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PMID:23207 | On the use of some phenothiazine derivatives in chickens. | 1. Perphenazine, promethazine and levomepromazine induced sedation in young pullets following intramuscular injections. 2. The best results were obtained with perphenazine at a dose rate of 1-5 mg/kg. | On the use of some phenothiazine derivatives in chickens. 1. Perphenazine, promethazine and levomepromazine induced sedation in young pullets following intramuscular injections. 2. The best results were obtained with perphenazine at a dose rate of 1-5 mg/kg. | [
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PMID:23209 | Further studies on the cardiolipin phosphodiesterase of Escherichia coli. | The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions. | Further studies on the cardiolipin phosphodiesterase of Escherichia coli. The cardiolipin phosphodiesterase of Escherichia coli was further characterized. This enzyme has a pH optimum of 7.0 and is Mg2+ dependent. Mn2+ and Co2+ could replace Mg2+ but other divalent cations were inhibitory or without effect. The enzyme is not periplasmic and does not appear to be associated with membrane fractions prepared by different methods. It is recovered as a soluble protein in the cytosol fraction but could not be readily purified because of its instability. With cell-free systems, a requirement for ATP or ADP could be shown under certain defined conditions. Other nucleotides were less effective or ineffective in stimulating the phosphodiesterase. The cells displayed the highest activity during the middle to late exponential stage but no marked requirement for ATP was apparent when the phosphodiesterase was obtained from such freshly grown cells. If, however, cells were starved for several hours in saline medium, the cardiolipin phosphodiesterase level fell and a requirement for added ATP could be shown. The cardiolipin phosphodiesterase is an enzyme distinct from cardiolipin synthase. The assay conditions are quite different from each of these enzymes as are their subcellular distributions. | [
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PMID:23211 | Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices. | In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor. | Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices. In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor. | [
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PMID:23212 | The effects of changes in inspired oxygen concentration on the experimental production of pulmonary edema in the dog with chronic left ventricular overload. | A twofold increase in left ventricular output was achieved by suturing a Telfon graft between the aorta and left atrium in dogs. Three weeks after surgery the animals were anesthetized and found to have left ventricular end-diastolic pressures averaging 36 mmHg with markedly elevated right ventricular systolic pressures (RVSP). Oxygen breathing resulted in a decrease in left ventricular pressures, RVSP, and arterial pressure in those animals which survived hypoxia. Fifty percent of the shunted dogs subsequently developed fatal pulmonary edema when allowed to breathe 10% oxygen in nitrogen. These animals showed no change in left ventricular function or pulmonary artery pressure (RVSP) in response to pure oxygen administration. It is suggested that there is a gradation of hemodynamic response to pure oxygen depending on the severity of left ventricular overload. In the severest case the 'fixing' of pulmonary hypertension may be due to neurohumoral mechanisms. The subsequent development of pulmonary edema in these animals with hypoxia either involves a change in permeability or a redistribution of hydrostatic pressure within the pulmonary vasculature. | The effects of changes in inspired oxygen concentration on the experimental production of pulmonary edema in the dog with chronic left ventricular overload. A twofold increase in left ventricular output was achieved by suturing a Telfon graft between the aorta and left atrium in dogs. Three weeks after surgery the animals were anesthetized and found to have left ventricular end-diastolic pressures averaging 36 mmHg with markedly elevated right ventricular systolic pressures (RVSP). Oxygen breathing resulted in a decrease in left ventricular pressures, RVSP, and arterial pressure in those animals which survived hypoxia. Fifty percent of the shunted dogs subsequently developed fatal pulmonary edema when allowed to breathe 10% oxygen in nitrogen. These animals showed no change in left ventricular function or pulmonary artery pressure (RVSP) in response to pure oxygen administration. It is suggested that there is a gradation of hemodynamic response to pure oxygen depending on the severity of left ventricular overload. In the severest case the 'fixing' of pulmonary hypertension may be due to neurohumoral mechanisms. The subsequent development of pulmonary edema in these animals with hypoxia either involves a change in permeability or a redistribution of hydrostatic pressure within the pulmonary vasculature. | [
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PMID:23213 | Symposium on intensive care: 3. Upper gastrointestinal bleeding in the intensive care unit. | Bleeding from hemorrhagic erosions in the stomach or duodenum of seriously ill patients is associated with a high mortality. While the pathogenesis of such lesions is by no means certain, it is known that they are universal after shock, sepsis or severe burns. Fiberoptic endoscopy has become the most valuable means of diagnosis. This should be preceded by gastric irrigaiton, which usually sufficies to control bleeding caused by acetylsalicylic acid or alcohol, or both. Neutralization of gastric acidity is essential. The histamine HI-receptor antagonist, cimetidine, was used in 27 patients with erosive gastritis, and bleeding ceased in 24. There is a prospect that sugh agents will obviate the necessity of total gastrectomy in the occasional resistant cases in favour of conservative surgery. | Symposium on intensive care: 3. Upper gastrointestinal bleeding in the intensive care unit. Bleeding from hemorrhagic erosions in the stomach or duodenum of seriously ill patients is associated with a high mortality. While the pathogenesis of such lesions is by no means certain, it is known that they are universal after shock, sepsis or severe burns. Fiberoptic endoscopy has become the most valuable means of diagnosis. This should be preceded by gastric irrigaiton, which usually sufficies to control bleeding caused by acetylsalicylic acid or alcohol, or both. Neutralization of gastric acidity is essential. The histamine HI-receptor antagonist, cimetidine, was used in 27 patients with erosive gastritis, and bleeding ceased in 24. There is a prospect that sugh agents will obviate the necessity of total gastrectomy in the occasional resistant cases in favour of conservative surgery. | [
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PMID:23218 | A monolayer (pi,deltaV) study of the ionic properties of alanylphosphatidylglycerol: effects of pH and ions. | 1,2-Didodecanolyl-sn-glycero-3-phosphoryl-1'-(3'-O-L-alanyl)-sn-glycerol (Ala-PG) has been synthetized. Its ionic properties have been studied at the air-water interface through film compressions and surface potential measurements as a function of subphase pH and ionic content (NaCl, Na2MoO4, CaCl2). The existence of the polar head in a loop conformation allowing for interactions between phosphate and amino groups is suggested. Ionic properties of Ala--PG clearly depended on subphase ionic strength but no specific interactions between either cations or anions in the subphase and phosphate or amino groups in the film could be detected. Results are interpreted in terms of ion-pair interactions at the interface between these two groups and anions and cations from the subphase. Occurrence of charge separation between these two groups, induced by increasing subphase ionic strength, is postulated. Since the molecular packing appeared independent of the subphase ionic content over a large domain of pH (3--7) and surface pressure (pi greater than 5 dyne/cm) and since the lipid can be considered as zwitterionic or slightly positive below pH 5--6, it is suggested that in the parent bacteria, grown under acidic conditions, Ala--PG could play a role in maintaining the membrane integrity and in preventing the passive diffusion of protons. | A monolayer (pi,deltaV) study of the ionic properties of alanylphosphatidylglycerol: effects of pH and ions. 1,2-Didodecanolyl-sn-glycero-3-phosphoryl-1'-(3'-O-L-alanyl)-sn-glycerol (Ala-PG) has been synthetized. Its ionic properties have been studied at the air-water interface through film compressions and surface potential measurements as a function of subphase pH and ionic content (NaCl, Na2MoO4, CaCl2). The existence of the polar head in a loop conformation allowing for interactions between phosphate and amino groups is suggested. Ionic properties of Ala--PG clearly depended on subphase ionic strength but no specific interactions between either cations or anions in the subphase and phosphate or amino groups in the film could be detected. Results are interpreted in terms of ion-pair interactions at the interface between these two groups and anions and cations from the subphase. Occurrence of charge separation between these two groups, induced by increasing subphase ionic strength, is postulated. Since the molecular packing appeared independent of the subphase ionic content over a large domain of pH (3--7) and surface pressure (pi greater than 5 dyne/cm) and since the lipid can be considered as zwitterionic or slightly positive below pH 5--6, it is suggested that in the parent bacteria, grown under acidic conditions, Ala--PG could play a role in maintaining the membrane integrity and in preventing the passive diffusion of protons. | [
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PMID:23221 | [Purification of the glutamate decarboxylase from human brain]. | A method of purifying the glutamate decarboxylase from human brain is described. The enzyme was purified 8 000 fold in regard to the initial homogenate and appears homogenous by electrophoresis, both in denaturing and non-denaturing conditions. The molecular weight of the native enzyme and its subunits indicate that GAD from human brain is formed by two similar if non identical polypeptide chains. The Km for glutamate and pyridoxal phosphate found for the human enzyme, respectively 1,2.10(-3) M and 0,13.10(-6) M, are close to the Km found for the Mouse enzyme. | [Purification of the glutamate decarboxylase from human brain]. A method of purifying the glutamate decarboxylase from human brain is described. The enzyme was purified 8 000 fold in regard to the initial homogenate and appears homogenous by electrophoresis, both in denaturing and non-denaturing conditions. The molecular weight of the native enzyme and its subunits indicate that GAD from human brain is formed by two similar if non identical polypeptide chains. The Km for glutamate and pyridoxal phosphate found for the human enzyme, respectively 1,2.10(-3) M and 0,13.10(-6) M, are close to the Km found for the Mouse enzyme. | [
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PMID:23223 | Endotoxemia and large intestinal blood flow in subhuman primates. | The hemodynamic effects of Escherichia coli endotoxin (LD80) were measured in the large intestine of anesthetized Rhesus monkeys to determine whether this organ contributes to the pathogenesis of experimental shock. Inferior mesenteric arterial blood flow (IMF) was measured with an electromagnetic flowmeter. Pressures within the aorta (AP) and portal vein (PP) were recorded. Distribution of colon blood flow was measured with radioactive microspheres: Ce, Sr, and Cr were injected into the left heart. Reference blood samples were obtained from a femoral artery. Mean control IMF was 22.9 +/- 2.2 (SE) ml/min. Aortic pressure was 113 +/- 11 mm Hg, and PP was 6 +/- 1 mm Hg. Arterial blood pH was 7.43 +/- 0.02; pO2 and pCO2 were 93.4 and 37.1 mm Hg, respectively. All parameters were measured at hourly intervals for 4 hr. Neither IMF nor its distribution within the colon changed during the entire observation period. Aortic pressure fell to a low of 60 +/- 6 mm Hg (p less than 0.02) at 3 hr; PP, pO2 and pCO2 were unchanged by endotoxin. Arterial blood pH fell to 7.315 +/- 0.020 at 4 hr (p less than 0.01). These observations indicate that the colon is not a "target organ" of endotoxic shock in subhuman primates, despite considerable hypotension and metabolic disturbances subsequent to near lethal endotoxemia. | Endotoxemia and large intestinal blood flow in subhuman primates. The hemodynamic effects of Escherichia coli endotoxin (LD80) were measured in the large intestine of anesthetized Rhesus monkeys to determine whether this organ contributes to the pathogenesis of experimental shock. Inferior mesenteric arterial blood flow (IMF) was measured with an electromagnetic flowmeter. Pressures within the aorta (AP) and portal vein (PP) were recorded. Distribution of colon blood flow was measured with radioactive microspheres: Ce, Sr, and Cr were injected into the left heart. Reference blood samples were obtained from a femoral artery. Mean control IMF was 22.9 +/- 2.2 (SE) ml/min. Aortic pressure was 113 +/- 11 mm Hg, and PP was 6 +/- 1 mm Hg. Arterial blood pH was 7.43 +/- 0.02; pO2 and pCO2 were 93.4 and 37.1 mm Hg, respectively. All parameters were measured at hourly intervals for 4 hr. Neither IMF nor its distribution within the colon changed during the entire observation period. Aortic pressure fell to a low of 60 +/- 6 mm Hg (p less than 0.02) at 3 hr; PP, pO2 and pCO2 were unchanged by endotoxin. Arterial blood pH fell to 7.315 +/- 0.020 at 4 hr (p less than 0.01). These observations indicate that the colon is not a "target organ" of endotoxic shock in subhuman primates, despite considerable hypotension and metabolic disturbances subsequent to near lethal endotoxemia. | [
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PMID:23219 | [Technical note on the isolation of arboviruses by inoculation in young mice: preparation of the mosquito brei]. | Studying the effects of centrifugation and deep freezing on the quantity of yellow fever virus in a grinded pool of mosquitoes, the authors followed the mortality rate of inoculated baby mice with twenty five artificially infected mosquitoes treated in four different ways. The statistical analysis of the results show that centrifugation and deep freezing have both an effect on the titer of virus and that the addition of the two treatments have an effect superior to the addition of the separate effects of each of them. The authors propose a new technic for the preparation of pools of mosquitoes, without centrifugation or deep freezing. | [Technical note on the isolation of arboviruses by inoculation in young mice: preparation of the mosquito brei]. Studying the effects of centrifugation and deep freezing on the quantity of yellow fever virus in a grinded pool of mosquitoes, the authors followed the mortality rate of inoculated baby mice with twenty five artificially infected mosquitoes treated in four different ways. The statistical analysis of the results show that centrifugation and deep freezing have both an effect on the titer of virus and that the addition of the two treatments have an effect superior to the addition of the separate effects of each of them. The authors propose a new technic for the preparation of pools of mosquitoes, without centrifugation or deep freezing. | [
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PMID:23225 | Simple, refined fluorometric method for measuring cystyl-amino peptidase activity. | Cystyl-amino peptidase (EC 3.4.11.3) activity in serum or plasma was measured fluorometrically using L-cystine-di-beta-naphthylamide in the absence and presence of thiol such as mercaptoethanol. In the presence of thiol, L-cystine-di-beta-naphthylamide is converted to L-cysteine-beta-naphthylamide, and the enzyme activity to hydrolyze L-cysteine-beta-naphthylamide can be measured, while in the absence of thiol, the enzyme activity to hydrolyze L-cystine-di-beta-naphthylamide is determined. Thiol added did not affect various aminopeptidase activities. The present method is able to measure the enzyme activity hydrolyzing L-cystine-di-beta-naphthylamide and L-cysteine-beta-naphthylamide simultaneously and separately using only L-cysteine-di-beta-naphthylamide. This method is simple, sensitive and useful in clinical routine work, assessing placental function for the evaluation of the pregnant status. | Simple, refined fluorometric method for measuring cystyl-amino peptidase activity. Cystyl-amino peptidase (EC 3.4.11.3) activity in serum or plasma was measured fluorometrically using L-cystine-di-beta-naphthylamide in the absence and presence of thiol such as mercaptoethanol. In the presence of thiol, L-cystine-di-beta-naphthylamide is converted to L-cysteine-beta-naphthylamide, and the enzyme activity to hydrolyze L-cysteine-beta-naphthylamide can be measured, while in the absence of thiol, the enzyme activity to hydrolyze L-cystine-di-beta-naphthylamide is determined. Thiol added did not affect various aminopeptidase activities. The present method is able to measure the enzyme activity hydrolyzing L-cystine-di-beta-naphthylamide and L-cysteine-beta-naphthylamide simultaneously and separately using only L-cysteine-di-beta-naphthylamide. This method is simple, sensitive and useful in clinical routine work, assessing placental function for the evaluation of the pregnant status. | [
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PMID:23226 | Influence of thiocyanate ions on starch-iodine reaction used for the estimation of alpha-amylase activity. | A strong simulating effect of thiocyanate ions during the estimation of alpha-amylase by the amyloclastic method is shown. Thiocyanate ions themselves catalyse the decolorization of the starch-iodine complex. This may lead to wrong values during determination of alpha-amylase by this method. The rate of thiocyanate-iodine reaction depends upon the hydrogen ion concentration of the iodine reagent. | Influence of thiocyanate ions on starch-iodine reaction used for the estimation of alpha-amylase activity. A strong simulating effect of thiocyanate ions during the estimation of alpha-amylase by the amyloclastic method is shown. Thiocyanate ions themselves catalyse the decolorization of the starch-iodine complex. This may lead to wrong values during determination of alpha-amylase by this method. The rate of thiocyanate-iodine reaction depends upon the hydrogen ion concentration of the iodine reagent. | [
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PMID:23227 | A protein-binding assay for measurement of biotin in physiological fluids. | A sensitive and convenient protein-binding assay for biotin in physiological fluids is described. The method is based upon the binding of an iodinated biotin conjugate by avidin followed by separation of bound and free conjugate by charcoal absorption. Adult plasma biotin levels averaged 1.26 pmol/ml, a value comparable to that determined by microbiological assays for biotin. | A protein-binding assay for measurement of biotin in physiological fluids. A sensitive and convenient protein-binding assay for biotin in physiological fluids is described. The method is based upon the binding of an iodinated biotin conjugate by avidin followed by separation of bound and free conjugate by charcoal absorption. Adult plasma biotin levels averaged 1.26 pmol/ml, a value comparable to that determined by microbiological assays for biotin. | [
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PMID:23228 | Calibration of a turbidimetric assay of serum lipase activity. | Serum lipase activity has been measured by the turbidimetric method of Shihabi and Bishop, using an olive oil suspension as substrate (Shihabi, Z.K. and Bishop, C. (1971) Clin. Chem. 17, 1150-1153). For this method we developed a new calibration procedure, which can be carried out with great precision. Moreover, the technique is simple and fast and therefore suitable for routine use in clinical chemical laboratories. The new calibration procedure consists of the continuous titration of fatty acids, liberated by lipase from the sample, under the same reaction conditions as those used in the turbidimetric assay. A close correlation has been found with the results from the method of Shihabi and Bishop, in which method calibration has been carried out by making use of the linear relationship between the olive oil concentration and the absorbance of the suspension. In our method, the numerical results are twice those obtained by the method of Shihabi and Bishop. A possible explanation for this phenomenon is given. | Calibration of a turbidimetric assay of serum lipase activity. Serum lipase activity has been measured by the turbidimetric method of Shihabi and Bishop, using an olive oil suspension as substrate (Shihabi, Z.K. and Bishop, C. (1971) Clin. Chem. 17, 1150-1153). For this method we developed a new calibration procedure, which can be carried out with great precision. Moreover, the technique is simple and fast and therefore suitable for routine use in clinical chemical laboratories. The new calibration procedure consists of the continuous titration of fatty acids, liberated by lipase from the sample, under the same reaction conditions as those used in the turbidimetric assay. A close correlation has been found with the results from the method of Shihabi and Bishop, in which method calibration has been carried out by making use of the linear relationship between the olive oil concentration and the absorbance of the suspension. In our method, the numerical results are twice those obtained by the method of Shihabi and Bishop. A possible explanation for this phenomenon is given. | [
