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PMID:5178 | A quantitative comparison of the formation of synapses in the rat superior cervical sympathetic ganglion by its own and by foreign nerve fibres. | The rat superior cervical sympathetic ganglion (SCG) has about 36,000 neurones in a volume of about 1 cu.mm. There are about 8.8 X 10(6) synapses, and 6000-9000 preganglionic axons. Section of the preganglionic chain causes a loss of 93% of the synapses. In the denervated SCG there are 0.6 X 10(6) remaining ('intrinsic') synapses, and a proportion of the synaptic sites are identifiable as vacated synaptic thickenings (3 X 10(6) per SCG, as compared with 0.5 X 10(6) in the normal intact SCG). After deducting the intrinsic synapses, this indicates that each preganglionic axon forms about 1100 (900-1400) synapses. After freezing the preganglionic chain, subsequent axonal regeneration restores synapse numbers to 85% of normal (7.5 X 10(6) synapses per SCG). After anastomotic repair by suture of the cut ends of the preganglionic chain (a necessary control for the foreign nerve anastomoses), the SCG contains only 60% of the normal complement of synapses (5.2 X 10(6) synapses per SCG). The results of this anastomosis are very variable. However, in individual ganglia the numbers of synapses are directly correlated with the numbers of axons which reach the SCG. After deducting the intrinsic synapses it can be calculated that each axon forms about 700 synapses. This is probably an underestimation of the numbers which would be achieved at longer survival times. After anastomosis of the vagal nerve into the denervated SCG there are about 4.4 X 10(6) synapses per SCG. Morphologically the majority have axon terminals with large dense cored vesicles, and it is likely that these belong to the axons of the parasympathetic preganglionic neurones in the dorsal motor nucleus of the vagus. A smaller population of axon terminals are devoid of large dense cored vesicles; their origin is unknown. The dorsal motor nucleus of the vagus has between 1000 and 2000 neurones. After deducting the intrinsic synapses, this indicates that each axon may form up to 1900-3800 synapses. To the extent that other, unidentified vagal fibres also contribute to the synapses found after this anastomosis, this figure is an overestimate. After anastomosis of the hypoglossal nerve into the denervated SCG, there are 1.5 X 10(6) synapses per SCG. A morphologically distinctive type of axon terminal is found, and it is argued that this may belong to a special category of skeletomotor neurones located in the caudoventral part of the hypoglossal nucleus and distinguished by pseudocholinesterase staining. There are about 600 of these neurones, which would indicate that they form about 1500 synapses per axon (after deducting the numbers of intrinsic synapses). The majority of the hypoglossal neurones do not form intraganglionic synapses; this suggests that although the possession of a cholinergic mechanism may be necessary for axons to be able to form ganglionic synapses, it is not in itself sufficient. For each of the types of anastomosis, the numbers of vacated thickenings are inversely proportional to the numbers of synapses... | A quantitative comparison of the formation of synapses in the rat superior cervical sympathetic ganglion by its own and by foreign nerve fibres. The rat superior cervical sympathetic ganglion (SCG) has about 36,000 neurones in a volume of about 1 cu.mm. There are about 8.8 X 10(6) synapses, and 6000-9000 preganglionic axons. Section of the preganglionic chain causes a loss of 93% of the synapses. In the denervated SCG there are 0.6 X 10(6) remaining ('intrinsic') synapses, and a proportion of the synaptic sites are identifiable as vacated synaptic thickenings (3 X 10(6) per SCG, as compared with 0.5 X 10(6) in the normal intact SCG). After deducting the intrinsic synapses, this indicates that each preganglionic axon forms about 1100 (900-1400) synapses. After freezing the preganglionic chain, subsequent axonal regeneration restores synapse numbers to 85% of normal (7.5 X 10(6) synapses per SCG). After anastomotic repair by suture of the cut ends of the preganglionic chain (a necessary control for the foreign nerve anastomoses), the SCG contains only 60% of the normal complement of synapses (5.2 X 10(6) synapses per SCG). The results of this anastomosis are very variable. However, in individual ganglia the numbers of synapses are directly correlated with the numbers of axons which reach the SCG. After deducting the intrinsic synapses it can be calculated that each axon forms about 700 synapses. This is probably an underestimation of the numbers which would be achieved at longer survival times. After anastomosis of the vagal nerve into the denervated SCG there are about 4.4 X 10(6) synapses per SCG. Morphologically the majority have axon terminals with large dense cored vesicles, and it is likely that these belong to the axons of the parasympathetic preganglionic neurones in the dorsal motor nucleus of the vagus. A smaller population of axon terminals are devoid of large dense cored vesicles; their origin is unknown. The dorsal motor nucleus of the vagus has between 1000 and 2000 neurones. After deducting the intrinsic synapses, this indicates that each axon may form up to 1900-3800 synapses. To the extent that other, unidentified vagal fibres also contribute to the synapses found after this anastomosis, this figure is an overestimate. After anastomosis of the hypoglossal nerve into the denervated SCG, there are 1.5 X 10(6) synapses per SCG. A morphologically distinctive type of axon terminal is found, and it is argued that this may belong to a special category of skeletomotor neurones located in the caudoventral part of the hypoglossal nucleus and distinguished by pseudocholinesterase staining. There are about 600 of these neurones, which would indicate that they form about 1500 synapses per axon (after deducting the numbers of intrinsic synapses). The majority of the hypoglossal neurones do not form intraganglionic synapses; this suggests that although the possession of a cholinergic mechanism may be necessary for axons to be able to form ganglionic synapses, it is not in itself sufficient. For each of the types of anastomosis, the numbers of vacated thickenings are inversely proportional to the numbers of synapses... | [
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PMID:5179 | Properties of soluble and particulate cysteine sulfinate decarboxylase of the adult and the developing rat brain. | Some properties of cysteine sulfinate decarboxylase (CSD) activity were studied in the pellet and supernatant of a 18,000 X g centrifugation of isotonic sucrose rat brain homogenates. About 50% of the enzyme activity was found associated to the particulate fraction and 16% of the activity remained particle bound after hypo-osmotic shock of the 18,000 X g pellet. The activity of the 18,000 X g supernatant showed a lower dependence on exogenous pyridoxal phosphate (PLP) than the activity in the particulate fraction. The CSD activity of these fractions also differed in optimal pH and in apparent kinetic constants. The enzyme associated to particles showed the highest Vmax and the lowest Km. The activity and kinetic characteristics of CSD were studied during brain postnatal development. In the newborn brain only a small amount of the enzyme activity was found associated to the 18,000 X g pellet. CSD in brain homogenates of immature rats was less dependent on free PLP and showed a higher Km and a lower Vmax as compared with the enzyme in the adult brain. Between birth and adulthood the enzyme activity increased more than 10-fold in the particulate fraction and 2-fold in the soluble fraction. It is concluded that the differences observed in CSD activity between newborn and adult brain are due to an increase of the particulate form of the enzyme during postnatal development. | Properties of soluble and particulate cysteine sulfinate decarboxylase of the adult and the developing rat brain. Some properties of cysteine sulfinate decarboxylase (CSD) activity were studied in the pellet and supernatant of a 18,000 X g centrifugation of isotonic sucrose rat brain homogenates. About 50% of the enzyme activity was found associated to the particulate fraction and 16% of the activity remained particle bound after hypo-osmotic shock of the 18,000 X g pellet. The activity of the 18,000 X g supernatant showed a lower dependence on exogenous pyridoxal phosphate (PLP) than the activity in the particulate fraction. The CSD activity of these fractions also differed in optimal pH and in apparent kinetic constants. The enzyme associated to particles showed the highest Vmax and the lowest Km. The activity and kinetic characteristics of CSD were studied during brain postnatal development. In the newborn brain only a small amount of the enzyme activity was found associated to the 18,000 X g pellet. CSD in brain homogenates of immature rats was less dependent on free PLP and showed a higher Km and a lower Vmax as compared with the enzyme in the adult brain. Between birth and adulthood the enzyme activity increased more than 10-fold in the particulate fraction and 2-fold in the soluble fraction. It is concluded that the differences observed in CSD activity between newborn and adult brain are due to an increase of the particulate form of the enzyme during postnatal development. | [
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PMID:5180 | [Tissue lactates, pH and blood gasses in hibernating dormouse: changes during periodic arousals]. | Periodic arousal in the garden dormouse is accompagnied by a rise in plasma and muscle lactate levels and a diminution of muscle glycogen. In addition, the decrease of arterial blood pH and O2 concentration agrees well with these results. All of which are in good agreement with the general stimulation of adrenergic activity observed at the onset of rewarming. | [Tissue lactates, pH and blood gasses in hibernating dormouse: changes during periodic arousals]. Periodic arousal in the garden dormouse is accompagnied by a rise in plasma and muscle lactate levels and a diminution of muscle glycogen. In addition, the decrease of arterial blood pH and O2 concentration agrees well with these results. All of which are in good agreement with the general stimulation of adrenergic activity observed at the onset of rewarming. | [
