, on uptake and release of [3H]noradrenaline (3H-NA), [3H]dopamine (3H-DA), [3H]-5-hydroxytryptamine(3H-5-HT) and [3H]gamma-amino-butyric acid (3H-GABA) in synaptosomes from different areas of the rat brain were investigated in a comparative study with the tricyclic antidepressant imipramine. Mianserin and imipramine were inhibitors of 3H-NA uptake in the hypothalamus, but could not increase 3H-NA release from noradrenergic nerve endings. This behaviour was similar to that of (+)-amphetamine. Both mianserin and imipramine had essentially no effect on 3H-DA transport mechanisms in striatal synaptosomes. (+)-Amphetamine, in contrast, strongly affected both 3H-DA uptake and release. Imipramine was stronger than mianserin in inhibiting 3H-5-HT accumulation by striatal synaptosomes. In contrast, mianserin stimulated 3H-5-HT release whereas imipramine was ineffective. Mianserin had virtually no inhibitory activity on 3H-5-HT uptake by rat blood platelets. Imipramine was a modest inhibitor of 3H-GABA accumulation by whole brain synaptosomes; mianserin had no effect. Both drugs did not alter 3H-GABA release. These results indicate that mianserin interferes in a way different from that to tricyclic antidepressants with the neurotransmitter transport mechanisms at the presynaptic level. | Comparative study of the effects of mianserin, a tetracyclic antidepressant, and of imipramine on uptake and release of neurotransmitters in synaptosomes. The effects of mianserin, a tetracyclic antidepressant, on uptake and release of [3H]noradrenaline (3H-NA), [3H]dopamine (3H-DA), [3H]-5-hydroxytryptamine(3H-5-HT) and [3H]gamma-amino-butyric acid (3H-GABA) in synaptosomes from different areas of the rat brain were investigated in a comparative study with the tricyclic antidepressant imipramine. Mianserin and imipramine were inhibitors of 3H-NA uptake in the hypothalamus, but could not increase 3H-NA release from noradrenergic nerve endings. This behaviour was similar to that of (+)-amphetamine. Both mianserin and imipramine had essentially no effect on 3H-DA transport mechanisms in striatal synaptosomes. (+)-Amphetamine, in contrast, strongly affected both 3H-DA uptake and release. Imipramine was stronger than mianserin in inhibiting 3H-5-HT accumulation by striatal synaptosomes. In contrast, mianserin stimulated 3H-5-HT release whereas imipramine was ineffective. Mianserin had virtually no inhibitory activity on 3H-5-HT uptake by rat blood platelets. Imipramine was a modest inhibitor of 3H-GABA accumulation by whole brain synaptosomes; mianserin had no effect. Both drugs did not alter 3H-GABA release. These results indicate that mianserin interferes in a way different from that to tricyclic antidepressants with the neurotransmitter transport mechanisms at the presynaptic level. | [
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PMID:7645 | Adrenergic responses of the rabbit stomach serosal strip and their modification by monoamine oxidase inhibitors and anti-adrenergic drugs. | The rabbit stomach serosal strip, was found to contract to adrenaline and noradrenaline but not to isoprenaline. The contractile response could be totally abolished by phenoxybenzamine but was not influenced by propranolol, indicating that the preparation has almost exclusively alpha-adrenoceptors. The responses to adrenaline and noradrenaline were markedly potentiated in the presence of monoamine oxidase inhibitors, guanethidine or reserpine, indicating the presence of MAO activity in the tissue and possible catecholamine stores. The functional state of the latter has not been conclusively established, since tyramine, an indirectly acting amine, was unable to elicit a response qualitatively similar to that of adrenaline, even in the presence of nialamide or tranylcypromine. | Adrenergic responses of the rabbit stomach serosal strip and their modification by monoamine oxidase inhibitors and anti-adrenergic drugs. The rabbit stomach serosal strip, was found to contract to adrenaline and noradrenaline but not to isoprenaline. The contractile response could be totally abolished by phenoxybenzamine but was not influenced by propranolol, indicating that the preparation has almost exclusively alpha-adrenoceptors. The responses to adrenaline and noradrenaline were markedly potentiated in the presence of monoamine oxidase inhibitors, guanethidine or reserpine, indicating the presence of MAO activity in the tissue and possible catecholamine stores. The functional state of the latter has not been conclusively established, since tyramine, an indirectly acting amine, was unable to elicit a response qualitatively similar to that of adrenaline, even in the presence of nialamide or tranylcypromine. | [
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