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PMID:23229 | The effect of sickle cell haemoglobin polymerization on hydrogen ion dissociation. | An increase in hydrogen ion dissociation was found on sickling due to sickle cell haemoglobin polymerization. Since a decrease in pH favours sickling, this might enhance the sickling process in a vicious cycle. | The effect of sickle cell haemoglobin polymerization on hydrogen ion dissociation. An increase in hydrogen ion dissociation was found on sickling due to sickle cell haemoglobin polymerization. Since a decrease in pH favours sickling, this might enhance the sickling process in a vicious cycle. | [
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PMID:23230 | The mechanism underlying increased tryptaminuria after alcohol ingestion. | It is demonstrated that the increased tryptaminuria which follows alcohol ingestion can be abolished by the concurrent ingestion of alkali even when the amount of alkali consumed is insufficient to increase urinary pH to 6.5 (above which pH a fall in urinary tryptamine may be anticipated, from previous studies, to occur). It is suggested that this increased tryptaminuria is largely if not wholly dependent on the metabolic acidosis induced by alcohol which the concurrent ingestion of alkali abolishes. | The mechanism underlying increased tryptaminuria after alcohol ingestion. It is demonstrated that the increased tryptaminuria which follows alcohol ingestion can be abolished by the concurrent ingestion of alkali even when the amount of alkali consumed is insufficient to increase urinary pH to 6.5 (above which pH a fall in urinary tryptamine may be anticipated, from previous studies, to occur). It is suggested that this increased tryptaminuria is largely if not wholly dependent on the metabolic acidosis induced by alcohol which the concurrent ingestion of alkali abolishes. | [
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PMID:23231 | A benign deficiency of typeB beta-galactosidase in human liver. | The type A or 'acid' and type B or 'neutral' beta-galactosidase activities have been measured in post-mortem liver samples from individuals dying of non-genetic diseases and patients dying of ganglioside storage disease other than GM1 gangliosidosis. The type A activities fell within the established normal range in all samples. The type B activities showed a biomodal distribution suggesting the occurrence of two distinct populations of human individuals. The greater proportion had activities within the range 11.67 pkat/mg of protein (+/- 3.33, S.D.), while others had lower activities in the range 0.48 pkat/mg of protein (+/- 0.38, S.D.). No clinical symptoms were associated with the much lower type B beta-galactosidase activities and it appears that this beta-galactosidase deficiency could be found in the original tissues. Methods of screening for type B beta-galactosidase deficiency are described and the significance of this enzyme deficiency is discussed. | A benign deficiency of typeB beta-galactosidase in human liver. The type A or 'acid' and type B or 'neutral' beta-galactosidase activities have been measured in post-mortem liver samples from individuals dying of non-genetic diseases and patients dying of ganglioside storage disease other than GM1 gangliosidosis. The type A activities fell within the established normal range in all samples. The type B activities showed a biomodal distribution suggesting the occurrence of two distinct populations of human individuals. The greater proportion had activities within the range 11.67 pkat/mg of protein (+/- 3.33, S.D.), while others had lower activities in the range 0.48 pkat/mg of protein (+/- 0.38, S.D.). No clinical symptoms were associated with the much lower type B beta-galactosidase activities and it appears that this beta-galactosidase deficiency could be found in the original tissues. Methods of screening for type B beta-galactosidase deficiency are described and the significance of this enzyme deficiency is discussed. | [
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PMID:23232 | Acquired enzymopathy of erythrocyte glucose-6-phosphate dehydrogenase in acute viral hepatitis. | Erythrocyte glucose-6-phosphate dehydrogenase from patients with acute viral hepatitis has been purified and characterized. The enzyme showed decreased activity (relative to protein), reduced affinity for glucose 6-phosphate and was inactivated at 45 degrees C or at low pH values. The activity and properties of normal erythrocyte enzyme, incubated in vitro with blood plasma of patients, showed a similar pattern of modifications. Incubation with bilirubin affected the enzyme stability, but not its activity or affinity for substrate. | Acquired enzymopathy of erythrocyte glucose-6-phosphate dehydrogenase in acute viral hepatitis. Erythrocyte glucose-6-phosphate dehydrogenase from patients with acute viral hepatitis has been purified and characterized. The enzyme showed decreased activity (relative to protein), reduced affinity for glucose 6-phosphate and was inactivated at 45 degrees C or at low pH values. The activity and properties of normal erythrocyte enzyme, incubated in vitro with blood plasma of patients, showed a similar pattern of modifications. Incubation with bilirubin affected the enzyme stability, but not its activity or affinity for substrate. | [
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PMID:23233 | Lysosomal glycosidase activities in human hair roots. | The levels of three lysosomal glycosidase, alpha-D-mannosidase, a-L-fucosidase and beta-D-hexosaminidase have been determined in normal hair roots and in hair roots obtained from a patient with mannosidosis. The most active glycosidase in normal hair roots was beta-D-hexosaminidase, followed by alpha-L-fucosidase and alpha-D-mannosidase. There was no alpha-D-mannosidase activity in the hair roots of the patient with mannosidosis. The significance of these results is discussed in relation to the detection of lysosomal storage diseases. | Lysosomal glycosidase activities in human hair roots. The levels of three lysosomal glycosidase, alpha-D-mannosidase, a-L-fucosidase and beta-D-hexosaminidase have been determined in normal hair roots and in hair roots obtained from a patient with mannosidosis. The most active glycosidase in normal hair roots was beta-D-hexosaminidase, followed by alpha-L-fucosidase and alpha-D-mannosidase. There was no alpha-D-mannosidase activity in the hair roots of the patient with mannosidosis. The significance of these results is discussed in relation to the detection of lysosomal storage diseases. | [
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PMID:23265 | Peptidases in germinating barley grain: properties, localization and possible functions. | Barley grain contains about 10% insoluble reserve proteins. When the grain germinates the reserve proteins are hydrolysed to amino acids and transported to the growing tissues of the seedling. In the resting grain most of the reserve proteins are 'packed' into the non-living storage tissue, the starchy endosperm. During germination the internal pH of the starchy endosperm is about 5, and it contains high activities of proteinases (secreted by the living aleurone cells) and carboxypeptidases, all with pH optima between 4 and 6. As a whole the starchy endosperm of a germinating grain resembles a giant secondary lysosome. Adjacent to the starchy endosperm is a specialized absorptive and processing tissue, the scutellum. This organ contains very high activities of the 'acid carboxypeptidases' and also two 'alkaline peptidase': a leucine aminopeptidase and a dipeptidase, both pH optima at 8 to 10. The high peptidase activities in the scutellum suggest that the hydrolysis products of the reserve proteins are absorbed from the starchy endosperm as a mixture of amino acids and small peptides, which are hydrolysed to amino acids in the scutellum before transport to the growing seedling tissues. | Peptidases in germinating barley grain: properties, localization and possible functions. Barley grain contains about 10% insoluble reserve proteins. When the grain germinates the reserve proteins are hydrolysed to amino acids and transported to the growing tissues of the seedling. In the resting grain most of the reserve proteins are 'packed' into the non-living storage tissue, the starchy endosperm. During germination the internal pH of the starchy endosperm is about 5, and it contains high activities of proteinases (secreted by the living aleurone cells) and carboxypeptidases, all with pH optima between 4 and 6. As a whole the starchy endosperm of a germinating grain resembles a giant secondary lysosome. Adjacent to the starchy endosperm is a specialized absorptive and processing tissue, the scutellum. This organ contains very high activities of the 'acid carboxypeptidases' and also two 'alkaline peptidase': a leucine aminopeptidase and a dipeptidase, both pH optima at 8 to 10. The high peptidase activities in the scutellum suggest that the hydrolysis products of the reserve proteins are absorbed from the starchy endosperm as a mixture of amino acids and small peptides, which are hydrolysed to amino acids in the scutellum before transport to the growing seedling tissues. | [
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PMID:23267 | Hepatic mono-oxygenase activity and hepatocellular morphology in chickens treated with 3-methylcholanthrene. | Hepatic drug metabolism in the chicken was investigated. White leghorn chickens were administered 20 mg of 3-methylcholanthrene (3MC) per kg 72 and 48 hr before killing. Levels of hepatic cytochrome P-450 were increased approximately 4-fold. In vitro ethylmorphine N-demethylase (ND) activity was enhanced approximately 1.7-fold, aniline hydroxylase (AH) was increased 2.5-fold, aryl hydrocarbon hydroxylase was increased 20-fold, and NADPH-cytochrome c reductase was unchanged. The Vmax was increased for both ND and AH activities, but the KM for demethylation was depressed whereas that for hydroxylation of aniline was increased. The metabolism of hexobarbital in vivo was not enhanced by 3MC treatment. In brief, the distinctive features of the hepatic mono-oxygenase system of the 3MC-treated chicken were: (a) enhanced ethylmorphine N-demethylase activity, (b) a shift in the Soret peak in the CO-difference spectrum of reduced cytochrome P-450 from the control value of 452 nm to 449 nm, and (c) proliferation and pronounced vesiculation of the hepatic endoplasmic reticulum as revealed by electron-microscopic examination. | Hepatic mono-oxygenase activity and hepatocellular morphology in chickens treated with 3-methylcholanthrene. Hepatic drug metabolism in the chicken was investigated. White leghorn chickens were administered 20 mg of 3-methylcholanthrene (3MC) per kg 72 and 48 hr before killing. Levels of hepatic cytochrome P-450 were increased approximately 4-fold. In vitro ethylmorphine N-demethylase (ND) activity was enhanced approximately 1.7-fold, aniline hydroxylase (AH) was increased 2.5-fold, aryl hydrocarbon hydroxylase was increased 20-fold, and NADPH-cytochrome c reductase was unchanged. The Vmax was increased for both ND and AH activities, but the KM for demethylation was depressed whereas that for hydroxylation of aniline was increased. The metabolism of hexobarbital in vivo was not enhanced by 3MC treatment. In brief, the distinctive features of the hepatic mono-oxygenase system of the 3MC-treated chicken were: (a) enhanced ethylmorphine N-demethylase activity, (b) a shift in the Soret peak in the CO-difference spectrum of reduced cytochrome P-450 from the control value of 452 nm to 449 nm, and (c) proliferation and pronounced vesiculation of the hepatic endoplasmic reticulum as revealed by electron-microscopic examination. | [
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PMID:23269 | N-Acetylation of drugs. A genetically controlled reciprocal relationship between drug N-acetylating enzymes of rabbit liver and peripheral blood cells. | The reciprocal relation between liver isoniazid N-acetyltransferase and blood p-aminobenzoic and N-acetyltransferase previously reported is confirmed and found to be expressed in erythrocytes and lymphocytes of genetically rapid and slow isoniazid-acetylator rabbits. Both erythrocytes and lymphocytes from slow acetylator rabbits contained 2.5-3.3 times as much p-aminobenzoic acid N-acetyltransferase activity as the same cells from rapid acetylator rabbits. Mechanisms which might account for the reciprocal association between liver and blood N-acetyltransferases are considered. | N-Acetylation of drugs. A genetically controlled reciprocal relationship between drug N-acetylating enzymes of rabbit liver and peripheral blood cells. The reciprocal relation between liver isoniazid N-acetyltransferase and blood p-aminobenzoic and N-acetyltransferase previously reported is confirmed and found to be expressed in erythrocytes and lymphocytes of genetically rapid and slow isoniazid-acetylator rabbits. Both erythrocytes and lymphocytes from slow acetylator rabbits contained 2.5-3.3 times as much p-aminobenzoic acid N-acetyltransferase activity as the same cells from rapid acetylator rabbits. Mechanisms which might account for the reciprocal association between liver and blood N-acetyltransferases are considered. | [
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PMID:23272 | Identification of the metabolites of trichlorocarbanilide in the rat. | The metabolism and excretion of 14C-labeled 3,4,4'-trichlorodiphenylurea has been studied in the rat after oral and iv administration. More than 80% of the administered radioactivity was excreted in the feces and urine over 5 days. Five isolated metabolites were characterized by mass spectrometry and by comparative thin-layer chromatography with synthesized compounds. Metabolites found include 2'-hydroxy-, 3'-hydroxy-, 6-hydroxy-, 2',6-dihydroxy- and 3',6-dihydroxy-3,4,4'-trichlorodiphenylurea. | Identification of the metabolites of trichlorocarbanilide in the rat. The metabolism and excretion of 14C-labeled 3,4,4'-trichlorodiphenylurea has been studied in the rat after oral and iv administration. More than 80% of the administered radioactivity was excreted in the feces and urine over 5 days. Five isolated metabolites were characterized by mass spectrometry and by comparative thin-layer chromatography with synthesized compounds. Metabolites found include 2'-hydroxy-, 3'-hydroxy-, 6-hydroxy-, 2',6-dihydroxy- and 3',6-dihydroxy-3,4,4'-trichlorodiphenylurea. | [
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PMID:23274 | Absorption, metabolism, and excretion of 14C-tosifen in the dog and rat. | 14C-tosifen [N-2-(1-phenylpropyl)-N'-p-tolyl sulfonylurea] was readily absorbed in both rats and dogs. The rates of absorption, metabolism, and urinary excretion were higher in the rat than in the dog. More of the drug was excreted via the feces than in the urine of the rat, whereas in the dog, the drug was primarily excreted into the urine. The parent drug was the major radioactive component in the plasma of both species. In the urine, however, only a negligible amount of tosifen was found. The major urinary metabolite was a hydroxymethyl derivative which accounted for about 60% and 40% of the total radioactivity in the urine of the dog and rat, respectively. | Absorption, metabolism, and excretion of 14C-tosifen in the dog and rat. 14C-tosifen [N-2-(1-phenylpropyl)-N'-p-tolyl sulfonylurea] was readily absorbed in both rats and dogs. The rates of absorption, metabolism, and urinary excretion were higher in the rat than in the dog. More of the drug was excreted via the feces than in the urine of the rat, whereas in the dog, the drug was primarily excreted into the urine. The parent drug was the major radioactive component in the plasma of both species. In the urine, however, only a negligible amount of tosifen was found. The major urinary metabolite was a hydroxymethyl derivative which accounted for about 60% and 40% of the total radioactivity in the urine of the dog and rat, respectively. | [
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PMID:23271 | The metabolism of alpha-aminobenzo(b)thiophene-3-propionic acid (the sulfur analog of tryptophan) in the rat. | The biotransformation of 3H-labeled alpha-aminobenzo[b]thiophene-3-propionic acid (the sulfur analog of tryptophan) was investigated in rats. Forty-eight hours after ip administration, 80% of the radioactive dose was recovered in the urine and 5% in the feces; tissue levels of radioactivity accounted for the remainder of the administered dose. The urinary metabolites were identified by a combination of thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry, and were quantitated by thin-layer chromatography and liquid-scintillation counting to give, as percentages of the administered dose, unchanged alpha-aminobenzo[b]thiophene-3-propionic acid (20.6%), benzo[b]thiophene-3-acetic acid (1.3%), benzo[b]thiophene-3-pyruvic acid (14.2%), benzo[b]thiophene-3-lactic acid (4.9%), and N-(benzo[b]thiophene-3-acetyl)glycine (46.2%). | The metabolism of alpha-aminobenzo(b)thiophene-3-propionic acid (the sulfur analog of tryptophan) in the rat. The biotransformation of 3H-labeled alpha-aminobenzo[b]thiophene-3-propionic acid (the sulfur analog of tryptophan) was investigated in rats. Forty-eight hours after ip administration, 80% of the radioactive dose was recovered in the urine and 5% in the feces; tissue levels of radioactivity accounted for the remainder of the administered dose. The urinary metabolites were identified by a combination of thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry, and were quantitated by thin-layer chromatography and liquid-scintillation counting to give, as percentages of the administered dose, unchanged alpha-aminobenzo[b]thiophene-3-propionic acid (20.6%), benzo[b]thiophene-3-acetic acid (1.3%), benzo[b]thiophene-3-pyruvic acid (14.2%), benzo[b]thiophene-3-lactic acid (4.9%), and N-(benzo[b]thiophene-3-acetyl)glycine (46.2%). | [
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PMID:23276 | Excretion of phenytoin into semen of rabbits and man. Comparison with plasma levels. | The concentration of phenytoin (diphenylhydantoin, DPH) was measured in plasma and semen of rabbits and man. In the rabbit, a single iv injection of DPH (4.64 mg) resulted in a concentration-time curve for DPH in semen parallel to the concentration-time curve for DPH in plasma (t1/2beta = 171 +/- 29 min). A semen/plasma drug concentration ratio of 0.20 was maintained for at least 8 hr, demonstrating that DPH concentrations in semen are directly proportional to DPH concentrations in plasma. In epileptic subjects maintained on oral DPH the mean drug concentration in semen was 2.31 microgram/ml while that in plasma was 13.8 microgram/ml. The mean semen-plasma DPH concentration ratio in man was 0.17; this closely approximates the observed ratio in rabbits. | Excretion of phenytoin into semen of rabbits and man. Comparison with plasma levels. The concentration of phenytoin (diphenylhydantoin, DPH) was measured in plasma and semen of rabbits and man. In the rabbit, a single iv injection of DPH (4.64 mg) resulted in a concentration-time curve for DPH in semen parallel to the concentration-time curve for DPH in plasma (t1/2beta = 171 +/- 29 min). A semen/plasma drug concentration ratio of 0.20 was maintained for at least 8 hr, demonstrating that DPH concentrations in semen are directly proportional to DPH concentrations in plasma. In epileptic subjects maintained on oral DPH the mean drug concentration in semen was 2.31 microgram/ml while that in plasma was 13.8 microgram/ml. The mean semen-plasma DPH concentration ratio in man was 0.17; this closely approximates the observed ratio in rabbits. | [
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PMID:23278 | Absorption and disposition of oxarbazole in man and laboratory animals. | Oxarbazole (9-benzoyl-1,2,3,4-tetrahydro-6-methoxycarbazole-3-carboxylic acid) was absorbed by human volunteers, rats, dogs, guinea pigs, and monkeys. In all species of laboratory animals studied, the major urinary metabolite was the product of O-demethylation, 9-benzoyl-1,2,3,4-tetrahydro-6-hydroxycarbazole-3-carboxylic acid; this metabolite was conjugated in all species except the guinea pig. The dog and monkey excreted small quantities of a conjugate of 1,2,3,4-tetrahydro-6-methoxycarbazole-3-carboxylic acid in the urine. Enterohepatic circulation was demonstrated in bile duct-cannulated rats, in which almost 90% of the radioactivity of a dose of 14C-oxarbazole had been excreted into the bile within 24 hr. At the time of peak blood radioactivity, intact oxarbazole was the major constituent circulating in the bloodstream of rats and monkeys that had received 14C-oxarbazole orally. The clearance of either intact oxarbazole in man and guinea pig, or undifferentiated radioactivity in rat, dog, and monkey, did not follow the kinetics of a simple model. | Absorption and disposition of oxarbazole in man and laboratory animals. Oxarbazole (9-benzoyl-1,2,3,4-tetrahydro-6-methoxycarbazole-3-carboxylic acid) was absorbed by human volunteers, rats, dogs, guinea pigs, and monkeys. In all species of laboratory animals studied, the major urinary metabolite was the product of O-demethylation, 9-benzoyl-1,2,3,4-tetrahydro-6-hydroxycarbazole-3-carboxylic acid; this metabolite was conjugated in all species except the guinea pig. The dog and monkey excreted small quantities of a conjugate of 1,2,3,4-tetrahydro-6-methoxycarbazole-3-carboxylic acid in the urine. Enterohepatic circulation was demonstrated in bile duct-cannulated rats, in which almost 90% of the radioactivity of a dose of 14C-oxarbazole had been excreted into the bile within 24 hr. At the time of peak blood radioactivity, intact oxarbazole was the major constituent circulating in the bloodstream of rats and monkeys that had received 14C-oxarbazole orally. The clearance of either intact oxarbazole in man and guinea pig, or undifferentiated radioactivity in rat, dog, and monkey, did not follow the kinetics of a simple model. | [