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PMID:5181 | Study of the natural antihistamine-like substance in bile in mammals. | A natural antihistamine substance (NAS) present in bile has been investigated. It was found that the antihistamine activity was not due to proteins, lipids, pigments, or amino acids. On ion exchange chromatography and thin-layer chromatography, this activity was associated with bile acids. Many bile acids could, in varying degrees, inhibit this histamine induced guinea pig ileum contraction, desoxycholic acid being the most potent. However NAS activity could be separated from bile acids and their conjugates using a different solvent system. Furthermore, NAS showed a higher antihistamine activity than bile acids. This substance seems to be responsible for 15-20% of the activity of whole bile. The substance has not yet been identified. | Study of the natural antihistamine-like substance in bile in mammals. A natural antihistamine substance (NAS) present in bile has been investigated. It was found that the antihistamine activity was not due to proteins, lipids, pigments, or amino acids. On ion exchange chromatography and thin-layer chromatography, this activity was associated with bile acids. Many bile acids could, in varying degrees, inhibit this histamine induced guinea pig ileum contraction, desoxycholic acid being the most potent. However NAS activity could be separated from bile acids and their conjugates using a different solvent system. Furthermore, NAS showed a higher antihistamine activity than bile acids. This substance seems to be responsible for 15-20% of the activity of whole bile. The substance has not yet been identified. | [
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PMID:5182 | Spin labels as probes for tetraphenylboron ion interaction with liposomes. | The effects of tetraphenylboron (TFB) on the molecular organization of lipids within phosphatidylcholine (PC) liposomes were investigated using the spin-labeled method. Perturbations at the surface of the lipid were probed using stearamide and cholestane spin labels; perturbations in the hydrophobic-portion were probed with spin-labeled amphiphilic fatty esters. | Spin labels as probes for tetraphenylboron ion interaction with liposomes. The effects of tetraphenylboron (TFB) on the molecular organization of lipids within phosphatidylcholine (PC) liposomes were investigated using the spin-labeled method. Perturbations at the surface of the lipid were probed using stearamide and cholestane spin labels; perturbations in the hydrophobic-portion were probed with spin-labeled amphiphilic fatty esters. | [
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PMID:5183 | The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolases. | A cell fractionation scheme was developed for studying the distribution of certain hydrolases, especially phosphohydrolases in a Crithidia sp. (Trypanosomatidae). Whilst between 26-56% of the total cellular hydrolase activities were soluble (probably of flagellar pocket origin), a certain percentage, 5-40%, was sedimentable. A particulate fraction obtained after isopycnic density gradient centrifugation (p = 1.187-1.241), designated fraction FA/FB, was enriched in various acid hydrolases (relative specific activities 1.33-6.24) and displayed latent phosphohydrolase activities. The density gradient distributions of this hydrolytic enzymes were compared with reference to one another and malate dehydrogenase (mitochondrial marker). From the results obtained it appears that the sedimentable acid hydrolases of Crithidia are associated with a heterogeneous population of subcellular particles. Cytochemical observations on the FA/FB fraction supported this finding and revealed the association of acid phosphatase reaction product with subcellular elements resembling multivesicular bodies. | The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolases. A cell fractionation scheme was developed for studying the distribution of certain hydrolases, especially phosphohydrolases in a Crithidia sp. (Trypanosomatidae). Whilst between 26-56% of the total cellular hydrolase activities were soluble (probably of flagellar pocket origin), a certain percentage, 5-40%, was sedimentable. A particulate fraction obtained after isopycnic density gradient centrifugation (p = 1.187-1.241), designated fraction FA/FB, was enriched in various acid hydrolases (relative specific activities 1.33-6.24) and displayed latent phosphohydrolase activities. The density gradient distributions of this hydrolytic enzymes were compared with reference to one another and malate dehydrogenase (mitochondrial marker). From the results obtained it appears that the sedimentable acid hydrolases of Crithidia are associated with a heterogeneous population of subcellular particles. Cytochemical observations on the FA/FB fraction supported this finding and revealed the association of acid phosphatase reaction product with subcellular elements resembling multivesicular bodies. | [
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PMID:5184 | Acid proteases from species of Mucor. IV. Hydrogen-tritium exchange of Mucor miehei protease. | The kinetics of hydrogen-tritium exchange were studied in the range pH-3 for both the fully and partially tritiated protein. Exchange constants for an intermediate class and slow class of hydrogens were determined and found to give a parabolic curve characteristic of acid and base catalysis about the observed pHmin of 4.03. The anomalous rate retardation on the acid portion of the curve was attributed to electrostatic interactions which could be evaluated quantitatively from the titration data. Partial tritation and pH cross-over experiments indicated that the rank order was pH-independent thus eliminating the possiblitity of a major conformational change. Consequently, the data are most likely explicable in terms of restricted solvent accessibility. | Acid proteases from species of Mucor. IV. Hydrogen-tritium exchange of Mucor miehei protease. The kinetics of hydrogen-tritium exchange were studied in the range pH-3 for both the fully and partially tritiated protein. Exchange constants for an intermediate class and slow class of hydrogens were determined and found to give a parabolic curve characteristic of acid and base catalysis about the observed pHmin of 4.03. The anomalous rate retardation on the acid portion of the curve was attributed to electrostatic interactions which could be evaluated quantitatively from the titration data. Partial tritation and pH cross-over experiments indicated that the rank order was pH-independent thus eliminating the possiblitity of a major conformational change. Consequently, the data are most likely explicable in terms of restricted solvent accessibility. | [
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PMID:5185 | Some properties of acetylcholinesterase from rat retina. | Acetylcholinesterase (AChE, EC 3.1.1.7) of rat retina was studied with respect to its kinetic and other properties, and a comparison was made with the enzyme from brain. The subcellular distribution of the retinal AChE showed that the enzyme was concentrated in the synaptosomal-mitochondrial fraction although in the brain the AChE was distributed more evenly between the fractions studied. The enzyme from both retina and brain was easily solubilised and exhibited a Km of the order of 10(-4) M. The pH optimum was 8.3-8.6 for the AChE from both tissues for both the soluble and particulate enzyme. | Some properties of acetylcholinesterase from rat retina. Acetylcholinesterase (AChE, EC 3.1.1.7) of rat retina was studied with respect to its kinetic and other properties, and a comparison was made with the enzyme from brain. The subcellular distribution of the retinal AChE showed that the enzyme was concentrated in the synaptosomal-mitochondrial fraction although in the brain the AChE was distributed more evenly between the fractions studied. The enzyme from both retina and brain was easily solubilised and exhibited a Km of the order of 10(-4) M. The pH optimum was 8.3-8.6 for the AChE from both tissues for both the soluble and particulate enzyme. | [
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PMID:5188 | The metabolic activation of the carcinogen 1'-hydroxysafrole in vivo and in vitro and the electrophilic reactivities of possible ultimate carcinogens. | Administration of [2',3'-3H]-1'-hydroxysafrole to rats or mice resulted in the formation of hepatic DNA-, ribosomal RNA-, and protein-bound 3H derivatives. Alkaline digestion of the 3H-protein released 0.1 to 0.3% of the 3H as a derivative that was identified as 3'-methylmercaptoisosafrole by its cochromatography in five solvent systems with the synthetic compound. 1'-Hydroxysafrole was metabolized at a low rate by rat and mouse liver cytosols in a 3'-phosphoadenosine 5'-phosphosulfate-dependent reaction to a derivative (presumably the sulfuric acid ester) that was captured by its reaction with RNA. Likewise, 1'-hydroxysafrole was oxidized at a low rate by rat and mouse liver microsomes to 1'-hydroxysafrole-2',3'-oxide in a reduced nicotinamide adenine dinucleotide phosphate-dependent reaction. Both of these electrophilic metabolites are candidate ultimate carcinogenic derivatives of 1'-hydroxysafrole. The electrophilic reactivities of various safrole derivatives with nucleosides were determined to be in the order of 1'-oxosafrole greater than 1'-acetoxysafrole greater than 1'-acetoxysafrole-2',3'-oxide greater than 1'-hydroxysafrole-2',3'-oxide greater than safrole-2',3'-oxide greater than or equal to 1'-oxosafrole-2',3'-oxide. The major reactions were generally observed with guanosine. A major reaction product of 1'-acetoxysafrole and guanosine 5'-monophosphate yielded 3'-hydroxyisosafrole under very mild acidic conditions. These data further substantiate the previous characterization of this reaction product as O-6-(isosafrol-3'-yl)guanylic acid. The syntheses of 1'-oxosafrole, 2',3'-dehydrosafrole, [2',3'-3H]-1'-hydroxysafrole, and the 2',3'-oxed. | The metabolic activation of the carcinogen 1'-hydroxysafrole in vivo and in vitro and the electrophilic reactivities of possible ultimate carcinogens. Administration of [2',3'-3H]-1'-hydroxysafrole to rats or mice resulted in the formation of hepatic DNA-, ribosomal RNA-, and protein-bound 3H derivatives. Alkaline digestion of the 3H-protein released 0.1 to 0.3% of the 3H as a derivative that was identified as 3'-methylmercaptoisosafrole by its cochromatography in five solvent systems with the synthetic compound. 1'-Hydroxysafrole was metabolized at a low rate by rat and mouse liver cytosols in a 3'-phosphoadenosine 5'-phosphosulfate-dependent reaction to a derivative (presumably the sulfuric acid ester) that was captured by its reaction with RNA. Likewise, 1'-hydroxysafrole was oxidized at a low rate by rat and mouse liver microsomes to 1'-hydroxysafrole-2',3'-oxide in a reduced nicotinamide adenine dinucleotide phosphate-dependent reaction. Both of these electrophilic metabolites are candidate ultimate carcinogenic derivatives of 1'-hydroxysafrole. The electrophilic reactivities of various safrole derivatives with nucleosides were determined to be in the order of 1'-oxosafrole greater than 1'-acetoxysafrole greater than 1'-acetoxysafrole-2',3'-oxide greater than 1'-hydroxysafrole-2',3'-oxide greater than safrole-2',3'-oxide greater than or equal to 1'-oxosafrole-2',3'-oxide. The major reactions were generally observed with guanosine. A major reaction product of 1'-acetoxysafrole and guanosine 5'-monophosphate yielded 3'-hydroxyisosafrole under very mild acidic conditions. These data further substantiate the previous characterization of this reaction product as O-6-(isosafrol-3'-yl)guanylic acid. The syntheses of 1'-oxosafrole, 2',3'-dehydrosafrole, [2',3'-3H]-1'-hydroxysafrole, and the 2',3'-oxed. | [