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PMID:23281 | Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyls. | Various parameters have been measured in order to characterize the type of cytochrome(s) P-450 induced by a single ip injection of polybrominated biphenyls (PBB's), 150 mg/kg, given to female rats. The ratio of the 427/455-nm peaks in the microsomal ethylisocyanide difference spectra showed a gradual increase with time after treatment with PBB's. Inhibition studies of aryl hydrocarbon hydroxylase and ethoxycoumarin O-de-ethylase and alpha-naphthoflavone and metyrapone also showed changes in the qualitative nature of these enzymes after treatment with PBB's. Both the high- and low-affinity KM values for ethoxycoumarin O-de-ethylase decreased in magnitude from 0.240 and 0.083 micrometer at 24 hr to 0.188 and 0.042 micrometer, respectively, at 192 hr after treatment with PBB's. Sodium dodecyl sulfate-gel electrophoresis failed to demonstrate a 3-methylcholanthrene-like pattern of hemoprotein at any time after treatment with PBB's. We conclude that while PBB's have some of the properties of a mixed inducer they do not have all of the properties of both phenobarbital and 3-methylcholanthrene, and the PBB's may represent a new class of inducing agents. | Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyls. Various parameters have been measured in order to characterize the type of cytochrome(s) P-450 induced by a single ip injection of polybrominated biphenyls (PBB's), 150 mg/kg, given to female rats. The ratio of the 427/455-nm peaks in the microsomal ethylisocyanide difference spectra showed a gradual increase with time after treatment with PBB's. Inhibition studies of aryl hydrocarbon hydroxylase and ethoxycoumarin O-de-ethylase and alpha-naphthoflavone and metyrapone also showed changes in the qualitative nature of these enzymes after treatment with PBB's. Both the high- and low-affinity KM values for ethoxycoumarin O-de-ethylase decreased in magnitude from 0.240 and 0.083 micrometer at 24 hr to 0.188 and 0.042 micrometer, respectively, at 192 hr after treatment with PBB's. Sodium dodecyl sulfate-gel electrophoresis failed to demonstrate a 3-methylcholanthrene-like pattern of hemoprotein at any time after treatment with PBB's. We conclude that while PBB's have some of the properties of a mixed inducer they do not have all of the properties of both phenobarbital and 3-methylcholanthrene, and the PBB's may represent a new class of inducing agents. | [
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PMID:23279 | The disposition of threo-alpha-(2-piperidyl)-2-trifluoromethyl-6-(4-trifluoromethylphenyl)-4-pyridinemethanol phosphate in mice. | Following oral administration of the 14C-labeled title compound to male mice, the drug was well absorbed and rapidly distributed throughout the body. At least 90% of the radioactivity in the tissues at 2 hr after dosing was identified as parent compound by thin-layer chromatography. The peak plasma level of radioactivity occurred at 4 hr, and the t1/2 of elimination of parent drug was about 26 hr from the plasma and about 27 hr from the red blood cells. The major route of elimination of total radioactivity was fecal (84%), with only 5.5% in the urine at 240 hr. Only a trace of radioactivity (0.32%) was found in the expired air over a 192-hr period. | The disposition of threo-alpha-(2-piperidyl)-2-trifluoromethyl-6-(4-trifluoromethylphenyl)-4-pyridinemethanol phosphate in mice. Following oral administration of the 14C-labeled title compound to male mice, the drug was well absorbed and rapidly distributed throughout the body. At least 90% of the radioactivity in the tissues at 2 hr after dosing was identified as parent compound by thin-layer chromatography. The peak plasma level of radioactivity occurred at 4 hr, and the t1/2 of elimination of parent drug was about 26 hr from the plasma and about 27 hr from the red blood cells. The major route of elimination of total radioactivity was fecal (84%), with only 5.5% in the urine at 240 hr. Only a trace of radioactivity (0.32%) was found in the expired air over a 192-hr period. | [
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PMID:23280 | Age-dependent renal accumulation of cephaloridine in the rabbit. | The accumulation of cephaloridine in the renal cortex of the rabbit was studied in vitro and in vivo in rabbits of various ages. The cortical concentration of cephaloridine, the cortex/serum ratio, and the slice/medium ratio determined by incubation of cortical slices in cephaloridine-containing media rose from birth to adult levels at approximately 1 month of age. Pretreatment with procaine penicillin G stimulated the ability to accumulate cephaloridine in vitro and in vivo. The data indicate that the lack of susceptibility of immature rabbits to cephaloridine nephrotoxicity is due to the lack of development of the anionic transport system which is apparently necessary to achieve the high cortical concentrations of cephaloridine that result in renal injury. | Age-dependent renal accumulation of cephaloridine in the rabbit. The accumulation of cephaloridine in the renal cortex of the rabbit was studied in vitro and in vivo in rabbits of various ages. The cortical concentration of cephaloridine, the cortex/serum ratio, and the slice/medium ratio determined by incubation of cortical slices in cephaloridine-containing media rose from birth to adult levels at approximately 1 month of age. Pretreatment with procaine penicillin G stimulated the ability to accumulate cephaloridine in vitro and in vivo. The data indicate that the lack of susceptibility of immature rabbits to cephaloridine nephrotoxicity is due to the lack of development of the anionic transport system which is apparently necessary to achieve the high cortical concentrations of cephaloridine that result in renal injury. | [
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PMID:23288 | Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A. | 1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9. | Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A. 1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9. | [
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PMID:23290 | Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme. | The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed. | Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme. The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed. | [
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PMID:23291 | The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues. | Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+-influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+. | The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues. Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+-influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+. | [
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PMID:23292 | Induction and 'superinduction' of sialylation of membrane-bound gamma-glutamyltransferase during liver regeneration. | The present paper shows that in the regenerating rat liver the membrane-bound-gamma-glutamyltransferase exists in two molecular forms. Depending on the state of proliferation, a sialic-acid-rich enzyme (in the fetal or regenerating liver) or a sialic-acid-poor enzyme (in the adult or quiescent liver) could be detected. In regeneration liver (24 h after 2/3 resection) only the sialic-acid-rich or fetal enzyme could be found. Since total enzyme activity (adult + fetal type) remained unchanged, it is assumed that the adult type of gamma-glutamyltransferase was modified by sialylation during the initial phase of liver regeneration. This process of sialylation was prevented by inhibitors of RNA or protein synthesis such as D-galactosamine, actinomycin D or cycloheximide, provided that the inhibitor (D-galactosamine) was given within the first 8 h after partial hepatectomy. Sialylation was not impaired by inhibitors of DNA synthesis, e.g. hydroxyurea or cytosine arabinoside. Administration of actinomycin D during a defined phase of proliferation (24 to 48 h after partial hepatectomy) stimulated the transfer of sialic acid to gamma-glutamyltransferase, a finding which describes for the first time the so-called 'superinduction' of a sialylation process. | Induction and 'superinduction' of sialylation of membrane-bound gamma-glutamyltransferase during liver regeneration. The present paper shows that in the regenerating rat liver the membrane-bound-gamma-glutamyltransferase exists in two molecular forms. Depending on the state of proliferation, a sialic-acid-rich enzyme (in the fetal or regenerating liver) or a sialic-acid-poor enzyme (in the adult or quiescent liver) could be detected. In regeneration liver (24 h after 2/3 resection) only the sialic-acid-rich or fetal enzyme could be found. Since total enzyme activity (adult + fetal type) remained unchanged, it is assumed that the adult type of gamma-glutamyltransferase was modified by sialylation during the initial phase of liver regeneration. This process of sialylation was prevented by inhibitors of RNA or protein synthesis such as D-galactosamine, actinomycin D or cycloheximide, provided that the inhibitor (D-galactosamine) was given within the first 8 h after partial hepatectomy. Sialylation was not impaired by inhibitors of DNA synthesis, e.g. hydroxyurea or cytosine arabinoside. Administration of actinomycin D during a defined phase of proliferation (24 to 48 h after partial hepatectomy) stimulated the transfer of sialic acid to gamma-glutamyltransferase, a finding which describes for the first time the so-called 'superinduction' of a sialylation process. | [
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PMID:23293 | On the mechanism of substrate binding to the purine-transport system of Saccharomyces cerevisiae. | The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy-dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X-100. The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule. The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa = 1.8) may be attributed to the substrate, the other (pKa = 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group-specific inhibitors indicate that this amino acid residue contains a carboxyl group. The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex. | On the mechanism of substrate binding to the purine-transport system of Saccharomyces cerevisiae. The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy-dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X-100. The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule. The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa = 1.8) may be attributed to the substrate, the other (pKa = 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group-specific inhibitors indicate that this amino acid residue contains a carboxyl group. The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex. | [
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PMID:23295 | Haemodynamic response to graded exercise during chronic beta-adrenergic blockade with bunitrolol, an agent with intrinsic sympathomimetic activity. | The effect of chronic beta blockade on the haemodynamic response to graded exercise was studied in 18 hypertensive patients treated with bunitrolol, which has partial agonist activity. The patients first received a placebo for 5 to 12 days, then bunitrolol 30 mg daily for one week and subsequently the dose was doubled weekly as necessary up to 240 mg daily. At rest haemodynamic changes after beta blockade were only minor; heart rate decreased by 8% and no significant change was observed in stroke index, cardiac index, (a-v)O2 difference and VO2. The hypotensive effect was not significant and no significant change in mean pulmonary arterial and wedge pressure was observed. Maximal exercise capacity remained unchanged, because of haemodynamic responses. The maximal exercise heart rate was reduced by 25% during beta blockade, which was compensated by a 34% elevation in stroke index, whereas maximal cardiac index and (a-v)O2 difference remained unchanged. There was no consistent change in mean pulmonary artery pressure during maximal exercise, but the mean brachial artery pressure fell by 12%. | Haemodynamic response to graded exercise during chronic beta-adrenergic blockade with bunitrolol, an agent with intrinsic sympathomimetic activity. The effect of chronic beta blockade on the haemodynamic response to graded exercise was studied in 18 hypertensive patients treated with bunitrolol, which has partial agonist activity. The patients first received a placebo for 5 to 12 days, then bunitrolol 30 mg daily for one week and subsequently the dose was doubled weekly as necessary up to 240 mg daily. At rest haemodynamic changes after beta blockade were only minor; heart rate decreased by 8% and no significant change was observed in stroke index, cardiac index, (a-v)O2 difference and VO2. The hypotensive effect was not significant and no significant change in mean pulmonary arterial and wedge pressure was observed. Maximal exercise capacity remained unchanged, because of haemodynamic responses. The maximal exercise heart rate was reduced by 25% during beta blockade, which was compensated by a 34% elevation in stroke index, whereas maximal cardiac index and (a-v)O2 difference remained unchanged. There was no consistent change in mean pulmonary artery pressure during maximal exercise, but the mean brachial artery pressure fell by 12%. | [
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PMID:23296 | Circadian rhythm of urinary pH in man with and without chronic antacid administration. | In normal human volunteers, when urinary pH was plotted versus time, the circadian sine-wave type curve was not altered by chronic administration of a commercially available suspension containing a mixture of magnesium and aluminum hydroxides, although the antacid perturbed the entire curve in a more alkaline direction. A single dose of the antacid had little effect on urinary pH. There was a highly significant linear relationship between the change in hydrogen ion concentration during chronic antacid treatment and the initial control urinary hydrogen ion concentration, but there was no significant correlation between change in urinary pH and initial control urinary pH as has been previously reported. The above results were based on the evaluation of the hydrogen ion concentrations of 1562 separate urine samples collected from 24 normal subjects in a three treatment crossover study. It is recommended that: (1) research studies involving drug-drug interactions with antacids be designed to consider the effect of the antacid on the circadian rhythm of urinary pH, and (2) pH values not be averaged as commonly reported in the literature, but rather the pH values be converted to hydrogen ion concentrations before statistical analysis. | Circadian rhythm of urinary pH in man with and without chronic antacid administration. In normal human volunteers, when urinary pH was plotted versus time, the circadian sine-wave type curve was not altered by chronic administration of a commercially available suspension containing a mixture of magnesium and aluminum hydroxides, although the antacid perturbed the entire curve in a more alkaline direction. A single dose of the antacid had little effect on urinary pH. There was a highly significant linear relationship between the change in hydrogen ion concentration during chronic antacid treatment and the initial control urinary hydrogen ion concentration, but there was no significant correlation between change in urinary pH and initial control urinary pH as has been previously reported. The above results were based on the evaluation of the hydrogen ion concentrations of 1562 separate urine samples collected from 24 normal subjects in a three treatment crossover study. It is recommended that: (1) research studies involving drug-drug interactions with antacids be designed to consider the effect of the antacid on the circadian rhythm of urinary pH, and (2) pH values not be averaged as commonly reported in the literature, but rather the pH values be converted to hydrogen ion concentrations before statistical analysis. | [
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PMID:23297 | Antidiuresis induced by beta1- and beta2-adrenergic agonists in ethanol-anesthetized rats. | The beta1- and beta2-components in antidiuresis and sodium retention induced by beta-adrenergic agonists were analysed in ethanol-anesthetized, water-diuretic rats. Intravenous infusions of isoprenaline, salbutamol and carbuterol did not affect insulin clearance but increased plasma renin concentration to the same same extent. Propranolol completely blocked the decreases in urine volume (V) and urinary sodium excretion (UNaV) induced by isoprenaline; practolol (beta1-blocker) inhibited only the decrease in UNaV and butaxamine (beta2-blocker) inhibited only the decrease in V. The ratios of doses of beta-agonists which decreased UNaV and by 50% (ED50 UNaV decrease/ED50 V decrease) were 0.34, 0.68, 1.56 and 2.36 for isoprenaline, tretoquinol, salbutamol and carbuterol, respectively. This increasing order of the ratios coincided with the order reported for the preponderance of the beta2- over beta1-component of these agonists. These results indicate that the decrease in UNaV induced by beta-agonists is related to beta1 stimulation, while the decrease in V is related to beta2 stimulation. | Antidiuresis induced by beta1- and beta2-adrenergic agonists in ethanol-anesthetized rats. The beta1- and beta2-components in antidiuresis and sodium retention induced by beta-adrenergic agonists were analysed in ethanol-anesthetized, water-diuretic rats. Intravenous infusions of isoprenaline, salbutamol and carbuterol did not affect insulin clearance but increased plasma renin concentration to the same same extent. Propranolol completely blocked the decreases in urine volume (V) and urinary sodium excretion (UNaV) induced by isoprenaline; practolol (beta1-blocker) inhibited only the decrease in UNaV and butaxamine (beta2-blocker) inhibited only the decrease in V. The ratios of doses of beta-agonists which decreased UNaV and by 50% (ED50 UNaV decrease/ED50 V decrease) were 0.34, 0.68, 1.56 and 2.36 for isoprenaline, tretoquinol, salbutamol and carbuterol, respectively. This increasing order of the ratios coincided with the order reported for the preponderance of the beta2- over beta1-component of these agonists. These results indicate that the decrease in UNaV induced by beta-agonists is related to beta1 stimulation, while the decrease in V is related to beta2 stimulation. | [
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PMID:23298 | Effects of baclofen on dopamine metabolism and interaction with neuroleptic effects. | Baclofen increased striatal levels of dopamine (DA), homovanillic (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) dose-dependently above 10 mg/kg i.p. The effect on the DA metabolites was shown to be caused only by the (-)-isomer. The HVA increase after 20 mg/kg i.p. was not antagonized by either scopolamine or picrotoxin. Repeated treatment produced a smaller increase in HVA than a single administration. Baclofen reduced both the disappearance of DA after alpha-methyl-p-tyrosine and the acceleration of the DA disappearance caused by neuroleptics in corpus striatum and in the mesolimbic area. The neuroleptic-induced increases in HVA and DOPAC and in DOPA accumulation after central decarboxylase inhibition were also reduced. Picrotoxin could not antagonize these effects of baclofen which therefore cannot be regarded as being garbergic. Baclofen effects on DA metabolism are similar to those reported for gamma-hydroxybutyric acid and are probably a consequence of inhibition of firing of DA neurons. | Effects of baclofen on dopamine metabolism and interaction with neuroleptic effects. Baclofen increased striatal levels of dopamine (DA), homovanillic (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) dose-dependently above 10 mg/kg i.p. The effect on the DA metabolites was shown to be caused only by the (-)-isomer. The HVA increase after 20 mg/kg i.p. was not antagonized by either scopolamine or picrotoxin. Repeated treatment produced a smaller increase in HVA than a single administration. Baclofen reduced both the disappearance of DA after alpha-methyl-p-tyrosine and the acceleration of the DA disappearance caused by neuroleptics in corpus striatum and in the mesolimbic area. The neuroleptic-induced increases in HVA and DOPAC and in DOPA accumulation after central decarboxylase inhibition were also reduced. Picrotoxin could not antagonize these effects of baclofen which therefore cannot be regarded as being garbergic. Baclofen effects on DA metabolism are similar to those reported for gamma-hydroxybutyric acid and are probably a consequence of inhibition of firing of DA neurons. | [