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PMID:5189 | Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrololate reductases. | Four cultured mammalian cell lines, differing in intrinsic resistance to methotrexate over a 70-fold range, have been compared with respect to several biochemical factors that might influence response to the drug. Cellular activity of the enzymes dihydrofolate reductase and thymidylate synthetase and the total levels of folate cofactors did not vary by more than a factor of 2 among the cell lines. All the cell types were able to transport extracellular methotrexate efficiently across the cell membrane, and at comparable rates. A kinetic study of highly purified dihydrofolate reductases from the four sources revealed small differences in the Km values for dihydrofolate and reduced nicotinamide adenine dinucleotide phosphate. A study was made of the inhibition of the four dihydrofolate reductases by methotrexate, and Ki values were obtained by fitting the Zone B equation of Goldstein (Goldstein, A., J. Gen. Physiol., 27: 529-580, 1944) to the resulting data. Values Ki determined by this method correlated with intrinsic resistance of the cell lines and showed a 25-fold range from the most sensitive to the most resistant line. It is concluded that the response of a cell to methotrexate is significantly influenced by the dissociation constant of its dihydrofolate reductase-methotrexate complex. | Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrololate reductases. Four cultured mammalian cell lines, differing in intrinsic resistance to methotrexate over a 70-fold range, have been compared with respect to several biochemical factors that might influence response to the drug. Cellular activity of the enzymes dihydrofolate reductase and thymidylate synthetase and the total levels of folate cofactors did not vary by more than a factor of 2 among the cell lines. All the cell types were able to transport extracellular methotrexate efficiently across the cell membrane, and at comparable rates. A kinetic study of highly purified dihydrofolate reductases from the four sources revealed small differences in the Km values for dihydrofolate and reduced nicotinamide adenine dinucleotide phosphate. A study was made of the inhibition of the four dihydrofolate reductases by methotrexate, and Ki values were obtained by fitting the Zone B equation of Goldstein (Goldstein, A., J. Gen. Physiol., 27: 529-580, 1944) to the resulting data. Values Ki determined by this method correlated with intrinsic resistance of the cell lines and showed a 25-fold range from the most sensitive to the most resistant line. It is concluded that the response of a cell to methotrexate is significantly influenced by the dissociation constant of its dihydrofolate reductase-methotrexate complex. | [
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PMID:5190 | Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver. | Aflatoxicol, R0, was isolated from Mt. Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1. Its identity was confirmed by mass, infrared, and ultraviolet spectrometry. The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1. Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2. In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B. subtilis. Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated. The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1. The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites. The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium. The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma. | Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver. Aflatoxicol, R0, was isolated from Mt. Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1. Its identity was confirmed by mass, infrared, and ultraviolet spectrometry. The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1. Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2. In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B. subtilis. Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated. The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1. The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites. The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium. The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma. | [
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PMID:5191 | Xylanase (hemicellulase) activity in cell-free rumen fluid. | It has been shown that there is hemicellulase (xylanase) activity in cell-free filtrates of rumen liquor. This activity changes during the feeding cycle. The optimal pH and temperature for this activity have been found, as have the substrate-to-enzyme ratios. Many reagents, particularly heavy metal ions and phenols, inhibit the activity, but the activity is enhanced by reducing agents. No activity towards monosaccharides, disaccharides, or glycosides was found. The xylanase component was not stable, due to proteolytic enzymes in the rumen liquor, but could be purified by a variety of methods to give more-stable enzymes. | Xylanase (hemicellulase) activity in cell-free rumen fluid. It has been shown that there is hemicellulase (xylanase) activity in cell-free filtrates of rumen liquor. This activity changes during the feeding cycle. The optimal pH and temperature for this activity have been found, as have the substrate-to-enzyme ratios. Many reagents, particularly heavy metal ions and phenols, inhibit the activity, but the activity is enhanced by reducing agents. No activity towards monosaccharides, disaccharides, or glycosides was found. The xylanase component was not stable, due to proteolytic enzymes in the rumen liquor, but could be purified by a variety of methods to give more-stable enzymes. | [
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PMID:5194 | Antitumor activity of equinatoxin. | Equinatoxin, a highly basic protein extracted from Actinia equina, causes an increase in the survival time of mice bearing the ascitic form of Ehrlich carcinoma, whereas it has no effect on L1210 leukaemia. When tested for in vitro cytotoxicity by the dye exclusion test, it shows a potent activity on both tumour cell lines, with ED50 of a few ng/ml. Higher concentrations produce an extensive lysis of the cells. The cytotoxic effects of Equinatoxin are inhibited by phospholipids, thus suggesting that its mechanism of action may be related to interactions with lipids or other charged components of cell membrane. The observed lack of in vivo activity against L1210 leukaemia presumably is due to poor systemic absorption of the protein and/or neutralization by serum factors. | Antitumor activity of equinatoxin. Equinatoxin, a highly basic protein extracted from Actinia equina, causes an increase in the survival time of mice bearing the ascitic form of Ehrlich carcinoma, whereas it has no effect on L1210 leukaemia. When tested for in vitro cytotoxicity by the dye exclusion test, it shows a potent activity on both tumour cell lines, with ED50 of a few ng/ml. Higher concentrations produce an extensive lysis of the cells. The cytotoxic effects of Equinatoxin are inhibited by phospholipids, thus suggesting that its mechanism of action may be related to interactions with lipids or other charged components of cell membrane. The observed lack of in vivo activity against L1210 leukaemia presumably is due to poor systemic absorption of the protein and/or neutralization by serum factors. | [
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PMID:5199 | [Effect of ionic strength on the protection of DNA by histone F1 against the enzymatic activity of DNase I]. | Studying the effect of ionic strength on DNA protection by histone F1 against DNase I has shown a maximum protection near 0,1 M NaCl. At this ionic strength, different results have been obtained by measuring the initial velocity or the amount of DNA hydrolysed at the end of the reaction. | [Effect of ionic strength on the protection of DNA by histone F1 against the enzymatic activity of DNase I]. Studying the effect of ionic strength on DNA protection by histone F1 against DNase I has shown a maximum protection near 0,1 M NaCl. At this ionic strength, different results have been obtained by measuring the initial velocity or the amount of DNA hydrolysed at the end of the reaction. | [
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PMID:5200 | [Disadvantages of acid precipitation of proteins during the assay of blood glucose by the enzymatic glucose oxidase-peroxidase system]. | In the enzymatic procedure for blood sugar by means of glucose oxidase, acid protein precipitation of blood by perchloric acid or trichloracetic acid liberated oxidizing substances, which enhanced the coloration density in oxidizing the reduced chromogen of the reaction mixture, independently of the hydrogen peroxide generated from glucose, and would give false high values of glycemia, if additional precautions had not been taken. These substances, increasing considerably with times and temperature of blood conservation, would be of peroxide nature, and would accumulate in red blood cells during their exposure to air. | [Disadvantages of acid precipitation of proteins during the assay of blood glucose by the enzymatic glucose oxidase-peroxidase system]. In the enzymatic procedure for blood sugar by means of glucose oxidase, acid protein precipitation of blood by perchloric acid or trichloracetic acid liberated oxidizing substances, which enhanced the coloration density in oxidizing the reduced chromogen of the reaction mixture, independently of the hydrogen peroxide generated from glucose, and would give false high values of glycemia, if additional precautions had not been taken. These substances, increasing considerably with times and temperature of blood conservation, would be of peroxide nature, and would accumulate in red blood cells during their exposure to air. | [
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PMID:5201 | Evidence for a renal alpha-adrenergic receptor inhibiting renin release. | The mechanism by which clonidine suppresses renin release was investigated in conscious rats. This suppression was studied by means of selected autonomic interventions in conjunction with changes in sodium balance. Serum renin activity and direct arterial pressure were monitored. Clonidine administration suppressed basal (by 68-85%), diuretic-induced (by 89%), and sympathetic nervous system-mediated (by 75-100%) renin release. Cholinergic, ganglionic, and peripheral sympathetic neuronal blockade did not prevent this inhibitory effect of clonidine. These results indicate a peripheral site of action for suppression of renin release by clonidine. The alpha-adrenergic blocking drug phentolamine prevented clonidine suppression of renin release in sodium-depleted rats and was partially effective in normal rats. Phentolamine blocked the decrease in renin caused by clonidine in ganglion-blocked rats. Clozapine, a new neuroleptic agent with alpha-adrenergic blocking activity, or phenoxybenzamine blocked the effect of clonidine on renin release in both sodium-depleted and normal rats. After ganglionic blockade in sodium-depleted rats, clonidine caused a significantly greater suppression of renin release than did an equipressor dose of methoxamine. These data, combined with hemodynamic correlates, suggest that clonidine inhibits renin release by activation of an intrarenal alpha-adrenergic receptor. | Evidence for a renal alpha-adrenergic receptor inhibiting renin release. The mechanism by which clonidine suppresses renin release was investigated in conscious rats. This suppression was studied by means of selected autonomic interventions in conjunction with changes in sodium balance. Serum renin activity and direct arterial pressure were monitored. Clonidine administration suppressed basal (by 68-85%), diuretic-induced (by 89%), and sympathetic nervous system-mediated (by 75-100%) renin release. Cholinergic, ganglionic, and peripheral sympathetic neuronal blockade did not prevent this inhibitory effect of clonidine. These results indicate a peripheral site of action for suppression of renin release by clonidine. The alpha-adrenergic blocking drug phentolamine prevented clonidine suppression of renin release in sodium-depleted rats and was partially effective in normal rats. Phentolamine blocked the decrease in renin caused by clonidine in ganglion-blocked rats. Clozapine, a new neuroleptic agent with alpha-adrenergic blocking activity, or phenoxybenzamine blocked the effect of clonidine on renin release in both sodium-depleted and normal rats. After ganglionic blockade in sodium-depleted rats, clonidine caused a significantly greater suppression of renin release than did an equipressor dose of methoxamine. These data, combined with hemodynamic correlates, suggest that clonidine inhibits renin release by activation of an intrarenal alpha-adrenergic receptor. | [
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PMID:5202 | Effects of regional ischemia on metabolism of glucose and fatty acids. Relative rates of aerobic and anaerobic energy production during myocardial infarction and comparison with effects of anoxia. | The rate of coronary flow reaching the oxygen-linited heart appears to be crucial in determining the myocardial tissue metabolic response. The tissue metabolic response to anoxia, well studied in hearts perfused with anoxic media, differs in many important ways from the response to ischemia. In regional ischemia (developing infarction) there is still a residual oxygen uptake which is reduced approximately to the same extent as the delivery of O2; there is also decreased delivery of substrates and decreased removal of CO2, H+, and lactate, with increased concentrations of these metabolites. Contents of hexose monophosphates rise rather than fall in anoxia. Measurements of glycolytic intermediates show an initial burst of accelerated glycolytic flux lasting less than 1 minute after coronary artery ligation; thereafter rates of flux decrease to control values or even less at 120 minutes. Relative inhibition of phosphofructokinase (PFK) activity may be explained by a slow rate of fall of ATP and a developing intracellular acidosis. In this model, glucose accounts for a greater part of the residual oxidative metabolism than does free fatty acid (FFA). | Effects of regional ischemia on metabolism of glucose and fatty acids. Relative rates of aerobic and anaerobic energy production during myocardial infarction and comparison with effects of anoxia. The rate of coronary flow reaching the oxygen-linited heart appears to be crucial in determining the myocardial tissue metabolic response. The tissue metabolic response to anoxia, well studied in hearts perfused with anoxic media, differs in many important ways from the response to ischemia. In regional ischemia (developing infarction) there is still a residual oxygen uptake which is reduced approximately to the same extent as the delivery of O2; there is also decreased delivery of substrates and decreased removal of CO2, H+, and lactate, with increased concentrations of these metabolites. Contents of hexose monophosphates rise rather than fall in anoxia. Measurements of glycolytic intermediates show an initial burst of accelerated glycolytic flux lasting less than 1 minute after coronary artery ligation; thereafter rates of flux decrease to control values or even less at 120 minutes. Relative inhibition of phosphofructokinase (PFK) activity may be explained by a slow rate of fall of ATP and a developing intracellular acidosis. In this model, glucose accounts for a greater part of the residual oxidative metabolism than does free fatty acid (FFA). | [
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PMID:5203 | Control of ionic permeabilities in normal and ischemic heart. | The major ionic conductances underlying electrical activity in cardiac tissues are described. The participation of electrogenic active transport in electrical phenomenon and the influence of metabolic inhibition on cardiac action potentials are briefly summarized. Some electrophysiological effects of lactate and acidosis, such as might be induced by ischemia, are described. In dog Purkinje fibers, lactate (20 mM pH 7.0) may induce transient periods of arrhythmias. Acidosis decreases rapid sodium conductance, slow calcium-sodium conductance, and anomalous and delayed rectifications in frog atrial fibers. CO2-induced acidosis (20% CO2, pH 6.6) may alter the repolarization phase of the action potential in dog Purkinje fibers, presumably because it decreases potassium conductance. Alterations consist of partial depolarizations (humps) that result in reexcitation of the fibers and lead to a maintained depolarization. It is proposed that acidosis induces a decrease in potassium conductance that can be responsible for ectopic foci causing arrhythmias during ischemia. | Control of ionic permeabilities in normal and ischemic heart. The major ionic conductances underlying electrical activity in cardiac tissues are described. The participation of electrogenic active transport in electrical phenomenon and the influence of metabolic inhibition on cardiac action potentials are briefly summarized. Some electrophysiological effects of lactate and acidosis, such as might be induced by ischemia, are described. In dog Purkinje fibers, lactate (20 mM pH 7.0) may induce transient periods of arrhythmias. Acidosis decreases rapid sodium conductance, slow calcium-sodium conductance, and anomalous and delayed rectifications in frog atrial fibers. CO2-induced acidosis (20% CO2, pH 6.6) may alter the repolarization phase of the action potential in dog Purkinje fibers, presumably because it decreases potassium conductance. Alterations consist of partial depolarizations (humps) that result in reexcitation of the fibers and lead to a maintained depolarization. It is proposed that acidosis induces a decrease in potassium conductance that can be responsible for ectopic foci causing arrhythmias during ischemia. | [
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PMID:5204 | Cutaneous and muscular rasodilation in the canine hindlimb evoked by central stimulation. | Using stereotaxic procedures, we electrically stimulated specific sites in the hypothalamus and midbrain of anesthetized dogs pretreated with guanethidine and atropine methonitrate. A tract in which stimulation caused noncholinergic dilator responses in the hindlimbs was identified. The course of this trace was different from that subserving cholinergic vasodilation in the hindlimb musculature. In a number of experiments we studied the proportional distribution of blood flow to leg and paw. Responses restricted to the paw were regarded as occurring mainly in cutaneous vessels; those restricted to the leg were regarded as occurring mainly in the skeletal muscle vessels. Some dilator responses in both beds were abolished by intra-arterial administration of antihistamines: other dilator responses were abolished by intra-arterial injections of dopamine antagonists. Centrally evoked dilation of leg and paw vessels by noncholinergic pathways suggests physiological roles for these fibers in the regulation of cardiovascular function. | Cutaneous and muscular rasodilation in the canine hindlimb evoked by central stimulation. Using stereotaxic procedures, we electrically stimulated specific sites in the hypothalamus and midbrain of anesthetized dogs pretreated with guanethidine and atropine methonitrate. A tract in which stimulation caused noncholinergic dilator responses in the hindlimbs was identified. The course of this trace was different from that subserving cholinergic vasodilation in the hindlimb musculature. In a number of experiments we studied the proportional distribution of blood flow to leg and paw. Responses restricted to the paw were regarded as occurring mainly in cutaneous vessels; those restricted to the leg were regarded as occurring mainly in the skeletal muscle vessels. Some dilator responses in both beds were abolished by intra-arterial administration of antihistamines: other dilator responses were abolished by intra-arterial injections of dopamine antagonists. Centrally evoked dilation of leg and paw vessels by noncholinergic pathways suggests physiological roles for these fibers in the regulation of cardiovascular function. | [
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PMID:5205 | Spinal adrenergic mechanisms regulating sympathetic outflow to blood vessels. | The role of descending spinal catecholamine (CA)-containing fibers in cardiovascular regulation was examined. Monoamine-containing fibers were visualized by fluorescence histochemical methods at the cervical, thoracic, and lumbar spinal segments. Two major groups of descending CA-containing axons were located in the lateral funiculus at the midcervical spinal level. At the thoracolumbar spinal segments CA-containing terminals were concentrated mostly around cells of the intermediolateral nucleus. In addition a distinct CA-containing fasciculus was observed at the level of the sympathetic nucleus which appeared to course toward the opposite site of the cord. To evaluate the role of the efferent CA-containing fibers, changes in heart rate, arterial pressure, femoral blood flow, and calculated peak vascular resistance were elicited by electrical stimulation of selected sites in the midcervical spinal cord. Although changes in femoral flow and arterial pressure were evoked from many midcervical sites, there was a distinct correlation between the magnitude of peak femoral resistance elevation and density of CA-containing axons activated. Furthermore, efferent spinal vasoconstrictor outflow to the hindlimbs crosses below the cervical level. This observation is in good agreement with fluorescence microscopy studies revealing CA-containing fibers which appear to decussate. Pharmacological studies also were undertaken to evaluate transmission in spinal vasopressor pathways. In these studies alpha-adrenergic receptor antagonists and agonists were perfused into the spinal subarachnoid space. Heart rate, arterial pressure, femoral flow, and femoral resistance responses elicited from efferent spinal pathways were significantly attenuated following superfusion of the spinal cord with the alpha-antagonists BE-2254 (HEAT) and phentolamine. It was shown that inhibition of the centrally evoked responses was due to actions of the alpha-antagonists at a spinal locus and not due to peripheral vascular alpha-receptor blockade. Spinal perfusion