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PMID:23300 | Inhibition of somato-sympathetic reflex via peripheral presynaptic alha-adrenoceptors. | In cats anaesthetized with chloralose--urethane (70/100 mg/kg) a somato-sympathetic reflex increase of blood pressure and heart rate was evoked by electrical stimulation of sensory fibres running within the tibial nerve. I.V. injection of 30 microgram/kg of the alpha-adrenoceptor agonist clonidine caused a reflex inhibition in intact and vagotomized animals. In contrast, injection of 2 microgram/kg clonidine into the vertebral artery elicited hypotension but no reflux inhibition. In small doses, phenylephrine which is known from in vitro experiments to prefer the postsynaptic alpha-adrenoceptor, potentiated the reflex increase in blood pressure by an additive effect on the vascular alpha-adrenoceptor, while oxymetazoline and tramazoline, which preferentially activate the presynaptic receptor, were inhibitory. The inhibitory effect of tramazoline on the reflex increase in blood pressure and heart rate was antagonized by the presynaptic alpha-adrenolytic drug yohimbine. The results suggest that peripheral presynaptic alpha-adrenoceptors are responsible for somato-sympathetic reflex inhibition. The importance of peripheral presynaptic alpha-adrenoceptors in the in vivo regulation of blood pressure is discussed. | Inhibition of somato-sympathetic reflex via peripheral presynaptic alha-adrenoceptors. In cats anaesthetized with chloralose--urethane (70/100 mg/kg) a somato-sympathetic reflex increase of blood pressure and heart rate was evoked by electrical stimulation of sensory fibres running within the tibial nerve. I.V. injection of 30 microgram/kg of the alpha-adrenoceptor agonist clonidine caused a reflex inhibition in intact and vagotomized animals. In contrast, injection of 2 microgram/kg clonidine into the vertebral artery elicited hypotension but no reflux inhibition. In small doses, phenylephrine which is known from in vitro experiments to prefer the postsynaptic alpha-adrenoceptor, potentiated the reflex increase in blood pressure by an additive effect on the vascular alpha-adrenoceptor, while oxymetazoline and tramazoline, which preferentially activate the presynaptic receptor, were inhibitory. The inhibitory effect of tramazoline on the reflex increase in blood pressure and heart rate was antagonized by the presynaptic alpha-adrenolytic drug yohimbine. The results suggest that peripheral presynaptic alpha-adrenoceptors are responsible for somato-sympathetic reflex inhibition. The importance of peripheral presynaptic alpha-adrenoceptors in the in vivo regulation of blood pressure is discussed. | [
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PMID:23301 | Chronic effects of nicotine on catecholamine synthesizing enzymes in rats. | The effects of chronic administration of nicotine upon catecholamine (CA) synthesizing enzymes of rat hypothalamus, striatum and adrenal medulla were studied. Nicotine 3 mg/kg/day for 14 days, increased tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) in hypothalamus and adrenal medulla but did change striatum TH. The data suggest that chronic nicotine administration can produce similar long-term alterations in both of the main CA forming enzymes in the hypothalamus and in adrenal medulla. | Chronic effects of nicotine on catecholamine synthesizing enzymes in rats. The effects of chronic administration of nicotine upon catecholamine (CA) synthesizing enzymes of rat hypothalamus, striatum and adrenal medulla were studied. Nicotine 3 mg/kg/day for 14 days, increased tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) in hypothalamus and adrenal medulla but did change striatum TH. The data suggest that chronic nicotine administration can produce similar long-term alterations in both of the main CA forming enzymes in the hypothalamus and in adrenal medulla. | [
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PMID:23306 | [The usefulness of ruthenium red in dye exclusion test on cultured cells (author's transl)]. | The reliability of statements and the reproducibility of results derived from experiments on cell cultures are dependent on the use of practicable methods for most objective assay of the state of the cells. The experiments were to clarify the following questions: 1. Is it possible to assay vital and nonvital cells by use of the dye exclusion test (FET)? 2. Are intact cells also stained by FET? 3. Will there be a possibility to make FET more sensitive to obtain more reliable statements? | [The usefulness of ruthenium red in dye exclusion test on cultured cells (author's transl)]. The reliability of statements and the reproducibility of results derived from experiments on cell cultures are dependent on the use of practicable methods for most objective assay of the state of the cells. The experiments were to clarify the following questions: 1. Is it possible to assay vital and nonvital cells by use of the dye exclusion test (FET)? 2. Are intact cells also stained by FET? 3. Will there be a possibility to make FET more sensitive to obtain more reliable statements? | [
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PMID:23321 | Testicular germ cell differentiation in vivo. | The effects of artificial cryptorchidism and surgical reversal on spermatogenesis were examined in mice. Only undifferentiated type A spermatogonia were present as germ cells in cryptorchid testes. The surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells as judged by testicular weight, histologic examination, and increase in the specific activity of lactate dehydrogenase-X. Leydig cell function was also examined by assessment of the weight of target tissues of androgen. They showed a unique change following the surgical reversal. Thirty days after surgical reversal, hyperfunction of the Leydig cells was observed, and the testes became normal after 60 days. | Testicular germ cell differentiation in vivo. The effects of artificial cryptorchidism and surgical reversal on spermatogenesis were examined in mice. Only undifferentiated type A spermatogonia were present as germ cells in cryptorchid testes. The surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells as judged by testicular weight, histologic examination, and increase in the specific activity of lactate dehydrogenase-X. Leydig cell function was also examined by assessment of the weight of target tissues of androgen. They showed a unique change following the surgical reversal. Thirty days after surgical reversal, hyperfunction of the Leydig cells was observed, and the testes became normal after 60 days. | [
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PMID:23322 | [Relationship between the anionic structure of organic acids and the response of cat taste receptors]. | With the aid of analysis of afferent impulse activity in the cat chorda tympani, it was shown that the effect of application of organic acids solutions of the same pH to the tongue could be represented as follows: propionic acid greater than lactic acid greater than pyruvic acid. These data confirm the suggestion that carboxylic acids without polar groups are the most effective, and an anion which has OH-group in alpha-position is more effective than an anion with keto-group. | [Relationship between the anionic structure of organic acids and the response of cat taste receptors]. With the aid of analysis of afferent impulse activity in the cat chorda tympani, it was shown that the effect of application of organic acids solutions of the same pH to the tongue could be represented as follows: propionic acid greater than lactic acid greater than pyruvic acid. These data confirm the suggestion that carboxylic acids without polar groups are the most effective, and an anion which has OH-group in alpha-position is more effective than an anion with keto-group. | [
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PMID:23331 | Molecular biology and molecular pathology of a newly described molecular disease--tyrosinemia II (the Richner-Hanhart syndrome). | A deficiency of hepatic tyrosine aminotransferase in humans is responsible for a syndrome of keratitis, palmar and plantar erosions and hyperkeratosis and mental retardation. Serum tyrosine increases due to the enzymatic deficiency leads to the deposition of tyrosine crystals in the eye and cornea. This deposition and possible lysosomal activation leads to inflammation in the cornea and the skin. The syndrome can be reproduced in animals who are fed a high tyrosine diet. The interaction of tyrosine crystals with membrane-bound particles can be studied in vitro with lysosomes and erythrocytes. | Molecular biology and molecular pathology of a newly described molecular disease--tyrosinemia II (the Richner-Hanhart syndrome). A deficiency of hepatic tyrosine aminotransferase in humans is responsible for a syndrome of keratitis, palmar and plantar erosions and hyperkeratosis and mental retardation. Serum tyrosine increases due to the enzymatic deficiency leads to the deposition of tyrosine crystals in the eye and cornea. This deposition and possible lysosomal activation leads to inflammation in the cornea and the skin. The syndrome can be reproduced in animals who are fed a high tyrosine diet. The interaction of tyrosine crystals with membrane-bound particles can be studied in vitro with lysosomes and erythrocytes. | [
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PMID:23333 | An in vitro study of functional maturation of murine thymus cells. | Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed. | An in vitro study of functional maturation of murine thymus cells. Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed. | [
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PMID:23334 | The further investigation on the gastric acid secretion in the primary hyperparathyroidism. | The gastric acid output was studied in the 11 patients of hyperparathyroidism before and after parathyroidectomy. The gastric acid output before operation was almost equal to the normal control in our hospital. After the correction of serum calcium by parathyroidectomy, the gastric acid output and serum gastrin were decreased. The decreased gastric acid output was recovered as the days passed since operation and approached to the preoperative level. The acid output in hyperparathyroidism was less in the case whose activity of alkaline phosphatase was more, which suggested that the calcium deposition on gastric mucosa might damage the parietal cell as the result of long lasting hypercalcemia. | The further investigation on the gastric acid secretion in the primary hyperparathyroidism. The gastric acid output was studied in the 11 patients of hyperparathyroidism before and after parathyroidectomy. The gastric acid output before operation was almost equal to the normal control in our hospital. After the correction of serum calcium by parathyroidectomy, the gastric acid output and serum gastrin were decreased. The decreased gastric acid output was recovered as the days passed since operation and approached to the preoperative level. The acid output in hyperparathyroidism was less in the case whose activity of alkaline phosphatase was more, which suggested that the calcium deposition on gastric mucosa might damage the parietal cell as the result of long lasting hypercalcemia. | [
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PMID:23335 | Effect of Ca2+, Mg2+, NaN3, cholinergic agents, and gastrointestinal hormones on the guanylate cyclase from guinea pig gastric mucosa. | Guanylate cyclase in the guinea pig fundic mucosa occurred in two enzymatic forms: a "soluble" form and a particulate form. The mean basal activity of the soluble fraction measured in the presence of 300 micrometer guanosine-5'-triphosphate and 5 mM MnCl2 was 72.6 +/- 5.3 pmoles of cyclic GMP per mg of protein per min. Guanylate cyclase activity was dependent on Mn2+; it was increased by sodium azide (NaN3), CaCl2, cysteine, secretin, and cholecystokinin, but it was not influenced by gastrin, histamine, cholinergic esters, prostaglandins E1 and A1. NaN3 (1 mM) decreased the apparent Km for MnCl2 and potentiated the effects of MgCl2. The activity of the particulate fraction represented about 14% of that of the supernatant fraction. The guanylate cyclase activity of that fraction was not modified by NaN3, gastrin, cholinergic agents, secretin, or cholecystokinin. Cysteine inhibited its activity. These data do not support the hypothesis that cyclic GMP acts as a second messenger for the action of cholinergic agents and gastrin in the guinea pig gastric mucosa. | Effect of Ca2+, Mg2+, NaN3, cholinergic agents, and gastrointestinal hormones on the guanylate cyclase from guinea pig gastric mucosa. Guanylate cyclase in the guinea pig fundic mucosa occurred in two enzymatic forms: a "soluble" form and a particulate form. The mean basal activity of the soluble fraction measured in the presence of 300 micrometer guanosine-5'-triphosphate and 5 mM MnCl2 was 72.6 +/- 5.3 pmoles of cyclic GMP per mg of protein per min. Guanylate cyclase activity was dependent on Mn2+; it was increased by sodium azide (NaN3), CaCl2, cysteine, secretin, and cholecystokinin, but it was not influenced by gastrin, histamine, cholinergic esters, prostaglandins E1 and A1. NaN3 (1 mM) decreased the apparent Km for MnCl2 and potentiated the effects of MgCl2. The activity of the particulate fraction represented about 14% of that of the supernatant fraction. The guanylate cyclase activity of that fraction was not modified by NaN3, gastrin, cholinergic agents, secretin, or cholecystokinin. Cysteine inhibited its activity. These data do not support the hypothesis that cyclic GMP acts as a second messenger for the action of cholinergic agents and gastrin in the guinea pig gastric mucosa. | [
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PMID:23336 | Characterization and development of cimetidine as a histamine H2-receptor antagonist. | The concept of two classes of histamine receptor, H1 and H2, is introduced and the chemical derivation of histamine H2-receptor antagonists is outlined briefly. Starting from the structure of histamine, chemical modification led eventually to burimamide, the first described histamine H2-receptor antagonist. Further stepwise modifications ultimately afforded metiamide and cimetidine. In vitro studies show that cimetidine is a specific competitive histamine H2-receptor antagonist. In vivo, it is a potent inhibitor of histamine-stimulated gastric acid secretion in rats and dogs after both intravenous and oral administration. It is equally potent as an inhibitor of pentagastrin-stimulated secretion. The evidence suggests that cimetidine inhibits gastric acid secretion through blockade of histamine H2-receptors in the gastric mucosa. Cimetidine has been shown to have low acute toxicity. Repeated dose studies of up to 24 months in rats and up to 12 months in dogs have been carried out and the results are presented and discussed. There is no known toxic effect which would limit the usefulness of cimetidine in man. | Characterization and development of cimetidine as a histamine H2-receptor antagonist. The concept of two classes of histamine receptor, H1 and H2, is introduced and the chemical derivation of histamine H2-receptor antagonists is outlined briefly. Starting from the structure of histamine, chemical modification led eventually to burimamide, the first described histamine H2-receptor antagonist. Further stepwise modifications ultimately afforded metiamide and cimetidine. In vitro studies show that cimetidine is a specific competitive histamine H2-receptor antagonist. In vivo, it is a potent inhibitor of histamine-stimulated gastric acid secretion in rats and dogs after both intravenous and oral administration. It is equally potent as an inhibitor of pentagastrin-stimulated secretion. The evidence suggests that cimetidine inhibits gastric acid secretion through blockade of histamine H2-receptors in the gastric mucosa. Cimetidine has been shown to have low acute toxicity. Repeated dose studies of up to 24 months in rats and up to 12 months in dogs have been carried out and the results are presented and discussed. There is no known toxic effect which would limit the usefulness of cimetidine in man. | [
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PMID:23338 | Effect of H2-receptor antagonists on gastric acid secretion and serum gastrin concentration: a review. | Cimetidine inhibits basal and nocturnal acid secretion and acid secretion stimulated by histamine, pentagastrin, caffeine, insulin, sham feeding, and food. Cinetidine (300 mg) inhibits basal acid secretion in duodenal ulcer patients by 95% for at least 5 hr. When taken at bedtime, cimetidine inhibits nocturnal acid secretion by greater than 80% for most of the night. Cimetidine markedly inhibits food-stimulated acid secretion and is more effective than anticholinergic drugs. However, to get adequate suppression of food-stimulated acid secretion throughout the day, cimetidine should be given with each meal. Cimetidine has no effect on nocturnal serum gastrin concentration, but, when stimulated by food, serum gastrin concentration is higher after cimetidine than after placebo. | Effect of H2-receptor antagonists on gastric acid secretion and serum gastrin concentration: a review. Cimetidine inhibits basal and nocturnal acid secretion and acid secretion stimulated by histamine, pentagastrin, caffeine, insulin, sham feeding, and food. Cinetidine (300 mg) inhibits basal acid secretion in duodenal ulcer patients by 95% for at least 5 hr. When taken at bedtime, cimetidine inhibits nocturnal acid secretion by greater than 80% for most of the night. Cimetidine markedly inhibits food-stimulated acid secretion and is more effective than anticholinergic drugs. However, to get adequate suppression of food-stimulated acid secretion throughout the day, cimetidine should be given with each meal. Cimetidine has no effect on nocturnal serum gastrin concentration, but, when stimulated by food, serum gastrin concentration is higher after cimetidine than after placebo. | [
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PMID:23345 | Ditazole activity and its interaction with urokinase on experimental thrombosis. | The effect of ditazole, a new antiaggregant oxazole derivative as well as its possible interaction with urokinase on the formation of electrically induced thrombus, was assayed in rabbits. The activity of ditazole in reducing thrombus weight was comparable to that of aspirin. In the ditazole- or aspirin-treated animals, the microscopical examination of the thrombus showed a reduction in the fibrin component, and well-isolated platelets not undergoing a viscous metamorphosis were present. Urokinase, administered in combination with these antiaggregant drugs, did not induce a further reduction in thrombus weight. However, this additional treatment did induce clearly visible lytic areas and histological modifications as observed with the antiaggregant drugs. These data suggest that the antiplatelet drug ditazole may be an effective antithrombotic agent in man and could facilitate the penetration of urokinase into the thrombus. | Ditazole activity and its interaction with urokinase on experimental thrombosis. The effect of ditazole, a new antiaggregant oxazole derivative as well as its possible interaction with urokinase on the formation of electrically induced thrombus, was assayed in rabbits. The activity of ditazole in reducing thrombus weight was comparable to that of aspirin. In the ditazole- or aspirin-treated animals, the microscopical examination of the thrombus showed a reduction in the fibrin component, and well-isolated platelets not undergoing a viscous metamorphosis were present. Urokinase, administered in combination with these antiaggregant drugs, did not induce a further reduction in thrombus weight. However, this additional treatment did induce clearly visible lytic areas and histological modifications as observed with the antiaggregant drugs. These data suggest that the antiplatelet drug ditazole may be an effective antithrombotic agent in man and could facilitate the penetration of urokinase into the thrombus. | [
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PMID:23346 | [Inhibitory effects of methyl o-(4-hydroxy-3-methoxycinnamoyl) reserpate (CD-3400) on the central nervous system (author's transl)]. | Effects of methyl o-(4-hydroxy-3-methoxycinnamoyl) reserpate (CD-3400) on the central nervous system in mice, rats and cats were investigated, and a comparison was made with such effects of reserpine and rescinamine. Inhibitory effects of CD-3400 on spontaneous motor activity and conditioned avoidance response were weaker and shorter than those of reserpine and rescinnamine. In the experiments of the inhibitory effects of the central actions such as ptosis, hypothermia, decrease in motor ability, potentiation of hexobarbital and taming, reserpine was found to be the most potent followed by rescinnamine and CD-3400, respectively. High doses of CD-3400 exhibited inhibitory effects on methamphetamine-induced hyperactivity in mice and this action was weaker than those of reserpine and rescinnamine. CD-3400, 80-160 mg/kg p.o., showed no significant effects on morphine-induced analgesia, while a slight inhibition was observed on the Straub-tail reaction using morphine. Reserpine, 0.5 mg/kg i.v., resulted in a drowsy pattern in the spontaneous EEG activity and the EEG arousal response was depressed, while with CD-3400, 5 mg/kg i.v., there was no drowsy pattern. CD-3400 as well as rescinnamine and reserpine remarkably depleted 5-HT levels in brain, heart and plasma and the potency of CD-3400, particularly in the brain, was weaker than the potency of reserpine and rescinnamine. These results indicate that CD-3400 is an antihypertensive agent with a low toxicity and a weak central action. | [Inhibitory effects of methyl o-(4-hydroxy-3-methoxycinnamoyl) reserpate (CD-3400) on the central nervous system (author's transl)]. Effects of methyl o-(4-hydroxy-3-methoxycinnamoyl) reserpate (CD-3400) on the central nervous system in mice, rats and cats were investigated, and a comparison was made with such effects of reserpine and rescinamine. Inhibitory effects of CD-3400 on spontaneous motor activity and conditioned avoidance response were weaker and shorter than those of reserpine and rescinnamine. In the experiments of the inhibitory effects of the central actions such as ptosis, hypothermia, decrease in motor ability, potentiation of hexobarbital and taming, reserpine was found to be the most potent followed by rescinnamine and CD-3400, respectively. High doses of CD-3400 exhibited inhibitory effects on methamphetamine-induced hyperactivity in mice and this action was weaker than those of reserpine and rescinnamine. CD-3400, 80-160 mg/kg p.o., showed no significant effects on morphine-induced analgesia, while a slight inhibition was observed on the Straub-tail reaction using morphine. Reserpine, 0.5 mg/kg i.v., resulted in a drowsy pattern in the spontaneous EEG activity and the EEG arousal response was depressed, while with CD-3400, 5 mg/kg i.v., there was no drowsy pattern. CD-3400 as well as rescinnamine and reserpine remarkably depleted 5-HT levels in brain, heart and plasma and the potency of CD-3400, particularly in the brain, was weaker than the potency of reserpine and rescinnamine. These results indicate that CD-3400 is an antihypertensive agent with a low toxicity and a weak central action. | [
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PMID:23347 | [High risk groups for gastrointestinal carcinoma]. | High-risk groups for gastrointestinal carcinoma are heterogenic in regard to etiopathology; familial predisposition and genetic defects (familial adenomatosis coli, tylosis palmaris et plantaris, Gardner syndrome, Peutz-Jeghers syndrome), occupational factors (asbestor exposure), surgical intervention (resected stomach, ureterosigmoidostomy), long lasting passage obstruction (oesophagus) or chronic inflammatory alteration of the mucosa (pernicious anemia, ulcerative colitis, Crohn's disease, glutenenteropathy). Although high-risk groups account only for about 5 per cent of all carcinomas, consequent follow-up examinations of these small collectives offer early diagnosis of carcinoma at a curable stage. | [High risk groups for gastrointestinal carcinoma]. High-risk groups for gastrointestinal carcinoma are heterogenic in regard to etiopathology; familial predisposition and genetic defects (familial adenomatosis coli, tylosis palmaris et plantaris, Gardner syndrome, Peutz-Jeghers syndrome), occupational factors (asbestor exposure), surgical intervention (resected stomach, ureterosigmoidostomy), long lasting passage obstruction (oesophagus) or chronic inflammatory alteration of the mucosa (pernicious anemia, ulcerative colitis, Crohn's disease, glutenenteropathy). Although high-risk groups account only for about 5 per cent of all carcinomas, consequent follow-up examinations of these small collectives offer early diagnosis of carcinoma at a curable stage. | [
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PMID:23348 | [Precancerous lesions in the gastrointestinal tract]. | In the large intestine, the pathologist has to differentiate between multiple polyps and polyposis (more than 100 polyps), further between adenomatosis (coli) and non-neoplastic (tumorlike) polyposis. Without prophylactic colectomy, in about 80% of adenomatosis patients an evolution of cancer is observed. Patients with extensive or total ulcerative colitis and a long history have an increased risk for developing carcinoma. Precancerous dysplasia can be demonstrated in rectoscopic and/or colonoscopic biopsies. Cancers complicating adenomatosis or ulcerative colitis account for only a very small proportion of large bowel carcinoma. The "adenoma-cancer sequence" is of greater importance. Colorectal polyps should be removed endoscopically whenever possible. Most gastric polyps are non-neoplastic and have no carcinomatous potential. The true adenoma and the so-called borderline lesion only can be considered as precursor of the gastric carcinoma. | [Precancerous lesions in the gastrointestinal tract]. In the large intestine, the pathologist has to differentiate between multiple polyps and polyposis (more than 100 polyps), further between adenomatosis (coli) and non-neoplastic (tumorlike) polyposis. Without prophylactic colectomy, in about 80% of adenomatosis patients an evolution of cancer is observed. Patients with extensive or total ulcerative colitis and a long history have an increased risk for developing carcinoma. Precancerous dysplasia can be demonstrated in rectoscopic and/or colonoscopic biopsies. Cancers complicating adenomatosis or ulcerative colitis account for only a very small proportion of large bowel carcinoma. The "adenoma-cancer sequence" is of greater importance. Colorectal polyps should be removed endoscopically whenever possible. Most gastric polyps are non-neoplastic and have no carcinomatous potential. The true adenoma and the so-called borderline lesion only can be considered as precursor of the gastric carcinoma. | [
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PMID:23351 | Drug refusal in schizophrenia: causes and prescribing hints. | Many discharged patients diagnosed as schizophrenic do not continue to take their prescribed antipsychotic medication. Reasons for reluctance to take drugs include the development of extrapyramidal symptoms, most notably akathisia and akinesia; a poor doctor-patient relationship; or the patient's preference to continue his schizophrenic existence. To improve drug compliance, the physician should ask the patient about his impressions of side-effects and should let the patient help determine the optimal dosage. | Drug refusal in schizophrenia: causes and prescribing hints. Many discharged patients diagnosed as schizophrenic do not continue to take their prescribed antipsychotic medication. Reasons for reluctance to take drugs include the development of extrapyramidal symptoms, most notably akathisia and akinesia; a poor doctor-patient relationship; or the patient's preference to continue his schizophrenic existence. To improve drug compliance, the physician should ask the patient about his impressions of side-effects and should let the patient help determine the optimal dosage. | [
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PMID:23352 | Characterisation of purine nucleoside phosphorylase from fibroblasts using ultra-microchemical methods. | A new technique to quantitate nucleoside phosphorylase (NP) activity in single or small numbers of counted visually selected cells is presented. Fibroblasts were cultivated on the plastic film bottom of culture dishes. After lyophilisation in situ, plastic film leaflets carrying a counted number of cells were cut out and tested for NP activity. Some properties of NP, including temperature stability, pH optimum and substrate affinity, have been studied. The data obtained suggest that Np might play a regulatory role in the purine interconversion pathway. | Characterisation of purine nucleoside phosphorylase from fibroblasts using ultra-microchemical methods. A new technique to quantitate nucleoside phosphorylase (NP) activity in single or small numbers of counted visually selected cells is presented. Fibroblasts were cultivated on the plastic film bottom of culture dishes. After lyophilisation in situ, plastic film leaflets carrying a counted number of cells were cut out and tested for NP activity. Some properties of NP, including temperature stability, pH optimum and substrate affinity, have been studied. The data obtained suggest that Np might play a regulatory role in the purine interconversion pathway. | [
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PMID:23358 | Bioavailability of digoxin: some pitfalls and problems. | The bioavailability of tablet formulations averages about 60% for digoxin, 75% for beta-acetyldigoxin, and 75% for beta-methyldigoxin. Bioavailability as a measure of the absolute amount reaching the systemic circulation should be calculated from steady state data. Only in steady state does the retarding effect of absorption disappear which diminishes the p.o./i.v. relation of data. For a screening test bioavailability may be calculated from 24-hour renal excretion values after a single dose, performing absolute and relative studies consecutively in cross-over arrangements. The absorption rate constant of digoxin and its derivatives is approximately 0.7 (h-1) which corresponds to an absorption half life of about 1 hour. The absorption rate constant can be calculated from plasma concentration values as well as from renal excretion rates 1 or 2 hours after dosing. | Bioavailability of digoxin: some pitfalls and problems. The bioavailability of tablet formulations averages about 60% for digoxin, 75% for beta-acetyldigoxin, and 75% for beta-methyldigoxin. Bioavailability as a measure of the absolute amount reaching the systemic circulation should be calculated from steady state data. Only in steady state does the retarding effect of absorption disappear which diminishes the p.o./i.v. relation of data. For a screening test bioavailability may be calculated from 24-hour renal excretion values after a single dose, performing absolute and relative studies consecutively in cross-over arrangements. The absorption rate constant of digoxin and its derivatives is approximately 0.7 (h-1) which corresponds to an absorption half life of about 1 hour. The absorption rate constant can be calculated from plasma concentration values as well as from renal excretion rates 1 or 2 hours after dosing. | [
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PMID:23362 | [Pityriasis versicolor in Greece and its predisposition factors]. | The age and seasonal incidence of 2610 patients with pityriasis versicolor in Greece were studied. Determination of the pH of Na and K ions of the sweat and the microbiol flora of the skin of pityriasis versicolor patients was undertaken. Besides these the effectiveness of a 1% selenium disulfid suspension was tested. The results indicate that the age groups of 20-29 and 30-30 years are mostly affected by the disease. A high incidence of the skin manifestation was noted during the summer and fall months. A difference of the pH of the sweat between pityriasis versicolor and healthy controls was observed, but no difference was found in the Na and K ions of the sweat among these two groups. Neither did the microbial flora from the skin lesions of patients and from corresponding sites of controls show any difference. The high relapse in this experiment indicates the relative ineffectiveness of selenium disulfid preparations when used as a 1% suspension in the treatment of pityriasis versicolor. | [Pityriasis versicolor in Greece and its predisposition factors]. The age and seasonal incidence of 2610 patients with pityriasis versicolor in Greece were studied. Determination of the pH of Na and K ions of the sweat and the microbiol flora of the skin of pityriasis versicolor patients was undertaken. Besides these the effectiveness of a 1% selenium disulfid suspension was tested. The results indicate that the age groups of 20-29 and 30-30 years are mostly affected by the disease. A high incidence of the skin manifestation was noted during the summer and fall months. A difference of the pH of the sweat between pityriasis versicolor and healthy controls was observed, but no difference was found in the Na and K ions of the sweat among these two groups. Neither did the microbial flora from the skin lesions of patients and from corresponding sites of controls show any difference. The high relapse in this experiment indicates the relative ineffectiveness of selenium disulfid preparations when used as a 1% suspension in the treatment of pityriasis versicolor. | [
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PMID:23363 | Preparation of peroxisomes from carp liver by zonal rotor density gradient centrifugation. | Peroxisomes from carp liver can be separated by isopycnic density gradient centrifugation in sucrose. Without reaching complete sedimentation equilibrium, the purification by this method is quite successful. There is a 40-fold enrichment of catalase, the peroxisomal marker, with a total yield of 27%. No pretreatment of animals is necessary for separation from lysosomes, which, besides high fragility, show lower buoyant densities than peroxisomes. The enzyme content of carp liver peroxisomes is similar to that of rat liver, with the exception of alpha-glycerophosphate dehydrogenase, which in this tissue is a completely soluble cytoplasmic enzyme. Total activities are much lower than in the rat, for the characteristic peroxisomal oxidases the difference being in the range of one order of magnitude. | Preparation of peroxisomes from carp liver by zonal rotor density gradient centrifugation. Peroxisomes from carp liver can be separated by isopycnic density gradient centrifugation in sucrose. Without reaching complete sedimentation equilibrium, the purification by this method is quite successful. There is a 40-fold enrichment of catalase, the peroxisomal marker, with a total yield of 27%. No pretreatment of animals is necessary for separation from lysosomes, which, besides high fragility, show lower buoyant densities than peroxisomes. The enzyme content of carp liver peroxisomes is similar to that of rat liver, with the exception of alpha-glycerophosphate dehydrogenase, which in this tissue is a completely soluble cytoplasmic enzyme. Total activities are much lower than in the rat, for the characteristic peroxisomal oxidases the difference being in the range of one order of magnitude. | [
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PMID:23364 | [Ultrastructural localization of acid and alkaline phosphatases in sterile septae of the anthozoa Pachycerianthus fimbriatus (author's transl)]. | Acid and alkaline phosphatase activity has been localized in the cells of the sterile septae of starved and fed anthozoa. Acid phosphatase is present in lysosomes, in Golgi cisternae and old phagosomes of starved animals. In fed animals, the reaction is more intense and the number of lysosomes is increased. New phagosomes are loaded with lead phosphate. In starved animals, the alkaline phosphatase activity has been observed on the plasma membranes and in the old phagosomes. | [Ultrastructural localization of acid and alkaline phosphatases in sterile septae of the anthozoa Pachycerianthus fimbriatus (author's transl)]. Acid and alkaline phosphatase activity has been localized in the cells of the sterile septae of starved and fed anthozoa. Acid phosphatase is present in lysosomes, in Golgi cisternae and old phagosomes of starved animals. In fed animals, the reaction is more intense and the number of lysosomes is increased. New phagosomes are loaded with lead phosphate. In starved animals, the alkaline phosphatase activity has been observed on the plasma membranes and in the old phagosomes. | [
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PMID:23365 | Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation. | Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells. | Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation. Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells. | [
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PMID:23371 | Retrospective study of 350 cases of equine cryptorchidism. | Equine cryptorchidism was examined by a review of the literature and a retrospective study of 350 horses over a 14-year period. The incidence of left vs right testis retention was nearly equal. On the left side, 75.2% of the retained testes were retained abdominally and 24.8% inguinally; on the right side, 41.8% of the retained testes were retained abdominally and 58.2% inguinally. Preoperative diagnosis by rectal palpation of the vaginal rings was considered a valuable technique, with 87.9% accuracy in 190 horses. Invasive and nonivasive surgical techniques for abdominal cryptorchidectomy and associated complications were compared. The results supported the technique of traction on gonadal structures outside the abdominal cavity (noninvasive) as superior to techniques requiring intraabdominal manipulation (invasive). | Retrospective study of 350 cases of equine cryptorchidism. Equine cryptorchidism was examined by a review of the literature and a retrospective study of 350 horses over a 14-year period. The incidence of left vs right testis retention was nearly equal. On the left side, 75.2% of the retained testes were retained abdominally and 24.8% inguinally; on the right side, 41.8% of the retained testes were retained abdominally and 58.2% inguinally. Preoperative diagnosis by rectal palpation of the vaginal rings was considered a valuable technique, with 87.9% accuracy in 190 horses. Invasive and nonivasive surgical techniques for abdominal cryptorchidectomy and associated complications were compared. The results supported the technique of traction on gonadal structures outside the abdominal cavity (noninvasive) as superior to techniques requiring intraabdominal manipulation (invasive). | [
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PMID:23372 | Occurrence and distribution of western equine encephalomyelitis in Florida. | Research and surveillance programs relating to the occurrence and distribution of western equine encephalomyelitis virus in Florida, conducted between 1955 and 1976, suggest that the virus is (1) an endemic arbordae, (2) transmitted in a continuous cycle throughout the year by Culiseta melanura mosquitoes, and (3) restricted to fresh water swamps and waterways in central, north, and northwest Florida. | Occurrence and distribution of western equine encephalomyelitis in Florida. Research and surveillance programs relating to the occurrence and distribution of western equine encephalomyelitis virus in Florida, conducted between 1955 and 1976, suggest that the virus is (1) an endemic arbordae, (2) transmitted in a continuous cycle throughout the year by Culiseta melanura mosquitoes, and (3) restricted to fresh water swamps and waterways in central, north, and northwest Florida. | [
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PMID:23377 | Spectrophotometric determination of strychnine and brucine in liquid galenicals. | A rapid method is presented for determining strychnine and brucine in liquid galenicals. At pH 5.0, both strychnine and brucine are complexed with methyl orange. After treatment with 0.1N NaOH, the liberated alkaloids are determined spectrophotometrically, using the 2-wavelength method of analysis. The method has been successfully applied to the analysis of 4 batches of nux vomica tincture, nux vomica acid, and nux vomica alkaline mixtures. The method has a relative standard deviation of 0.52%. | Spectrophotometric determination of strychnine and brucine in liquid galenicals. A rapid method is presented for determining strychnine and brucine in liquid galenicals. At pH 5.0, both strychnine and brucine are complexed with methyl orange. After treatment with 0.1N NaOH, the liberated alkaloids are determined spectrophotometrically, using the 2-wavelength method of analysis. The method has been successfully applied to the analysis of 4 batches of nux vomica tincture, nux vomica acid, and nux vomica alkaline mixtures. The method has a relative standard deviation of 0.52%. | [
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PMID:23378 | Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi. | In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms. | Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi. In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms. | [
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PMID:23379 | Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin. | Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis. | Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin. Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis. | [
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PMID:23384 | Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells. | The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid. | Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells. The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid. | [