with the alpha-receptor agonist norepinephrine potentiated cardiovascular responses. Likewise, loading with the norepinephrine precursor 3,4-dihydroxy-L-phenylalanine (L-dopa) enhanced vasoconstrictor responses evoked in the cross-perfused hindlimb of p-chlorophenylalanine-pretreated cats. These observations support the idea that bulbospinal CA-containing fibers relay excitatory impulses to preganglionic vasoconstrictor neurons and that spinal alpha-adrenergic receptor activation is necessary for transmission of this information. | Spinal adrenergic mechanisms regulating sympathetic outflow to blood vessels. The role of descending spinal catecholamine (CA)-containing fibers in cardiovascular regulation was examined. Monoamine-containing fibers were visualized by fluorescence histochemical methods at the cervical, thoracic, and lumbar spinal segments. Two major groups of descending CA-containing axons were located in the lateral funiculus at the midcervical spinal level. At the thoracolumbar spinal segments CA-containing terminals were concentrated mostly around cells of the intermediolateral nucleus. In addition a distinct CA-containing fasciculus was observed at the level of the sympathetic nucleus which appeared to course toward the opposite site of the cord. To evaluate the role of the efferent CA-containing fibers, changes in heart rate, arterial pressure, femoral blood flow, and calculated peak vascular resistance were elicited by electrical stimulation of selected sites in the midcervical spinal cord. Although changes in femoral flow and arterial pressure were evoked from many midcervical sites, there was a distinct correlation between the magnitude of peak femoral resistance elevation and density of CA-containing axons activated. Furthermore, efferent spinal vasoconstrictor outflow to the hindlimbs crosses below the cervical level. This observation is in good agreement with fluorescence microscopy studies revealing CA-containing fibers which appear to decussate. Pharmacological studies also were undertaken to evaluate transmission in spinal vasopressor pathways. In these studies alpha-adrenergic receptor antagonists and agonists were perfused into the spinal subarachnoid space. Heart rate, arterial pressure, femoral flow, and femoral resistance responses elicited from efferent spinal pathways were significantly attenuated following superfusion of the spinal cord with the alpha-antagonists BE-2254 (HEAT) and phentolamine. It was shown that inhibition of the centrally evoked responses was due to actions of the alpha-antagonists at a spinal locus and not due to peripheral vascular alpha-receptor blockade. Spinal perfusion with the alpha-receptor agonist norepinephrine potentiated cardiovascular responses. Likewise, loading with the norepinephrine precursor 3,4-dihydroxy-L-phenylalanine (L-dopa) enhanced vasoconstrictor responses evoked in the cross-perfused hindlimb of p-chlorophenylalanine-pretreated cats. These observations support the idea that bulbospinal CA-containing fibers relay excitatory impulses to preganglionic vasoconstrictor neurons and that spinal alpha-adrenergic receptor activation is necessary for transmission of this information. | [
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PMID:5207 | Cellular basis for increased sensitivity of vascular smooth muscle in spontaneously hypertensive rats. | There is evidence that hypersensitivity of vascular muscle to neurotransmitters contributes to the development of hypertension. Comparison of the caudal arteries of spontaneously hypertensive rats (SHR) and their genetically related Kyoto-Wistar normotensive control rats (KNR) showed that although there is no difference in membrane potential under unstimulated conditions, greater depolarization of the SHR vascular muscle cells by norepinephrine occurs at concentrations which cause greater contraction. The mechanism for the increased depolarization and resulting increase in contraction appears to be a lower intracellular potassium ion activity in SHR vascular muscle cells, which results in a lower contribution of potassium gradient to membrane potential. Experiments to determine the sensitivity of isolated, dispersed chick omphalomesenteric vascular muscle cells to neurotransmitters showed remarkably low thresholds to the neutransmitters norepinephrine, serotonin, and acetylcholine, but not potassium chloride. The high sensitivity of isolated cells to neurotransmitters suggests that factors in the intact vessel may cause thresholds to be high, possibly implying that alterations in a neurotrophic mechanism might be responsible for changes in vascular muscle sensitivity in situ. | Cellular basis for increased sensitivity of vascular smooth muscle in spontaneously hypertensive rats. There is evidence that hypersensitivity of vascular muscle to neurotransmitters contributes to the development of hypertension. Comparison of the caudal arteries of spontaneously hypertensive rats (SHR) and their genetically related Kyoto-Wistar normotensive control rats (KNR) showed that although there is no difference in membrane potential under unstimulated conditions, greater depolarization of the SHR vascular muscle cells by norepinephrine occurs at concentrations which cause greater contraction. The mechanism for the increased depolarization and resulting increase in contraction appears to be a lower intracellular potassium ion activity in SHR vascular muscle cells, which results in a lower contribution of potassium gradient to membrane potential. Experiments to determine the sensitivity of isolated, dispersed chick omphalomesenteric vascular muscle cells to neurotransmitters showed remarkably low thresholds to the neutransmitters norepinephrine, serotonin, and acetylcholine, but not potassium chloride. The high sensitivity of isolated cells to neurotransmitters suggests that factors in the intact vessel may cause thresholds to be high, possibly implying that alterations in a neurotrophic mechanism might be responsible for changes in vascular muscle sensitivity in situ. | [
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PMID:5208 | Partial purification of a high molecular weight renin from hog kidney. | Previous investigators have reported finding a high molecular weight (HMW) renin in various species which is activated by acidification. Two laboratories have reported that a decrease in molecular weight of the renin accompanies the acidification step. This report describes the partial purification of an HMW renin from hog kidney extracts which had previously been acidified to pH 2.5. The HMW renin, with a molecular weight of 57,000-59,000, constitutes only a small, relatively constant fraction of the total renin isolated. Omission of the acidification step or initial acidification to pH 1.6 does not change the amount of HMW renin significantly. The HMW renin attacks the protein substrate to produce angiotensin at about one-fourth the rate expected, based upon the rate at which it cleaves the tetradecapeptide substrate or a model nonapeptide substrate. It is inactivated by antiserum to hog kidney renin prepared in dogs. It is less stable than hog renin when stored at 0 degrees C. The HMW renin has a pH optimum between 6.5 and 7.0. It is not activated by acidification to pH 3, nor by tryptic or peptic digestion. We have been unable to activate HMW renin or to change its molecular weight by procedures reported by other investigators and therefore conclude that the HMW renin differs from material previously reported and may be an isoenzyme of renin. | Partial purification of a high molecular weight renin from hog kidney. Previous investigators have reported finding a high molecular weight (HMW) renin in various species which is activated by acidification. Two laboratories have reported that a decrease in molecular weight of the renin accompanies the acidification step. This report describes the partial purification of an HMW renin from hog kidney extracts which had previously been acidified to pH 2.5. The HMW renin, with a molecular weight of 57,000-59,000, constitutes only a small, relatively constant fraction of the total renin isolated. Omission of the acidification step or initial acidification to pH 1.6 does not change the amount of HMW renin significantly. The HMW renin attacks the protein substrate to produce angiotensin at about one-fourth the rate expected, based upon the rate at which it cleaves the tetradecapeptide substrate or a model nonapeptide substrate. It is inactivated by antiserum to hog kidney renin prepared in dogs. It is less stable than hog renin when stored at 0 degrees C. The HMW renin has a pH optimum between 6.5 and 7.0. It is not activated by acidification to pH 3, nor by tryptic or peptic digestion. We have been unable to activate HMW renin or to change its molecular weight by procedures reported by other investigators and therefore conclude that the HMW renin differs from material previously reported and may be an isoenzyme of renin. | [
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PMID:5209 | Vasodilator administration in the presence of beta-adrenergic blockade. | To explore the possibility that the presence of propranolol-induced beta-adrenergic blockade might have an adverse effect upon homeostatic circulatory reflexes activated by the administration of a potent vasodilator agent, arterial blood pressure and pulse rate response to rapid intravenous diazoxide injection was monitored before and after pretreatment with propranolol in ten hypertensive patients. It appeared that beta-adrenergic blockade had no clinically significant effect on the magnitude of hypotension or the degree of heart rate acceleration induced by the administration of the potent vasodilator diazoxide. This reflex vasodilator-induced cardio-acceleration after propranolol adminstration could be the result of incomplete blockade of endogenously released neurotransmitter, inhibition of the parasympathetic nervous system, or a direct pharmacologic action of diazoxide. Diazoxide administration to hypertensive patients in the presence of beta-adrenergic blockade was not associated with any clinically significant hemodynamic consequences. | Vasodilator administration in the presence of beta-adrenergic blockade. To explore the possibility that the presence of propranolol-induced beta-adrenergic blockade might have an adverse effect upon homeostatic circulatory reflexes activated by the administration of a potent vasodilator agent, arterial blood pressure and pulse rate response to rapid intravenous diazoxide injection was monitored before and after pretreatment with propranolol in ten hypertensive patients. It appeared that beta-adrenergic blockade had no clinically significant effect on the magnitude of hypotension or the degree of heart rate acceleration induced by the administration of the potent vasodilator diazoxide. This reflex vasodilator-induced cardio-acceleration after propranolol adminstration could be the result of incomplete blockade of endogenously released neurotransmitter, inhibition of the parasympathetic nervous system, or a direct pharmacologic action of diazoxide. Diazoxide administration to hypertensive patients in the presence of beta-adrenergic blockade was not associated with any clinically significant hemodynamic consequences. | [