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PMID:23385 | Toxicity, radiation sensitivity modification, and metabolic effects of dehydroascorbate and ascorbate in mammalian cells. | Dehydroascorbate, an electron affinic metabolite of vitamin C, sensitized Ehrlich ascites tumor cells, in vivo, to radiation and was selectively toxic to V79 Chinese hamster lung cells under hypoxic conditions (without radiation). The radiosensitization may involve both the electron affinic nature of dehydroascorbate as well as its ability to oxidize the intracellular NAD(P)H and non-protein sulfhydryl. Dehydroascorbate's oxidation of NAD(P)H required higher concentrations than other sulfhydryl oxidants such as N-ethylmaleimide and diamide. The oxidation of NAD(P)H by dehydroascorbate could be reversed by glucose. Hypoxic cell radiosensitization of V79 cells in tissue culture by dehydroascorbate could not be easily demonstrated because of the rapid breakdown and appreciable cytotoxicity of the drug at high concentration. The cytotoxicity was found to occur with both high and low densities of V79 cells. With low cell densities small amounts of oxygen did not reduce the cytotoxicity of dehydroascorbate, but virtually eliminated the cytotoxicity of nitroaromatic electron affinic compounds (metronidazole and Ro-07-0582). The cytotoxicity to dense cell suspensions was found to depend upon the type of buffer included in the reaction medium. The maximum cytotoxicity was obtained in buffer free saline. The reduced form of dehydroascorbate, vitamin C, was found to be toxic only under aerobic conditions. The aerobic cytotoxicity could be prevented by the addition of catalase to the growth medium or by an increase in cell density, suggesting it was caused entirely by the production of H2O2 from the oxidation of vitamin C. | Toxicity, radiation sensitivity modification, and metabolic effects of dehydroascorbate and ascorbate in mammalian cells. Dehydroascorbate, an electron affinic metabolite of vitamin C, sensitized Ehrlich ascites tumor cells, in vivo, to radiation and was selectively toxic to V79 Chinese hamster lung cells under hypoxic conditions (without radiation). The radiosensitization may involve both the electron affinic nature of dehydroascorbate as well as its ability to oxidize the intracellular NAD(P)H and non-protein sulfhydryl. Dehydroascorbate's oxidation of NAD(P)H required higher concentrations than other sulfhydryl oxidants such as N-ethylmaleimide and diamide. The oxidation of NAD(P)H by dehydroascorbate could be reversed by glucose. Hypoxic cell radiosensitization of V79 cells in tissue culture by dehydroascorbate could not be easily demonstrated because of the rapid breakdown and appreciable cytotoxicity of the drug at high concentration. The cytotoxicity was found to occur with both high and low densities of V79 cells. With low cell densities small amounts of oxygen did not reduce the cytotoxicity of dehydroascorbate, but virtually eliminated the cytotoxicity of nitroaromatic electron affinic compounds (metronidazole and Ro-07-0582). The cytotoxicity to dense cell suspensions was found to depend upon the type of buffer included in the reaction medium. The maximum cytotoxicity was obtained in buffer free saline. The reduced form of dehydroascorbate, vitamin C, was found to be toxic only under aerobic conditions. The aerobic cytotoxicity could be prevented by the addition of catalase to the growth medium or by an increase in cell density, suggesting it was caused entirely by the production of H2O2 from the oxidation of vitamin C. | [
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PMID:23386 | Phenylalanine hydroxylase in melanoma cells. | A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. | Phenylalanine hydroxylase in melanoma cells. A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. | [
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PMID:23388 | Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy. | A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses. | Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy. A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses. | [
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PMID:23390 | Determination of the metabolites of bezitramide in urine. I. Acidic metabolite. | Two methylation methods are compared in relation to the determination of low levels (less than microgram/ml) of the acidic metabolite of bezitramide in human urine. It was necessary to use alkali flame ionisation detector, which specifically detects nitrogen-containing compounds. Several difficulties associated with the use of this detector are described. | Determination of the metabolites of bezitramide in urine. I. Acidic metabolite. Two methylation methods are compared in relation to the determination of low levels (less than microgram/ml) of the acidic metabolite of bezitramide in human urine. It was necessary to use alkali flame ionisation detector, which specifically detects nitrogen-containing compounds. Several difficulties associated with the use of this detector are described. | [
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PMID:23391 | Electron spin resonance studies of erythrocytes from patients with Duchenne muscular dystrophy. | The membrane organization of the erythrocytes from patients with Duchenne muscular dystrophy was studied by means of electron spin resonance. The fluidity of the membrane near the polar region of Duchenne muscular dystrophy erythrocytes was similar to that of normal erythrocytes. The membrane environment in the nonpolar region, however, was quite different from that of normal erythrocytes, judged by the spectra with 2-(14-carboxytetradecyl) - 2 - ethyl - 4,4 - dimethyl - 3 - oxazolidinyloxyl as probe. The temperature dependence of the ratio of the line height of central field to that at the low field showed two inflection points in normal erythrocytes at pH 7.4 (13.5 degrees -16.5 degrees and 37.5 degrees -40.5 degrees C, respectively) but the inflection point in the lower temperature range was not detected in Duchenne muscular dystrophy erythrocytes. When pH was varied, an abrupt decrease in the ratio was observed at pH 5.9-5.6 in normal erythrocytes whereas there was a gradual decrease over the range of pH from 6.6 to 5.0 in Duchenne muscular dystrophy erythrocytes. The rate of reduction of the radical 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl by ascorbate in normal erythrocytes was faster than that in Duchenne muscular dystrophy erythrocytes. Treatment of both erythrocytes with phloretin markedly reduced the rate of reduction by ascorbate and eliminated the difference in the two types of erythrocyte. These results indicate that in Duchenne muscular dystrophy the erythrocyte membrane is involved as well as the muscle cell. | Electron spin resonance studies of erythrocytes from patients with Duchenne muscular dystrophy. The membrane organization of the erythrocytes from patients with Duchenne muscular dystrophy was studied by means of electron spin resonance. The fluidity of the membrane near the polar region of Duchenne muscular dystrophy erythrocytes was similar to that of normal erythrocytes. The membrane environment in the nonpolar region, however, was quite different from that of normal erythrocytes, judged by the spectra with 2-(14-carboxytetradecyl) - 2 - ethyl - 4,4 - dimethyl - 3 - oxazolidinyloxyl as probe. The temperature dependence of the ratio of the line height of central field to that at the low field showed two inflection points in normal erythrocytes at pH 7.4 (13.5 degrees -16.5 degrees and 37.5 degrees -40.5 degrees C, respectively) but the inflection point in the lower temperature range was not detected in Duchenne muscular dystrophy erythrocytes. When pH was varied, an abrupt decrease in the ratio was observed at pH 5.9-5.6 in normal erythrocytes whereas there was a gradual decrease over the range of pH from 6.6 to 5.0 in Duchenne muscular dystrophy erythrocytes. The rate of reduction of the radical 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl by ascorbate in normal erythrocytes was faster than that in Duchenne muscular dystrophy erythrocytes. Treatment of both erythrocytes with phloretin markedly reduced the rate of reduction by ascorbate and eliminated the difference in the two types of erythrocyte. These results indicate that in Duchenne muscular dystrophy the erythrocyte membrane is involved as well as the muscle cell. | [
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PMID:23392 | Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding. | Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine. | Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding. Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine. | [
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PMID:23398 | The two dimensional structure of tetrazolium-mucopolysaccharide complexes deduced from reactivity studies. | The formation of complexes between mucopolysaccharides and tetrazolium salts has been studied both by turbidimetry and nephelometry. The technique of laser nephelometry allows the detection of colloidal aggregates in systems which may, by turbidimetric methods, be ambiguous. The results indicate that binding of tetrazolium salts to polyanions can result in soluble as well as insoluble complexes; the monotetrazolium salts form soluble complexes and the ditetrazolium compounds form insoluble complexes. The insoluble complexes are stable at relatively low pH, and are disrupted during reduction to the formazan. Complex formation is decreased at high ratios of heparin to tetrazolium, and divalent cations, even at high concentration, do not precipitate mucopolysaccharides. It is concluded that stable ionic interactions with spatial charge separation are responsible for the cross linking of the mucopolysaccharides and the formaiton of large insoluble aggregates. | The two dimensional structure of tetrazolium-mucopolysaccharide complexes deduced from reactivity studies. The formation of complexes between mucopolysaccharides and tetrazolium salts has been studied both by turbidimetry and nephelometry. The technique of laser nephelometry allows the detection of colloidal aggregates in systems which may, by turbidimetric methods, be ambiguous. The results indicate that binding of tetrazolium salts to polyanions can result in soluble as well as insoluble complexes; the monotetrazolium salts form soluble complexes and the ditetrazolium compounds form insoluble complexes. The insoluble complexes are stable at relatively low pH, and are disrupted during reduction to the formazan. Complex formation is decreased at high ratios of heparin to tetrazolium, and divalent cations, even at high concentration, do not precipitate mucopolysaccharides. It is concluded that stable ionic interactions with spatial charge separation are responsible for the cross linking of the mucopolysaccharides and the formaiton of large insoluble aggregates. | [
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PMID:23399 | Fractionation of cocksfoot (Dactylis glomerata) pollen by preparative isoelectric focussing. | The technique of isoelectric focussing in polyacrylamide gel has been developed to a preparative mode. Using the improved technique cocksfoot (Dactylis glomerata) pollen has been split into many components, and the allergenic reactions of the preparations have been studied by passive transfer in animal skin and by RAST. | Fractionation of cocksfoot (Dactylis glomerata) pollen by preparative isoelectric focussing. The technique of isoelectric focussing in polyacrylamide gel has been developed to a preparative mode. Using the improved technique cocksfoot (Dactylis glomerata) pollen has been split into many components, and the allergenic reactions of the preparations have been studied by passive transfer in animal skin and by RAST. | [
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PMID:23401 | Oxygen uptake and lung function in mice infected with Streptococcus pneumoniae, influenza virus, or Mycoplasma pulmonis. | Model systems of respiratory infection in mice were established with Streptococcus pneumoniae, influenza virus, and Mycoplasma pulmonis. The LT50 for S. pneumoniae was 2 1/2 days, for lethal influenza 6 days, and for M. pulmonis 5 days. Morbidity in sublethal influenza infections reached a peak during days 5 to 10, with recovery indicated by the third week. The course of each pulmonary infection was followed by use of the animal's maximal ability to consume oxygen (VO2max by determining the weight, compliance, and stability of the excised lung, and in some cases by following O2 consumption of minced tissue. Depression of VO2max began early in each infection; reductions ranged from 9% at the peak of sublethal influenza infection to 50% 12 to 48 hr before the LT50 of fatal infections. The depressions were not relieved by 100% O2. The noninvasive VO2max test, evoked by cold air, was simple, rapid, and reproducible and appeared to serve as a quantitative measure of over-all function during infection. Each type of infection caused an increase in lung weight, with the largest noted during fatal Mycoplasma illness and lethal influenza. The effects on lungs by influenza and M. pulmonis infections were similar but could be differentiated from those with S. pneumoniae. With sublethal influenza, CL was reduced 30% between days 5 to 10, with recovery by the third week. Ctis was not affected. M. pulmonis infections and lethal influenza caused depressions in CL of over 60% by day 4 but only a 30% decrease in Ctis. The data suggest that the decreased compliance in influenza and M. pulmonis infections was due primarily to increased surface tension. In contrast, S. pneumoniae did not affect compliance. | Oxygen uptake and lung function in mice infected with Streptococcus pneumoniae, influenza virus, or Mycoplasma pulmonis. Model systems of respiratory infection in mice were established with Streptococcus pneumoniae, influenza virus, and Mycoplasma pulmonis. The LT50 for S. pneumoniae was 2 1/2 days, for lethal influenza 6 days, and for M. pulmonis 5 days. Morbidity in sublethal influenza infections reached a peak during days 5 to 10, with recovery indicated by the third week. The course of each pulmonary infection was followed by use of the animal's maximal ability to consume oxygen (VO2max by determining the weight, compliance, and stability of the excised lung, and in some cases by following O2 consumption of minced tissue. Depression of VO2max began early in each infection; reductions ranged from 9% at the peak of sublethal influenza infection to 50% 12 to 48 hr before the LT50 of fatal infections. The depressions were not relieved by 100% O2. The noninvasive VO2max test, evoked by cold air, was simple, rapid, and reproducible and appeared to serve as a quantitative measure of over-all function during infection. Each type of infection caused an increase in lung weight, with the largest noted during fatal Mycoplasma illness and lethal influenza. The effects on lungs by influenza and M. pulmonis infections were similar but could be differentiated from those with S. pneumoniae. With sublethal influenza, CL was reduced 30% between days 5 to 10, with recovery by the third week. Ctis was not affected. M. pulmonis infections and lethal influenza caused depressions in CL of over 60% by day 4 but only a 30% decrease in Ctis. The data suggest that the decreased compliance in influenza and M. pulmonis infections was due primarily to increased surface tension. In contrast, S. pneumoniae did not affect compliance. | [
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PMID:23402 | Glucose 6-phosphate dehydrogenase variants: Gd (+) Alexandra associated with neonatal jaundice and Gd (-) Camperdown in a young man with lamellar cataracts. | Two male subjects are described, with unusual clinical presentations and with hitherto undescribed G6PD variants. The first, of Italian extraction, suffered from severe neonatal jaundice following maternal ingestion of fresh broad beans (Vicia fava) both prenatally and postnatally: the expression of the enzymatic defect was much more severe in the neonatal period than on retesting in adolescence, when biochemical characterization showed unique features which justify designation as a new variant Gd(+) Alexandra. The second patient, a boy of Maltese extraction who was found to have bilateral lamellar cataracts at the age of 4 years, was identified as G6PD deficient only as a result of a survey of children of Mediterranean origin with unexplained cataract formation; he has approximately 15% of normal enzyme activity, with another unique combination of biochemical characteristics which has led to its designation as Gd(-) Camperdown. Although this association may be coincidental, it prompts further attention to the possibility that under certain circumstances G6PD deficiency may favor cataract formation. The two cases illustrate the value of characterization of the mutant enzyme whenever unexpected clinical or laboratory results are obtained. | Glucose 6-phosphate dehydrogenase variants: Gd (+) Alexandra associated with neonatal jaundice and Gd (-) Camperdown in a young man with lamellar cataracts. Two male subjects are described, with unusual clinical presentations and with hitherto undescribed G6PD variants. The first, of Italian extraction, suffered from severe neonatal jaundice following maternal ingestion of fresh broad beans (Vicia fava) both prenatally and postnatally: the expression of the enzymatic defect was much more severe in the neonatal period than on retesting in adolescence, when biochemical characterization showed unique features which justify designation as a new variant Gd(+) Alexandra. The second patient, a boy of Maltese extraction who was found to have bilateral lamellar cataracts at the age of 4 years, was identified as G6PD deficient only as a result of a survey of children of Mediterranean origin with unexplained cataract formation; he has approximately 15% of normal enzyme activity, with another unique combination of biochemical characteristics which has led to its designation as Gd(-) Camperdown. Although this association may be coincidental, it prompts further attention to the possibility that under certain circumstances G6PD deficiency may favor cataract formation. The two cases illustrate the value of characterization of the mutant enzyme whenever unexpected clinical or laboratory results are obtained. | [
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PMID:23403 | Isolation and characterization of a phospholipase A2 from an inflammatory exudate. | Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed. | Isolation and characterization of a phospholipase A2 from an inflammatory exudate. Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed. | [
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PMID:23404 | CO2-inhibition of the amplitude of bending of triton-demembranated sea urcin sperm flagella. | Demembranated sea urchin spermatozoa were reactivated in solutions containing KHCO3 and observed in a covered well slide. Although KHCO3 itself causes a small inhibition of flagellar beat frequency, the results confirm previous observations of a direct inhibition of flagellar bend angle by CO2 with no effect of CO2 on frequency. Observation of the effect of pH on the inhibition of bend angle in solutions containing KHCO3 indicates that a given concentration of OH- has a similar effect to the same concentration of HCO3-, as would be expected if CO2- inhibition results from reaction of CO2 with protein-NH3+ groups to form carbamates. CO2 may interfere with a control mechanism which selectively suppresses dynein cross-bridge activity in order to generate rhythmic bending. This control mechanism may incorporate a feedback control involving a measure of flagellar amplitude, which fails to operate successfully when the amplitude is reduced below a critical level. | CO2-inhibition of the amplitude of bending of triton-demembranated sea urcin sperm flagella. Demembranated sea urchin spermatozoa were reactivated in solutions containing KHCO3 and observed in a covered well slide. Although KHCO3 itself causes a small inhibition of flagellar beat frequency, the results confirm previous observations of a direct inhibition of flagellar bend angle by CO2 with no effect of CO2 on frequency. Observation of the effect of pH on the inhibition of bend angle in solutions containing KHCO3 indicates that a given concentration of OH- has a similar effect to the same concentration of HCO3-, as would be expected if CO2- inhibition results from reaction of CO2 with protein-NH3+ groups to form carbamates. CO2 may interfere with a control mechanism which selectively suppresses dynein cross-bridge activity in order to generate rhythmic bending. This control mechanism may incorporate a feedback control involving a measure of flagellar amplitude, which fails to operate successfully when the amplitude is reduced below a critical level. | [
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PMID:23405 | Differences in the outcomes of acute episodes of care provided by various types of family practitioners. | This study was designed to compare the outcomes achieved in a series of acute care episodes by different levels of family practice providers working in the clinic setting. The study utilizes a method which depends upon the provider to estimate level of function expected and earliest date of recovery for each episode. When the patients are viewed as a single group, those patients treated by the medex appear to fare considerably better and those seen by a faculty member do worse; however, when each functional status group is examined separately, only the asymptomatic but clinically ill patients (45 cases) show a statistically significant difference in outcomes among the providers, with the medex having good results and the faculty poor results. | Differences in the outcomes of acute episodes of care provided by various types of family practitioners. This study was designed to compare the outcomes achieved in a series of acute care episodes by different levels of family practice providers working in the clinic setting. The study utilizes a method which depends upon the provider to estimate level of function expected and earliest date of recovery for each episode. When the patients are viewed as a single group, those patients treated by the medex appear to fare considerably better and those seen by a faculty member do worse; however, when each functional status group is examined separately, only the asymptomatic but clinically ill patients (45 cases) show a statistically significant difference in outcomes among the providers, with the medex having good results and the faculty poor results. | [
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PMID:23406 | Sodium and calcium movements in dog red blood cells. | Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed. | Sodium and calcium movements in dog red blood cells. Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed. | [
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PMID:23407 | The stimulating effect of fatty acids and amino acid derivatives on the labellar sugar receptor of the fleshfly. | Seven D-amino acids, including D-valine, D-phenylalanine, D-leucine, D-isoleucine, D-tryptophan, D-methionine, and D-alpha-aminobutyric acid, are markedly less stimulative than the corresponding L-isomers that can stimulate the labellar sugar receptor of the fleshfly. A distinct effect of len;th of the amino acid side chain is clearly observed. Esterification and amidation of the alpha-carboxyl group, as well as substitution by hydroxyl and methyl groups, result in extremely decreased responses. Amino acids whose amino groups are located at a position other than the alpha are almost ineffective. With all these rigid stereospecificities of the sugar receptor for amino acids, certain replacement of the alpha-amino group with the hydroxyl or carbonyl group shows a slight increase of the response at neutral pH. Furthermore, certain fatty acids can stimulate the sugar receptor once the solutions are buffered at neutral pH. This observation was further supported by the presence of a remarkable similarity of stimulating effectiveness between amino acids that can stimulate the sugar receptor and those fatty acids. The similarity was shown by testing the response concentration relationships, the stimulating effect of fatty acid derivatives, the effect of treatment with p-chloromercuribenzoate, the behavioral response, and so on. | The stimulating effect of fatty acids and amino acid derivatives on the labellar sugar receptor of the fleshfly. Seven D-amino acids, including D-valine, D-phenylalanine, D-leucine, D-isoleucine, D-tryptophan, D-methionine, and D-alpha-aminobutyric acid, are markedly less stimulative than the corresponding L-isomers that can stimulate the labellar sugar receptor of the fleshfly. A distinct effect of len;th of the amino acid side chain is clearly observed. Esterification and amidation of the alpha-carboxyl group, as well as substitution by hydroxyl and methyl groups, result in extremely decreased responses. Amino acids whose amino groups are located at a position other than the alpha are almost ineffective. With all these rigid stereospecificities of the sugar receptor for amino acids, certain replacement of the alpha-amino group with the hydroxyl or carbonyl group shows a slight increase of the response at neutral pH. Furthermore, certain fatty acids can stimulate the sugar receptor once the solutions are buffered at neutral pH. This observation was further supported by the presence of a remarkable similarity of stimulating effectiveness between amino acids that can stimulate the sugar receptor and those fatty acids. The similarity was shown by testing the response concentration relationships, the stimulating effect of fatty acid derivatives, the effect of treatment with p-chloromercuribenzoate, the behavioral response, and so on. | [
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PMID:23408 | On the effects of divalent cations and ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetate on action potential duration in frog heart. | Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions. | On the effects of divalent cations and ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetate on action potential duration in frog heart. Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions. | [
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PMID:23409 | Characterization of L-aspartate uptake by Streptomyces hydrogenans. | Multiple transport systems for L-aspartic acid exist in Steptomyces hydrogenans. The intracellular accumulation of L-aspartate against a concentration gradient was immediately inhibited by proton conductors, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol or nigericin. Transport activity was gradually lost when inhibitors of protein synthesis were added. L-Aspartate transport had two pH optima at 6.5 and 4.5. At pH 6.5, two saturable transport components with different Km and Vmax values could be resolved by kinetic studies. A high-affinity system (system I) preferred the L-isomers of the anionic forms of aspartic and glutamic acid. At the same pH, a second, low-affinity system (system II) operated, which was presumably less specific than system I and also able to accept, at high concentrations, neutral amino acids. At pH 4.5, the Lineweaver-Burk plot revealed only a single catalytic component, with Km and Vmax values similar to those of system II. Again, in contrast to system I, this component showed high affinity for neutral amino acids. The data suggest that L-aspartic acid and L-glutamic acid are transported by this system as neutral zwitterionic molecules. | Characterization of L-aspartate uptake by Streptomyces hydrogenans. Multiple transport systems for L-aspartic acid exist in Steptomyces hydrogenans. The intracellular accumulation of L-aspartate against a concentration gradient was immediately inhibited by proton conductors, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol or nigericin. Transport activity was gradually lost when inhibitors of protein synthesis were added. L-Aspartate transport had two pH optima at 6.5 and 4.5. At pH 6.5, two saturable transport components with different Km and Vmax values could be resolved by kinetic studies. A high-affinity system (system I) preferred the L-isomers of the anionic forms of aspartic and glutamic acid. At the same pH, a second, low-affinity system (system II) operated, which was presumably less specific than system I and also able to accept, at high concentrations, neutral amino acids. At pH 4.5, the Lineweaver-Burk plot revealed only a single catalytic component, with Km and Vmax values similar to those of system II. Again, in contrast to system I, this component showed high affinity for neutral amino acids. The data suggest that L-aspartic acid and L-glutamic acid are transported by this system as neutral zwitterionic molecules. | [
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PMID:23410 | The mode of action of N-(n-Dodecyl)diethanolamine with particular reference to the effect of protonation on uptake by Escherichia coli. | In a homologous series of N-(n-alkyl)diethanolamines antimicrobial activity was related to surface activity and increasing octanol-water partition coefficient. Maximum activity was exhibited by the dodecyl-, tetradecyl- and hexadecyl-derivatives. Dodecyldiethanolamine (DDE) displayed a broad spectrum of activity. Towards Escherichia coli NCIB8277, its bacteriostatic and bactericidal activity increased as the degree of protonation lessened, and may have been influenced by the formation of micelles. Uptake of DDE by washed suspensions of E. coli was more rapid and more extensive at pH 7.0 than pH 4.0. Within this pH range, bacterial uptake, the octanol-water partition coefficient (lipid solubility) and the proportion of unprotonated DDE all increased. Uptake isotherms at pH values in the range 4.0 to 8.0 are interpreted as signifying different uptake mechanisms for the protonated and unprotonated forms. | The mode of action of N-(n-Dodecyl)diethanolamine with particular reference to the effect of protonation on uptake by Escherichia coli. In a homologous series of N-(n-alkyl)diethanolamines antimicrobial activity was related to surface activity and increasing octanol-water partition coefficient. Maximum activity was exhibited by the dodecyl-, tetradecyl- and hexadecyl-derivatives. Dodecyldiethanolamine (DDE) displayed a broad spectrum of activity. Towards Escherichia coli NCIB8277, its bacteriostatic and bactericidal activity increased as the degree of protonation lessened, and may have been influenced by the formation of micelles. Uptake of DDE by washed suspensions of E. coli was more rapid and more extensive at pH 7.0 than pH 4.0. Within this pH range, bacterial uptake, the octanol-water partition coefficient (lipid solubility) and the proportion of unprotonated DDE all increased. Uptake isotherms at pH values in the range 4.0 to 8.0 are interpreted as signifying different uptake mechanisms for the protonated and unprotonated forms. | [
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PMID:23419 | Comparative digestibility of acid detergent fiber by laboratory albino and wild Polynesian rats. | The effects of adding 5%, 10%, and 15% acid detergent fiber to a nonfibrous basal diet were examined in a comparative feeding study with Polynesian rats (Rattus exulans) and laboratory rats. Digestibility coefficients for dry matter, crude protein, and gross energy declined significantly in both species as fiber content increased, but averaged significantly lower in the Polynesian than in the laboratory rat. Fiber digestibility was not significantly affected by fiber level but was by species, with the Polynesian rat having the higher digestibility coefficients. | Comparative digestibility of acid detergent fiber by laboratory albino and wild Polynesian rats. The effects of adding 5%, 10%, and 15% acid detergent fiber to a nonfibrous basal diet were examined in a comparative feeding study with Polynesian rats (Rattus exulans) and laboratory rats. Digestibility coefficients for dry matter, crude protein, and gross energy declined significantly in both species as fiber content increased, but averaged significantly lower in the Polynesian than in the laboratory rat. Fiber digestibility was not significantly affected by fiber level but was by species, with the Polynesian rat having the higher digestibility coefficients. | [
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PMID:23421 | Fetal hypertension and the development of increased pulmonary vascular smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. | Chronic pulmonary arterial hypertension was produced in six fetal lambs. In four (126 to 139 days' gestation) unilateral fetal renal artery constriction caused systemic arterial mean blood pressure elevations. In another fetus, constriction of the umbilical artery caused a systemic mean blood pressure elevation; in the sixth, partial occlusion of the ductus arteriosus caused isolated pulmonary arterial hypertension. The right lung of each fetus was perfused with fixative at the in vivo mean arterial pressure and the amount of smooth muscle in the fifth generation (resistance) vessels analyzed using the medial width/external diameter ratio. There was a significant increase in the medial width/external diameter ratio in the six experimental animals as compared to that in six normal fetuses. In separate fetuses the increased ratios were due to a decreased external diameter, increased smooth muscle, or both these factors. The total number of resistance vessels was counted in the right lung of each fetus and no significant difference from normal was observed. We postulate that either fetal systemic hypertension or constriction of the ductus arteriosus causes fetal pulmonary hypertension in utero and that this produces increased smooth muscle development in pulmonary arterial resistance vessels; this may be a pathogenic mechanism for the syndrome of persistent pulmonary hypertension of the newborn infant. | Fetal hypertension and the development of increased pulmonary vascular smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. Chronic pulmonary arterial hypertension was produced in six fetal lambs. In four (126 to 139 days' gestation) unilateral fetal renal artery constriction caused systemic arterial mean blood pressure elevations. In another fetus, constriction of the umbilical artery caused a systemic mean blood pressure elevation; in the sixth, partial occlusion of the ductus arteriosus caused isolated pulmonary arterial hypertension. The right lung of each fetus was perfused with fixative at the in vivo mean arterial pressure and the amount of smooth muscle in the fifth generation (resistance) vessels analyzed using the medial width/external diameter ratio. There was a significant increase in the medial width/external diameter ratio in the six experimental animals as compared to that in six normal fetuses. In separate fetuses the increased ratios were due to a decreased external diameter, increased smooth muscle, or both these factors. The total number of resistance vessels was counted in the right lung of each fetus and no significant difference from normal was observed. We postulate that either fetal systemic hypertension or constriction of the ductus arteriosus causes fetal pulmonary hypertension in utero and that this produces increased smooth muscle development in pulmonary arterial resistance vessels; this may be a pathogenic mechanism for the syndrome of persistent pulmonary hypertension of the newborn infant. | [
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PMID:23422 | Monitoring critically ill newborn infants with digital capillary blood samples: an alternative. | Capillary blood samples obtained from a warmed distal phalanx of the right hand were compared with either temporal or right radial arterial blood samples for PO2, PCO2, and pH in 33 critically ill newborn infants. The blood pressure and skin temperatures of each infant and the ambient oxygen concentration were recorded at the time the blood was sampled. Sixty-eight paired PO2 analyses yielded a regression line close to the line of identity. The mean difference between digital capillary and arterial PO2 was 11.3 +/- 1.4 mm Hg (r = 0.92). The results were similar for the paired PCO2 analyses (r = 0.84) and for the paired pH analyses (r = 0.94). The correlation between arterial PO2 and digital capillary PO2 deteriorated when the systolic blood pressure of the patient was below 35 mm Hg. There was no correlation between skin temperature and capillary-arterial PO2 differences. The frequency of retrolental fibroplasia leading to blindness was not different from that in nurseries that sample umbilical arterial blood routinely. | Monitoring critically ill newborn infants with digital capillary blood samples: an alternative. Capillary blood samples obtained from a warmed distal phalanx of the right hand were compared with either temporal or right radial arterial blood samples for PO2, PCO2, and pH in 33 critically ill newborn infants. The blood pressure and skin temperatures of each infant and the ambient oxygen concentration were recorded at the time the blood was sampled. Sixty-eight paired PO2 analyses yielded a regression line close to the line of identity. The mean difference between digital capillary and arterial PO2 was 11.3 +/- 1.4 mm Hg (r = 0.92). The results were similar for the paired PCO2 analyses (r = 0.84) and for the paired pH analyses (r = 0.94). The correlation between arterial PO2 and digital capillary PO2 deteriorated when the systolic blood pressure of the patient was below 35 mm Hg. There was no correlation between skin temperature and capillary-arterial PO2 differences. The frequency of retrolental fibroplasia leading to blindness was not different from that in nurseries that sample umbilical arterial blood routinely. | [
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PMID:23423 | Physicochemical properties of glycyrrhizic acid in aqueous media II: Effect on flocculation-deflocculation behavior of suspensions of sulfathiazole and graphite. | The flocculation-deflocculation behavior of sulfathiazole and graphite in aqueous solutions of glycyrrhizic acid was studied by measuring the sedimentation volume and turbidity of supernates. The dispersing effect of glycyrrhizic acid on suspension of sulfathiazole showed a maximum in the pH 3-4 region, the same pH region where the zeta-potential of sulfathiazole particles showed a negative maximum. The results were explained by the variation of degrees of ionization of glycyrrhizic acid and sulfathiazole with pH. With graphite suspensions, the pH region where the dispersing effect of glycyrrhizic acid showed a maximum shifted to a higher pH compared with sulfathiazole. This result can be attributed to the fact that graphite is a nonpolar substance so the surface properties are not affected by a pH change. Hence, the adsorption of glycyrrhizic acid occurs even in a fairly high pH range. | Physicochemical properties of glycyrrhizic acid in aqueous media II: Effect on flocculation-deflocculation behavior of suspensions of sulfathiazole and graphite. The flocculation-deflocculation behavior of sulfathiazole and graphite in aqueous solutions of glycyrrhizic acid was studied by measuring the sedimentation volume and turbidity of supernates. The dispersing effect of glycyrrhizic acid on suspension of sulfathiazole showed a maximum in the pH 3-4 region, the same pH region where the zeta-potential of sulfathiazole particles showed a negative maximum. The results were explained by the variation of degrees of ionization of glycyrrhizic acid and sulfathiazole with pH. With graphite suspensions, the pH region where the dispersing effect of glycyrrhizic acid showed a maximum shifted to a higher pH compared with sulfathiazole. This result can be attributed to the fact that graphite is a nonpolar substance so the surface properties are not affected by a pH change. Hence, the adsorption of glycyrrhizic acid occurs even in a fairly high pH range. | [
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PMID:23424 | Hydrolytic degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride. | Degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride was followed in basic medium (pH 7.5) to isolate and characterize all possible degradation products. IR, Raman, and NMR spectroscopy, TLC, and elemental analysis were used to identify the products. Degradation of base-hydrolyzed 2,6-dichlorobenzylthiopseudourea hydrochloride produced 2,6-dichlorobenzylthiol and cyanamide and was followed by oxidation (air) to produce bis(2,6-dichlorobenzyl) disulfide, dimerization to give cyanoguanidine, and hydrolysis to yield urea. The kinetics of hydrolysis at 22.5 degrees (pH 7.0 and 7.5) and at 37 degrees (pH 7.0) revealed a pseudo-first-order reaction with respect to the substrate. Apparent first-order rate constants and energy of activation, entropy of activation, and half-life values were determined. | Hydrolytic degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride. Degradation of 2,6-dichlorobenzylthiopseudourea hydrochloride was followed in basic medium (pH 7.5) to isolate and characterize all possible degradation products. IR, Raman, and NMR spectroscopy, TLC, and elemental analysis were used to identify the products. Degradation of base-hydrolyzed 2,6-dichlorobenzylthiopseudourea hydrochloride produced 2,6-dichlorobenzylthiol and cyanamide and was followed by oxidation (air) to produce bis(2,6-dichlorobenzyl) disulfide, dimerization to give cyanoguanidine, and hydrolysis to yield urea. The kinetics of hydrolysis at 22.5 degrees (pH 7.0 and 7.5) and at 37 degrees (pH 7.0) revealed a pseudo-first-order reaction with respect to the substrate. Apparent first-order rate constants and energy of activation, entropy of activation, and half-life values were determined. | [
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PMID:23425 | Improved colorimetric determination of aspirin and salicylic acid concentrations in human plasma. | An improvement in a previously described method for the determination of plasma salicylic acid and aspirin levels in humans is described. The procedure was simplified by employing only one plasma sample for both salicylates. More accurate estimation of salicylates, particularly aspirin, was achieved by using two different calibration curves. Salicylic acid was estimated by reaction with an aqueous solution of the Folin-Ciocalteu phenol reagent. Absorbance of the blue-colored complex, which formed on addition of sodium hydroxide, was measured at 670 nm. The influence of alkalinity in the formation of the colored complex is discussed. The average recovery of aspirin added to plasma was 94.61%; it was 214.72% by the previous method. | Improved colorimetric determination of aspirin and salicylic acid concentrations in human plasma. An improvement in a previously described method for the determination of plasma salicylic acid and aspirin levels in humans is described. The procedure was simplified by employing only one plasma sample for both salicylates. More accurate estimation of salicylates, particularly aspirin, was achieved by using two different calibration curves. Salicylic acid was estimated by reaction with an aqueous solution of the Folin-Ciocalteu phenol reagent. Absorbance of the blue-colored complex, which formed on addition of sodium hydroxide, was measured at 670 nm. The influence of alkalinity in the formation of the colored complex is discussed. The average recovery of aspirin added to plasma was 94.61%; it was 214.72% by the previous method. | [