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] |
PMID:5210 | A modified inactivation-inhibition method for determining the serum activity of alkaline phosphatase isoenzymes. | A procedure using heat inactivation and L-phenylalanine inhibition to quantitate the activities of bone, liver and intestinal alkaline phosphatase isoenzymes in human serum was confirmed by alkaline phosphatase isoenzyme analysis using an electrophoretic procedure. The results of this assay were compared with the radionuclear 85Sr test, and gamma-glutamyl transpeptidase activity in a group of patients with hepatobiliary and bone diseases. | A modified inactivation-inhibition method for determining the serum activity of alkaline phosphatase isoenzymes. A procedure using heat inactivation and L-phenylalanine inhibition to quantitate the activities of bone, liver and intestinal alkaline phosphatase isoenzymes in human serum was confirmed by alkaline phosphatase isoenzyme analysis using an electrophoretic procedure. The results of this assay were compared with the radionuclear 85Sr test, and gamma-glutamyl transpeptidase activity in a group of patients with hepatobiliary and bone diseases. | [
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PMID:5211 | A sensitive automated colorimetric method for the determination of serum gamma-glutamyl transpeptidase. | A highly sensitive and accurate automated method using a Technicon Autoanalyzer AAII was developed for the determination of human serum gamma-glutamyl transpeptidase. gamma-L-Glutamyl-p-nitroanilide added as substrate was split to p-nitroaniline and the gamma-glutamyl group was trapped by glycylglycine in Ammediol/HC1 buffer solution. Liberated p-nitroaniline was diazotized and converted by Tsuda Reagent (N,N-(diethyl)-N'-(1-naphthyl) ethylendiamine) to an azo-dye possessing an absorbance maximum at 550 nm. This new method of determination, using the conversion of p-nitroaniline to azo-dye, was found to be 7 times more sensitive as compared to the conventional direct p-nitroaniline determination method [1]. This method requires only 0.1 ml lerum and permits 60 determinations per hour. | A sensitive automated colorimetric method for the determination of serum gamma-glutamyl transpeptidase. A highly sensitive and accurate automated method using a Technicon Autoanalyzer AAII was developed for the determination of human serum gamma-glutamyl transpeptidase. gamma-L-Glutamyl-p-nitroanilide added as substrate was split to p-nitroaniline and the gamma-glutamyl group was trapped by glycylglycine in Ammediol/HC1 buffer solution. Liberated p-nitroaniline was diazotized and converted by Tsuda Reagent (N,N-(diethyl)-N'-(1-naphthyl) ethylendiamine) to an azo-dye possessing an absorbance maximum at 550 nm. This new method of determination, using the conversion of p-nitroaniline to azo-dye, was found to be 7 times more sensitive as compared to the conventional direct p-nitroaniline determination method [1]. This method requires only 0.1 ml lerum and permits 60 determinations per hour. | [
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PMID:5213 | Suppression of experimental allergic encephalomyelitis in guinea-pigs with poly-L-lysine. | Poly-L-lysin (PLL) given subcutaneously in a dose of 3 mg/day suppresses experimental allergic encephalomyelitis in guinea-pigs. The suppressive effect of PLL can still be demonstrated when administration is begun 8 days post-immunization. The effect is disease- and species-specific since PLL (3 mg/day) does not suppress experimental allergic orchitis in guinea-pigs, nor does it suppress EAE in rats (1-5 mg/day). PLL (0-2 mg/day) does not decrease the severity of graft-versus-host disease (GVH) in mice as judged by the spleen/body weight assay. | Suppression of experimental allergic encephalomyelitis in guinea-pigs with poly-L-lysine. Poly-L-lysin (PLL) given subcutaneously in a dose of 3 mg/day suppresses experimental allergic encephalomyelitis in guinea-pigs. The suppressive effect of PLL can still be demonstrated when administration is begun 8 days post-immunization. The effect is disease- and species-specific since PLL (3 mg/day) does not suppress experimental allergic orchitis in guinea-pigs, nor does it suppress EAE in rats (1-5 mg/day). PLL (0-2 mg/day) does not decrease the severity of graft-versus-host disease (GVH) in mice as judged by the spleen/body weight assay. | [
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PMID:5215 | Reactions to cigarettes as a function of nicotine and "tar". | Experiments carried out to examine the effects of nicotine and "tar" on the extent of and subjective reactions to cigarette smoking. It was confirmed that smokers rate commercial, low-nicotine cigarettes as less "strong" and less "satisfying" than their usual brands. Since such cigarettes deliver reduced amounts of tar as well as of nicotine, an experiment to distinguish between the two was carried out with special cigarettes. Ratings of "strength" were directly related to nicotine but were not affected by tar. The numbers of cigarettes smoked fell slightly as their estimated delivery of nicotine increased, but tar had no effect on this index. The urinary excretion of nicotine was correlated with the rated yields of nicotine for the different cigarettes, but there was also evidence that subjects tended to adjust their manner of smoking so as to titrate their doses of nicotine. The results are interpreted as indicating a role for nicotine, but not for tar, in the maintenance of cigarette smoking behavior, and as support for the view that less harmful cigarettes should have a high yield of nicotine relative to tar. | Reactions to cigarettes as a function of nicotine and "tar". Experiments carried out to examine the effects of nicotine and "tar" on the extent of and subjective reactions to cigarette smoking. It was confirmed that smokers rate commercial, low-nicotine cigarettes as less "strong" and less "satisfying" than their usual brands. Since such cigarettes deliver reduced amounts of tar as well as of nicotine, an experiment to distinguish between the two was carried out with special cigarettes. Ratings of "strength" were directly related to nicotine but were not affected by tar. The numbers of cigarettes smoked fell slightly as their estimated delivery of nicotine increased, but tar had no effect on this index. The urinary excretion of nicotine was correlated with the rated yields of nicotine for the different cigarettes, but there was also evidence that subjects tended to adjust their manner of smoking so as to titrate their doses of nicotine. The results are interpreted as indicating a role for nicotine, but not for tar, in the maintenance of cigarette smoking behavior, and as support for the view that less harmful cigarettes should have a high yield of nicotine relative to tar. | [
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PMID:5251 | Effect of beta-adrenergic agonists aerosolized by freon propellant on tracheal mucous velocity and cardiac output. | The present study was designed to assess the effects of two beta-adrenergic agonists, isoproterenol sulfate and carbuterol hydrochloride, and aerosolized Freon propellant (a mixture of Freon II, Freon 12, and Freon 114) on tracheal mucous velocity and cardiac output in anesthetized dogs. Five groups of ten animals each received the following dosages of aerosols: Freon, 20 puffs; isoproterenol, four puffs; carbuterol, four puffs; isoproterenol, 20 puffs; and carbuterol, 20 puffs. The puff was delivered by a standard metered aerosol; each puff of isoproterenol spray contained 75 mug of isoproterenol sulfate, and each puff of carbuterol spray contained 100 mug of carbuterol hydrochloride. Tracheal mucous velocity was not changed by receiving Freon, but administration of both isoproterenol and carbuterol caused a significant increase in this measurement, with peak increases ranging from 74 to 111 percent above control values. The duration of action for four and 20 puffs of isoproterenol and for four puffs of carbuterol was two hours. Twenty puffs of carbuterol increased tracheal mucous velocity for three hours. Administration of carbuterol effected a slightly larger increase in cardiac output than isoproterenol. The duration of action for the increased cardiac output was shorter than the duration of action for the increased tracheal mucous velocity. These studies indicate that beta-adrenergic agonists may have an important role in improving mucous transport in patients with chronic obstructive pulmonary disease in whom mucociliary clearance is depressed. | Effect of beta-adrenergic agonists aerosolized by freon propellant on tracheal mucous velocity and cardiac output. The present study was designed to assess the effects of two beta-adrenergic agonists, isoproterenol sulfate and carbuterol hydrochloride, and aerosolized Freon propellant (a mixture of Freon II, Freon 12, and Freon 114) on tracheal mucous velocity and cardiac output in anesthetized dogs. Five groups of ten animals each received the following dosages of aerosols: Freon, 20 puffs; isoproterenol, four puffs; carbuterol, four puffs; isoproterenol, 20 puffs; and carbuterol, 20 puffs. The puff was delivered by a standard metered aerosol; each puff of isoproterenol spray contained 75 mug of isoproterenol sulfate, and each puff of carbuterol spray contained 100 mug of carbuterol hydrochloride. Tracheal mucous velocity was not changed by receiving Freon, but administration of both isoproterenol and carbuterol caused a significant increase in this measurement, with peak increases ranging from 74 to 111 percent above control values. The duration of action for four and 20 puffs of isoproterenol and for four puffs of carbuterol was two hours. Twenty puffs of carbuterol increased tracheal mucous velocity for three hours. Administration of carbuterol effected a slightly larger increase in cardiac output than isoproterenol. The duration of action for the increased cardiac output was shorter than the duration of action for the increased tracheal mucous velocity. These studies indicate that beta-adrenergic agonists may have an important role in improving mucous transport in patients with chronic obstructive pulmonary disease in whom mucociliary clearance is depressed. | [
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PMID:5253 | Effect of nafenopin (SU-13437) on liver function. Influence on the hepatic transport of phenolphthalein glucuronide and chlorothiazide. | In rats treated with the hypolipidemic drug, nafenopin (NP), for 2 days the biliary excretion of phenolphthalein glucuronide (PPG) was markedly decreased, while in contrast that of chlorothiazide (CTZ) was enhanced. This suggests the existence of independent hepatic transport mechanisms for these two anions. For both PPG and CTZ blood disappearance curves showed an initial, rapid phase followed by a second, slow phase. The rapid phase for both compounds is affected only slightly by pretreatment with NP. Therefore, it is inferred that suppression of biliary excretion was attributable mainly to impairment of the liver-to-bile transport process. The increased bile flow induced by NP treatment was previously shown to be related to the biliary excretion of NP and its metabolites. Inhibition of NP choleresis by PPG may involve competition for biliary transport of these compounds. The marked hepatomegaly and choleresis seen after NP pretreatment was more evident in male rats than in females. | Effect of nafenopin (SU-13437) on liver function. Influence on the hepatic transport of phenolphthalein glucuronide and chlorothiazide. In rats treated with the hypolipidemic drug, nafenopin (NP), for 2 days the biliary excretion of phenolphthalein glucuronide (PPG) was markedly decreased, while in contrast that of chlorothiazide (CTZ) was enhanced. This suggests the existence of independent hepatic transport mechanisms for these two anions. For both PPG and CTZ blood disappearance curves showed an initial, rapid phase followed by a second, slow phase. The rapid phase for both compounds is affected only slightly by pretreatment with NP. Therefore, it is inferred that suppression of biliary excretion was attributable mainly to impairment of the liver-to-bile transport process. The increased bile flow induced by NP treatment was previously shown to be related to the biliary excretion of NP and its metabolites. Inhibition of NP choleresis by PPG may involve competition for biliary transport of these compounds. The marked hepatomegaly and choleresis seen after NP pretreatment was more evident in male rats than in females. | [
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PMID:5254 | Distribution and biliary excretion products of di-2-ethylhexyl phthalate in rainbow trout. | After 24-hr exposures of rainbow trout to 0.5 ppm of di-2-ethylhexyl [14C]phthalate, one-half of the radioactivity present in the fish was localized in the bile. The bile was pooled and fractionated by selective solvent extraction before and after beta-glucuronidase hydrolysis. Individual radioactive compounds were further separated and characterized by thin-layer chromatography. Gas chromatography-mass spectrometry was used to confirm the results of thin-layer chromatographic analysis. These procedures demonstrated that bile contained a number of DEHP metabolites, but only about 1% of unchanged DEHP. The major metabolite, mono-2-ethylhexyl phthalate glucuronide accounted for 72% of the total bile radioactivity. The remaining bile radioactivity was found to be present as phthalic acid glucuronide, mono-2-ethylhexyl phthalate and two partially characterized polar metabolites. | Distribution and biliary excretion products of di-2-ethylhexyl phthalate in rainbow trout. After 24-hr exposures of rainbow trout to 0.5 ppm of di-2-ethylhexyl [14C]phthalate, one-half of the radioactivity present in the fish was localized in the bile. The bile was pooled and fractionated by selective solvent extraction before and after beta-glucuronidase hydrolysis. Individual radioactive compounds were further separated and characterized by thin-layer chromatography. Gas chromatography-mass spectrometry was used to confirm the results of thin-layer chromatographic analysis. These procedures demonstrated that bile contained a number of DEHP metabolites, but only about 1% of unchanged DEHP. The major metabolite, mono-2-ethylhexyl phthalate glucuronide accounted for 72% of the total bile radioactivity. The remaining bile radioactivity was found to be present as phthalic acid glucuronide, mono-2-ethylhexyl phthalate and two partially characterized polar metabolites. | [
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PMID:5255 | Binding of radioactivity from (14C)thiourea to rat lung protein. | Binding of radioactivity from [14C]thiourea (TU) to rat lung protein was found to occur in vitro. Two binding sites are present. One possesses low affinity/high capacity while the other is characterized by high affinity/low capacity. In vitro binding of [14C]TU to lung protein can be antagonized by the presence of either unlabeled congeners (alpha-napthylthiourea or phenylthiourea) or thiol-containing compounds (cysteine, reduced glutathione). Conversely, depletion of lung-reduced glutathione by means of diethyl maleate administration results in elevated protein binding. Prior administration (24 hr) of a sublethal dose of TU (which renders tolerance to a subsequent lethal dose in vivo) results in a decrease in in vitro binding of radioactivity from [14C)TU to lung protein. In addition, immature rats, which are less sensitive to the edematogenic effect of TU, bind less radioactivity from [14C]TU to lung protein when the drug is administered in vivo. These results suggest a correlation between [14C]TU binding to lung protein and the pathophysiological effect of the drug in the lung. | Binding of radioactivity from (14C)thiourea to rat lung protein. Binding of radioactivity from [14C]thiourea (TU) to rat lung protein was found to occur in vitro. Two binding sites are present. One possesses low affinity/high capacity while the other is characterized by high affinity/low capacity. In vitro binding of [14C]TU to lung protein can be antagonized by the presence of either unlabeled congeners (alpha-napthylthiourea or phenylthiourea) or thiol-containing compounds (cysteine, reduced glutathione). Conversely, depletion of lung-reduced glutathione by means of diethyl maleate administration results in elevated protein binding. Prior administration (24 hr) of a sublethal dose of TU (which renders tolerance to a subsequent lethal dose in vivo) results in a decrease in in vitro binding of radioactivity from [14C)TU to lung protein. In addition, immature rats, which are less sensitive to the edematogenic effect of TU, bind less radioactivity from [14C]TU to lung protein when the drug is administered in vivo. These results suggest a correlation between [14C]TU binding to lung protein and the pathophysiological effect of the drug in the lung. | [
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PMID:5256 | Uptake and disposition of chlorinated biphenyls by isolated perfused rat liver. | The hepatic disposition of four chlorinated biphenyls was studied in isolated perfused rat liver preparations perfused with 30% rat blood. Biliary excretion of 14C-labeled 4-chlorobiphenyl (1-CB): 4,4'-dichlorobiphenyl (2-CB): 2,4,5,2',5'-pentachlorobiphenyl (5-CB) and 2,4,5,2',5'-hexachlorobiphenyl (6-CB) appeared to be inversely related to increased chlorination. Each of the compounds was rapidly taken up by the liver from the circulating perfusate. After 4 hr of perfusion, biliary excretion of 14C from 1-CB, 2-CB, 5-CB, and 6-CB was 48.2, 29.6, 20.5, and 1.3% of total dose, respectively. For 1-CB, this rate of biliary elimination represents a maximum perfusate/bile ratio of 107. About 97% of the label in the bile from 1-CB experiments was in the form of metabolites. Biliary excretion of 1-CB was biphasic: an initial rapid phase (t 1/2 = 13 min) was followed by a slower second phase (t 1/2 = 122 min). When the metabolites of 1-CB were added to the perfusate, biliary excretion of these metabolites was monophasic; t 1/2 = 120 min, which corresponded to the slower phase observed when the parent compound was presented to the liver. 1-CB disappeared from the perfusate via a biphasic process. Metabolites of 1-CB effused into the perfusate immediately after the initial rapid uptake of 1-CB by the liver, and the recirculating pool of metabolites was eliminated in the bile by the slow postsynthetic phase of biliary elimination. Less than 2% of 1-CB was present as the parent compound in the perfusion system after 4 hr of perfusion. These findings indicate that the metabolism of 1-CB is not the limiting factor in the hepatic disposition of 1-CB and that the circulating metabolites of 1-CB effusing from the liver might be the major source for urinary excretion. | Uptake and disposition of chlorinated biphenyls by isolated perfused rat liver. The hepatic disposition of four chlorinated biphenyls was studied in isolated perfused rat liver preparations perfused with 30% rat blood. Biliary excretion of 14C-labeled 4-chlorobiphenyl (1-CB): 4,4'-dichlorobiphenyl (2-CB): 2,4,5,2',5'-pentachlorobiphenyl (5-CB) and 2,4,5,2',5'-hexachlorobiphenyl (6-CB) appeared to be inversely related to increased chlorination. Each of the compounds was rapidly taken up by the liver from the circulating perfusate. After 4 hr of perfusion, biliary excretion of 14C from 1-CB, 2-CB, 5-CB, and 6-CB was 48.2, 29.6, 20.5, and 1.3% of total dose, respectively. For 1-CB, this rate of biliary elimination represents a maximum perfusate/bile ratio of 107. About 97% of the label in the bile from 1-CB experiments was in the form of metabolites. Biliary excretion of 1-CB was biphasic: an initial rapid phase (t 1/2 = 13 min) was followed by a slower second phase (t 1/2 = 122 min). When the metabolites of 1-CB were added to the perfusate, biliary excretion of these metabolites was monophasic; t 1/2 = 120 min, which corresponded to the slower phase observed when the parent compound was presented to the liver. 1-CB disappeared from the perfusate via a biphasic process. Metabolites of 1-CB effused into the perfusate immediately after the initial rapid uptake of 1-CB by the liver, and the recirculating pool of metabolites was eliminated in the bile by the slow postsynthetic phase of biliary elimination. Less than 2% of 1-CB was present as the parent compound in the perfusion system after 4 hr of perfusion. These findings indicate that the metabolism of 1-CB is not the limiting factor in the hepatic disposition of 1-CB and that the circulating metabolites of 1-CB effusing from the liver might be the major source for urinary excretion. | [
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PMID:5258 | (14C)Methylphenidate hydrochloride. Studies on disposition in rat brain. | Studies with [14C]methylphenidate-HCl were carried out in rats administered the drug intravenously or intraperitoneally. Levels of total radioactivity and parent drug were measured in plasma, brain, and certain brain regions up to 6 hr after dosing. The intravenous route of administration was characterized by rapid entry into brain; during the first half hour, brain levels were 3- to 6-fold higher than those obtained by the intraperitoneal route. At later time periods, brain and plasma concentration for both routes were comparable, and the decline of drug concentrations in brain paralleled that in plasma. No significant variance was found in regional distribution of drug in brain. Whereas the major fraction of circulating drug consisted of metabolites, predominantly unchanged drug was found in brain. After intravenous injection of [14C]ritalinic acid (the major metabolite of methylphenidate), no substantial concentrations were found in brain. | (14C)Methylphenidate hydrochloride. Studies on disposition in rat brain. Studies with [14C]methylphenidate-HCl were carried out in rats administered the drug intravenously or intraperitoneally. Levels of total radioactivity and parent drug were measured in plasma, brain, and certain brain regions up to 6 hr after dosing. The intravenous route of administration was characterized by rapid entry into brain; during the first half hour, brain levels were 3- to 6-fold higher than those obtained by the intraperitoneal route. At later time periods, brain and plasma concentration for both routes were comparable, and the decline of drug concentrations in brain paralleled that in plasma. No significant variance was found in regional distribution of drug in brain. Whereas the major fraction of circulating drug consisted of metabolites, predominantly unchanged drug was found in brain. After intravenous injection of [14C]ritalinic acid (the major metabolite of methylphenidate), no substantial concentrations were found in brain. | [