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PMID:23428 | An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. | 1. Intracellular pH (pH(i)) of surface fibres of the mouse soleus muscle was measured in vitro by recessed-tip pH-sensitive micro-electrodes. pH(i) was displaced in an acid direction by removal of external (NH(4))(2)SO(4) after a short exposure, and the mechanism of recovery from this acidification was investigated.2. Removal of external K caused a very slow acidification (probably due to the decreasing Na gradient) but had no effect on the rate of pH(i) recovery following acidification. This indicates that K(+)-H(+) exchange is not involved in the pH(i) regulating system.3. Short applications of 10(-4)M ouabain had no obvious effect on pH(i) and did not alter the rate of pH(i) recovery following acidification. This suggests that there is no direct connexion between the regulation of pH(i) and the Na pump.4. Reduction of external Ca from 10 to 1 mM caused a transient fall in pH(i), but the rate of pH(i) recovery following acidification was unaffected. This suggests that Ca(2+)-H(+) exchange is not involved in the pH(i) regulating system.5. An 11% reduction in external Na caused a significant slowing of pH(i) recovery following acidification. 90% or complete removal of external Na almost stopped pH(i) recovery. This suggests that Na(+)-H(+) exchange is involved in pH(i) regulation.6. Amiloride (10(-4)M) reversibly reduced the rate of pH(i) recovery to much the same extent as removal of external Na. Its effect was not additive to that of removal of external Na.7. Internal Na ion concentration ([Na(+)](i)), measured using Na(+)-sensitive micro-electrodes, fell on application of (NH(4))(2)SO(4) and increased on its removal. The increase transiently raised [Na(+)](i) above the level recorded before (NH(4))(2)SO(4) application. This overshoot of [Na(+)](i) was almost completely inhibited by amiloride. This is consistent with the involvement of Na(+)-H(+) exchange in the pH(i) regulating system.8. Removal of external CO(2) or application of SITS (10(-4)M) caused some slowing of the rate of pH(i) recovery following acidification by removal of (NH(4))(2)SO(4). The effect of SITS was additive to that of Na-free Ringer or amiloride. These results suggest that Cl(-)-HCO(3) (-) exchange is also involved in the pH(i) regulating system and that it is a separate mechanism. Under the conditions used, Cl(-)-HCO(3) (-) exchange formed about 20% of the pH(i) regulating system.9. Decreasing the temperature from 37 to 28 degrees C not only caused an increase in pH(i), but also considerably slowed the rate of pH(i) recovery following acidification. We have calculated a Q(10) for Na(+)-H(+) exchange of 1.4 and for Cl(-)-HCO(3) (-) exchange, 6.9.10. We conclude that the pH(i) regulating system is comprised of two separate ionic exchange mechanisms. The major mechanism is Na(+)-H(+) exchange, which is probably driven by the transmembrane Na gradient. The other mechanism is Cl(-)-HCO(3) (-) exchange, which probably requires metabolic energy. | An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. 1. Intracellular pH (pH(i)) of surface fibres of the mouse soleus muscle was measured in vitro by recessed-tip pH-sensitive micro-electrodes. pH(i) was displaced in an acid direction by removal of external (NH(4))(2)SO(4) after a short exposure, and the mechanism of recovery from this acidification was investigated.2. Removal of external K caused a very slow acidification (probably due to the decreasing Na gradient) but had no effect on the rate of pH(i) recovery following acidification. This indicates that K(+)-H(+) exchange is not involved in the pH(i) regulating system.3. Short applications of 10(-4)M ouabain had no obvious effect on pH(i) and did not alter the rate of pH(i) recovery following acidification. This suggests that there is no direct connexion between the regulation of pH(i) and the Na pump.4. Reduction of external Ca from 10 to 1 mM caused a transient fall in pH(i), but the rate of pH(i) recovery following acidification was unaffected. This suggests that Ca(2+)-H(+) exchange is not involved in the pH(i) regulating system.5. An 11% reduction in external Na caused a significant slowing of pH(i) recovery following acidification. 90% or complete removal of external Na almost stopped pH(i) recovery. This suggests that Na(+)-H(+) exchange is involved in pH(i) regulation.6. Amiloride (10(-4)M) reversibly reduced the rate of pH(i) recovery to much the same extent as removal of external Na. Its effect was not additive to that of removal of external Na.7. Internal Na ion concentration ([Na(+)](i)), measured using Na(+)-sensitive micro-electrodes, fell on application of (NH(4))(2)SO(4) and increased on its removal. The increase transiently raised [Na(+)](i) above the level recorded before (NH(4))(2)SO(4) application. This overshoot of [Na(+)](i) was almost completely inhibited by amiloride. This is consistent with the involvement of Na(+)-H(+) exchange in the pH(i) regulating system.8. Removal of external CO(2) or application of SITS (10(-4)M) caused some slowing of the rate of pH(i) recovery following acidification by removal of (NH(4))(2)SO(4). The effect of SITS was additive to that of Na-free Ringer or amiloride. These results suggest that Cl(-)-HCO(3) (-) exchange is also involved in the pH(i) regulating system and that it is a separate mechanism. Under the conditions used, Cl(-)-HCO(3) (-) exchange formed about 20% of the pH(i) regulating system.9. Decreasing the temperature from 37 to 28 degrees C not only caused an increase in pH(i), but also considerably slowed the rate of pH(i) recovery following acidification. We have calculated a Q(10) for Na(+)-H(+) exchange of 1.4 and for Cl(-)-HCO(3) (-) exchange, 6.9.10. We conclude that the pH(i) regulating system is comprised of two separate ionic exchange mechanisms. The major mechanism is Na(+)-H(+) exchange, which is probably driven by the transmembrane Na gradient. The other mechanism is Cl(-)-HCO(3) (-) exchange, which probably requires metabolic energy. | [
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PMID:23429 | The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. | 1. Intracellular pH (pH(i)), Cl(-) and Na(+) levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH(i) recovery from internal acidification investigated.2. Reducing the external HCO(3) (-) concentration greatly inhibited the rate of pH(i) recovery from HCl injection.3. Reducing external Cl(-) did not inhibit pH(i) recovery, but reducing internal Cl(-), by exposing the cell to sulphate Ringer, inhibited pH(i) recovery from CO(2) application.4. During pH(i) recovery from CO(2) application the internal Cl(-) concentration decreased. The measured fall in internal Cl(-) concentration averaged about 25% of the calculated increase in internal HCO(3) (-).5. Removal of external Na inhibited the pH(i) recovery from either CO(2) application or HCl injection.6. During the pH(i) recovery from acidification there was an increase in the internal Na(+) concentration ([Na(+)](i)). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na(+)](i) that occurred during pH(i) recovery from an injection of HCl was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH(i) recovery after HCl injection were not additive.9. It is concluded that the pH(i) regulating system involves tightly linked Cl(-)-HCO(3) (-) and Na(+)-H(+) exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO(3) (-) and the movement outward of Cl(-) and H(+) ions. | The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. 1. Intracellular pH (pH(i)), Cl(-) and Na(+) levels were recorded in snail neurones using ion-sensitive micro-electrodes, and the mechanism of the pH(i) recovery from internal acidification investigated.2. Reducing the external HCO(3) (-) concentration greatly inhibited the rate of pH(i) recovery from HCl injection.3. Reducing external Cl(-) did not inhibit pH(i) recovery, but reducing internal Cl(-), by exposing the cell to sulphate Ringer, inhibited pH(i) recovery from CO(2) application.4. During pH(i) recovery from CO(2) application the internal Cl(-) concentration decreased. The measured fall in internal Cl(-) concentration averaged about 25% of the calculated increase in internal HCO(3) (-).5. Removal of external Na inhibited the pH(i) recovery from either CO(2) application or HCl injection.6. During the pH(i) recovery from acidification there was an increase in the internal Na(+) concentration ([Na(+)](i)). The increase was larger than that occurring when the Na pump was inhibited by K-free Ringer.7. The increase in [Na(+)](i) that occurred during pH(i) recovery from an injection of HCl was about half of that produced by a similar injection of NaCl.8. The inhibitory effects of Na-free Ringer and of the anion exchange inhibitor SITS on pH(i) recovery after HCl injection were not additive.9. It is concluded that the pH(i) regulating system involves tightly linked Cl(-)-HCO(3) (-) and Na(+)-H(+) exchange, with Na entry down its concentration gradient probably providing the energy to drive the movement inwards of HCO(3) (-) and the movement outward of Cl(-) and H(+) ions. | [
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PMID:23430 | Further evidence for adrenergic transmission in the human vas deferens. | 1. Isolated portions of human vas deferens responded to field stimulation of the intramural nerve fibres or to exogenously applied noradrenaline with rhythmical contractions of both longitudinal and circular muscle layers. 2. In guinea-pig and rabbit vas deferens field stimulation produced an initial rapid 'twitch' response which was not found with human vasa. 3. Responses of the human vas to field stimulation were depressed by the alpha-adrenoceptor blocking agents phentolamine and yohimbine. 4. It is concluded that the motor innervation of the human vas deferens is adrenergic and the relevance of this to the physiological operation of the tissue is discussed. | Further evidence for adrenergic transmission in the human vas deferens. 1. Isolated portions of human vas deferens responded to field stimulation of the intramural nerve fibres or to exogenously applied noradrenaline with rhythmical contractions of both longitudinal and circular muscle layers. 2. In guinea-pig and rabbit vas deferens field stimulation produced an initial rapid 'twitch' response which was not found with human vasa. 3. Responses of the human vas to field stimulation were depressed by the alpha-adrenoceptor blocking agents phentolamine and yohimbine. 4. It is concluded that the motor innervation of the human vas deferens is adrenergic and the relevance of this to the physiological operation of the tissue is discussed. | [
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PMID:23431 | The effect of foreign cations, pH and pharmacological agents on the ionic permeability of an excitatory glutamate synapse. | 1. Voltage clamp studies of the post-synaptic membrane of the insect neuromuscular junction have shown that normal amplitude glutamate currents could be recorded for a limited time when external Na was completely replaced by Ca, Li, ammonium, methylamine and guanidine. No change in the reversal potential of the glutamate current was observed when Na was replaced by these ions. It is suggested that the glutamate ionic channel has a similar permeability to Na and to these foreign cations, although the foreign ions cause a longer-term block of the permeability increase or receptor function. 2. Procaine, pentobarbitone, 2-4-6-triaminopyrimidine, high and low pH and low temperature reduced the synaptic ionic permeability but did not alter the ratio of the conductance increase of Na to K (delta g Na/delta gK). 4-Aminopyridine and TEA did not reduce the synaptic ionic permeability. 3. The 3. The properties of the synaptic ionic channels resemble the tight junction transepithelial ionic channels of the mammalian gall-bladder, but are very different from the Na and K non-synaptic channels of axons. It is suggested that Na and K normally pass through a single relatively large channel containing strong proton accepting acidic ligands which render the channel cation selective. | The effect of foreign cations, pH and pharmacological agents on the ionic permeability of an excitatory glutamate synapse. 1. Voltage clamp studies of the post-synaptic membrane of the insect neuromuscular junction have shown that normal amplitude glutamate currents could be recorded for a limited time when external Na was completely replaced by Ca, Li, ammonium, methylamine and guanidine. No change in the reversal potential of the glutamate current was observed when Na was replaced by these ions. It is suggested that the glutamate ionic channel has a similar permeability to Na and to these foreign cations, although the foreign ions cause a longer-term block of the permeability increase or receptor function. 2. Procaine, pentobarbitone, 2-4-6-triaminopyrimidine, high and low pH and low temperature reduced the synaptic ionic permeability but did not alter the ratio of the conductance increase of Na to K (delta g Na/delta gK). 4-Aminopyridine and TEA did not reduce the synaptic ionic permeability. 3. The 3. The properties of the synaptic ionic channels resemble the tight junction transepithelial ionic channels of the mammalian gall-bladder, but are very different from the Na and K non-synaptic channels of axons. It is suggested that Na and K normally pass through a single relatively large channel containing strong proton accepting acidic ligands which render the channel cation selective. | [
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PMID:23433 | Study of the structural requirements for dopa potentiation and oxotremorine antagonism by L-prolyl-L-leucylglycinamide. | A number of analogs of the tripeptide L-prolyly-L-leucylglycinamide (1) were synthesized and evaluated in the Dopa potentiation and oxotremorine antagonism tests. The replacement of the glycinamide residue with either the glycine methylamide, glycine, aminoacetonitrile, amino-2-propanone, semicarbazide, or beta-alaninamide residues resulted in a loss of activity in both tests. A 1:1 mixture of L-prolyl-L-leucyl-(-)-thiazolidine-2-carboxamide (8) and L-prolyl-L-leucyl-(+)-thiazolidine-2-carboxamide (9) showed marked activity in the Dopa potentiation test but was unable to antagonize the tremors induced by oxotremorine. L-Prolyl-L-leucyl-L-prolinamide (11), on the other hand, was active in the oxotremorine antagonism test but inactive in the Dopa potentiation test. The replacement of the pyrrolidine ring of 1 with either a thiazolidine or cyclopentane ring system caused a loss of activity. The cyclopentanecarboxylic acid analogue 13, however, was found to have moderate activity in the serotonin potentiation test. | Study of the structural requirements for dopa potentiation and oxotremorine antagonism by L-prolyl-L-leucylglycinamide. A number of analogs of the tripeptide L-prolyly-L-leucylglycinamide (1) were synthesized and evaluated in the Dopa potentiation and oxotremorine antagonism tests. The replacement of the glycinamide residue with either the glycine methylamide, glycine, aminoacetonitrile, amino-2-propanone, semicarbazide, or beta-alaninamide residues resulted in a loss of activity in both tests. A 1:1 mixture of L-prolyl-L-leucyl-(-)-thiazolidine-2-carboxamide (8) and L-prolyl-L-leucyl-(+)-thiazolidine-2-carboxamide (9) showed marked activity in the Dopa potentiation test but was unable to antagonize the tremors induced by oxotremorine. L-Prolyl-L-leucyl-L-prolinamide (11), on the other hand, was active in the oxotremorine antagonism test but inactive in the Dopa potentiation test. The replacement of the pyrrolidine ring of 1 with either a thiazolidine or cyclopentane ring system caused a loss of activity. The cyclopentanecarboxylic acid analogue 13, however, was found to have moderate activity in the serotonin potentiation test. | [
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PMID:23434 | The ability of smooth and rough strains of Streptococcus pneumoniae to activate human complement by the alternative pathway. | Three capsulate pneumococcal strains of serotypes 1, 2 ans 3, and one non-capsulate strain of serotype 47, were found to activate human complement by the alternative pathway to a similar extent over the concentration range examined. Nevertheless, the capsulate strains, in contrast to the non-capsulate, are known to require complement attachment for phagocytosis and it is therefore postulated that the toxic by-products released cause the wave of oedema characteristic of pneumococcal lobar pneumonia. | The ability of smooth and rough strains of Streptococcus pneumoniae to activate human complement by the alternative pathway. Three capsulate pneumococcal strains of serotypes 1, 2 ans 3, and one non-capsulate strain of serotype 47, were found to activate human complement by the alternative pathway to a similar extent over the concentration range examined. Nevertheless, the capsulate strains, in contrast to the non-capsulate, are known to require complement attachment for phagocytosis and it is therefore postulated that the toxic by-products released cause the wave of oedema characteristic of pneumococcal lobar pneumonia. | [
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PMID:23435 | The kinetic mechanism of action of an uncoupler of oxidative phosphorylation. | The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study. | The kinetic mechanism of action of an uncoupler of oxidative phosphorylation. The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study. | [
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PMID:23437 | Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction. | The direct neurotoxic effects of E. coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction. Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs). Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates. The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency. No significant changes in MEPP amplitude were evident. The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations. Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high [K+]out. The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane. | Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction. The direct neurotoxic effects of E. coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction. Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs). Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates. The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency. No significant changes in MEPP amplitude were evident. The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations. Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high [K+]out. The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane. | [
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PMID:23439 | Biochemical and morphological characterization of mycobacteriophage R1. | Large-scale propagation of mycobacteriophage R1 in broth culture has allowed the isolation of quantities of virus sufficient for characterization of its nucleic acid and lipid components as well as investigation of its ultrastructural attributes. Analysis of R1 DNA indicates that it is double stranded and possesses a molecular weight of 2.5 X 10(7) and a guanine-plus-cytosine content of 65.7 +/- 0.5%. The lipid fraction of R1 accounts for 14% of the total dry weight of the virus, 20% of which was identified as free or esterified sterols. A rapid loss of viral titer occurred after seconds of exposure to organic solvents. This result suggests that the lipid fractions of R1 is essential for its infectivity. Electron microscopic investigation of solvent-extracted R1 showed extensive deterioration of its normal morphology, including nucleocapsid disintegration and base plate separation. Routine phosphotungstate preparations demonstrated a particle with an oval head and a noncontractile tail. Altering the pH of the phosphotungstate negative stain from neutrality damage the viral particles. Uranyl formate-contrasted specimens displayed an elongated hexagonal nucleocapsid with a neck region; the cross-striated tail possessed a starlike base plate. | Biochemical and morphological characterization of mycobacteriophage R1. Large-scale propagation of mycobacteriophage R1 in broth culture has allowed the isolation of quantities of virus sufficient for characterization of its nucleic acid and lipid components as well as investigation of its ultrastructural attributes. Analysis of R1 DNA indicates that it is double stranded and possesses a molecular weight of 2.5 X 10(7) and a guanine-plus-cytosine content of 65.7 +/- 0.5%. The lipid fraction of R1 accounts for 14% of the total dry weight of the virus, 20% of which was identified as free or esterified sterols. A rapid loss of viral titer occurred after seconds of exposure to organic solvents. This result suggests that the lipid fractions of R1 is essential for its infectivity. Electron microscopic investigation of solvent-extracted R1 showed extensive deterioration of its normal morphology, including nucleocapsid disintegration and base plate separation. Routine phosphotungstate preparations demonstrated a particle with an oval head and a noncontractile tail. Altering the pH of the phosphotungstate negative stain from neutrality damage the viral particles. Uranyl formate-contrasted specimens displayed an elongated hexagonal nucleocapsid with a neck region; the cross-striated tail possessed a starlike base plate. | [
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PMID:23440 | Transfection in pneumococcus: single-strand intermediates in the formation of infective centers. | Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection. | Transfection in pneumococcus: single-strand intermediates in the formation of infective centers. Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection. | [
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