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PMID:5257 | The absorption, distribution, and excretion in mice of a quinolinemethanol antimalarial, 2,8-bis(trifluoromethyl)-4-(1-hydroxy-3-(N-t-butylamino)propyl)quinoline phosphate (WR 184,806). | Approximately 75% of the radioactivity derived from WR 184806-14C was excreted in the feces with the remainder in the urine after po administration. At least 77-85% of the dose was absorbed by 2-8 hr; lungs, liver, skeletal muscle, kidneys, small intestine (less contents) and residual carcass were major sites of deposition of total radioactivity. Within these tissues the radioactivity present was predominantly as WR 184,806 rather than its metabolites. Peak blood plasma levels of WR 184,806 occurred at 2-4 and 7-10 hr. The peak erythrocyte level of WR 184,806 occurred at 6 hr. The presence in the urine and feces of unchanged WR 184,806 was confirmed by thin-layer chromatography and inverse isotope dilution. | The absorption, distribution, and excretion in mice of a quinolinemethanol antimalarial, 2,8-bis(trifluoromethyl)-4-(1-hydroxy-3-(N-t-butylamino)propyl)quinoline phosphate (WR 184,806). Approximately 75% of the radioactivity derived from WR 184806-14C was excreted in the feces with the remainder in the urine after po administration. At least 77-85% of the dose was absorbed by 2-8 hr; lungs, liver, skeletal muscle, kidneys, small intestine (less contents) and residual carcass were major sites of deposition of total radioactivity. Within these tissues the radioactivity present was predominantly as WR 184,806 rather than its metabolites. Peak blood plasma levels of WR 184,806 occurred at 2-4 and 7-10 hr. The peak erythrocyte level of WR 184,806 occurred at 6 hr. The presence in the urine and feces of unchanged WR 184,806 was confirmed by thin-layer chromatography and inverse isotope dilution. | [
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PMID:5259 | Oxisuran metabolism in pigs. | The fate of oxisuran in the pig was studied with 14C-labeled drug to quantify its biotransformation and disposition. Despite interanimal differences, it was clear that the compound absorbed rapidly and biotransformed extensively to metabolites which were excreted principally in urine rather than in feces. Direct attacks upon oxisuran involved reduction to diastereo-isomeric oxisuran alcohol sulfoxides and oxidation to oxisuran sulfone. Subsequently, the oxisuran alcohol sulfoxides were reduced to a sulfide and oxidized to a sulfone. Phase II reactions appeared to be limited to sulfation of oxisuran alcohol sulfoxides and oxisuran alcohol sulfone. Pharmacokinetics were investigated for oxisuran and its metabolites. | Oxisuran metabolism in pigs. The fate of oxisuran in the pig was studied with 14C-labeled drug to quantify its biotransformation and disposition. Despite interanimal differences, it was clear that the compound absorbed rapidly and biotransformed extensively to metabolites which were excreted principally in urine rather than in feces. Direct attacks upon oxisuran involved reduction to diastereo-isomeric oxisuran alcohol sulfoxides and oxidation to oxisuran sulfone. Subsequently, the oxisuran alcohol sulfoxides were reduced to a sulfide and oxidized to a sulfone. Phase II reactions appeared to be limited to sulfation of oxisuran alcohol sulfoxides and oxisuran alcohol sulfone. Pharmacokinetics were investigated for oxisuran and its metabolites. | [
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PMID:5261 | Qualitative metabolic fate of phenoxybenzamine in rat, dog, and man. Use of 15N-labeling. | Administration of an equimolar mixture of unlabeled and 15N-labeled phenoxybenzamine to rats and dogs facilitated identification of urinary metabolites by gas chromatography/chemical-ionization mass spectrometry by virtue of the the conspicuous equal-intensity ion pairs produced. By use of this technique N-benzyl-N-phenoxyisopropylamine (III), N-benzyl-N-(p-hydroxy-phenoxyisopropyl)amine (IV), and 2-benzylamino-1-propanol (VI) were identified as metabolites in rats. Phenoxyisopropylamine (V) as well as III and IV were identified in dogs. Compound IV was identified in humans under clinical treatment with phenoxybenzamine. The metabolites were screened for cardiovascular activity in rats. Compound III had weak alpha-adrenergic blocking activity and V elicited a hypertensive response. | Qualitative metabolic fate of phenoxybenzamine in rat, dog, and man. Use of 15N-labeling. Administration of an equimolar mixture of unlabeled and 15N-labeled phenoxybenzamine to rats and dogs facilitated identification of urinary metabolites by gas chromatography/chemical-ionization mass spectrometry by virtue of the the conspicuous equal-intensity ion pairs produced. By use of this technique N-benzyl-N-phenoxyisopropylamine (III), N-benzyl-N-(p-hydroxy-phenoxyisopropyl)amine (IV), and 2-benzylamino-1-propanol (VI) were identified as metabolites in rats. Phenoxyisopropylamine (V) as well as III and IV were identified in dogs. Compound IV was identified in humans under clinical treatment with phenoxybenzamine. The metabolites were screened for cardiovascular activity in rats. Compound III had weak alpha-adrenergic blocking activity and V elicited a hypertensive response. | [
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PMID:5260 | Metabolism of trans-stilbene in rabbits and rats. | The biotransformation of intramuscularly administered 14C-labeled trans-stilbene was investigated in both rabbits and rats. In rabbits radioactivity was excreted (78.8% in 17 days) almost exclusively in the urine. In rats, feces accounted for more than half of the total excretion (80.3% in 19 days). Hydroxylated metabolites in urine and feces were identified by thin-layer chromatography and quantitated by liquid scintillation counting. They were 4-hydroxy and 4,4'-dihydroxystilbene, and the two monomethyl ethers of 3,4-dihydroxystilbene and 4,4'-dihydroxybibenzyl. In addition, four polyhydroxylated metabolites of stilbene and bibenzyl were identified in rat urine by comparison of gas-liquid chromatography retention times of their silyl derivatives with synthetic reference compounds. They were 3,4,4'-trihydroxystilbene, and 3,4-dihydroxy-, 3,4,4'=trihydroxy-, and 3,4,3',4'-tetrahydroxybibenzyl. Oxidative cleavage of trans-stilbene to benzoic acid and 4-hydroxy- and 3,4-dihydroxybenzoic acid was established by the presence of both the free acids and conjugates of these compounds in both rabbit and rat urine. Their glycine conjugates were identified by thin-layer chromatography and quantitated by reverse isotope dilution procedures from unhydrolyzed rabbit and rat urine. Percentages obtained indicate that cleavage of trans-stilbene is more extensive in rabbits (9.5%) than in rats (2.7%). | Metabolism of trans-stilbene in rabbits and rats. The biotransformation of intramuscularly administered 14C-labeled trans-stilbene was investigated in both rabbits and rats. In rabbits radioactivity was excreted (78.8% in 17 days) almost exclusively in the urine. In rats, feces accounted for more than half of the total excretion (80.3% in 19 days). Hydroxylated metabolites in urine and feces were identified by thin-layer chromatography and quantitated by liquid scintillation counting. They were 4-hydroxy and 4,4'-dihydroxystilbene, and the two monomethyl ethers of 3,4-dihydroxystilbene and 4,4'-dihydroxybibenzyl. In addition, four polyhydroxylated metabolites of stilbene and bibenzyl were identified in rat urine by comparison of gas-liquid chromatography retention times of their silyl derivatives with synthetic reference compounds. They were 3,4,4'-trihydroxystilbene, and 3,4-dihydroxy-, 3,4,4'=trihydroxy-, and 3,4,3',4'-tetrahydroxybibenzyl. Oxidative cleavage of trans-stilbene to benzoic acid and 4-hydroxy- and 3,4-dihydroxybenzoic acid was established by the presence of both the free acids and conjugates of these compounds in both rabbit and rat urine. Their glycine conjugates were identified by thin-layer chromatography and quantitated by reverse isotope dilution procedures from unhydrolyzed rabbit and rat urine. Percentages obtained indicate that cleavage of trans-stilbene is more extensive in rabbits (9.5%) than in rats (2.7%). | [
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PMID:5267 | Differential dependence on NADPH concentrations of two microsomal corticosteroid 21-hydroxylation reactions. | The dependency on NADPH concentrations of the bovine adrenal cortex microsomal 21-hydroxylation of progesterone and of 17-hydroxyprogesterone was investigated. The average Km for NADPH in the 21-hydroxylation of progesterone is 10.4 muM while that for the 21-hydroxylation of 17-hydroxyprogesterone is 0.6 muM. The optimal NADPH concentrations for these two hydroxylations are, in average, one order of magnitude apart. The different affinities of the two 21-hydroxylating activities with respect to NADPH may indicate the presence of independent 21-hydroxylation reactions for these two steroid substrates. This evidence is consistent with the observed genetic data in human congenital adrenal hyperplasia. | Differential dependence on NADPH concentrations of two microsomal corticosteroid 21-hydroxylation reactions. The dependency on NADPH concentrations of the bovine adrenal cortex microsomal 21-hydroxylation of progesterone and of 17-hydroxyprogesterone was investigated. The average Km for NADPH in the 21-hydroxylation of progesterone is 10.4 muM while that for the 21-hydroxylation of 17-hydroxyprogesterone is 0.6 muM. The optimal NADPH concentrations for these two hydroxylations are, in average, one order of magnitude apart. The different affinities of the two 21-hydroxylating activities with respect to NADPH may indicate the presence of independent 21-hydroxylation reactions for these two steroid substrates. This evidence is consistent with the observed genetic data in human congenital adrenal hyperplasia. | [
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PMID:5262 | Identification of the urinary metabolites of 14C-bumetanide in the rat and their excretion by rats and dogs. | Unchanged bumetanide, 3-(n-butylamino)-4-phenoxy-5-sulfamoylbenzoic acid, and five metabolites were excreted in the urine of rats given 50 mg of 14C-labeled drug per kg intravenously. The metabolites, which were identified by mass spectrometry and/or nuclear magnetic resonance spectroscopy, arose by metabolic alteration of the n-butyl sidechain. In metabolites I-V, the butylamino group was converted to -NHCH2CH2CH2COOH, -NHCH2CH2CH2CH2OH,, -NHCH2CH2CHOHCH3, -NHCH2CH2CHOHCH2OH and -NH2, respectively. The total urinary and fecal excretion of labeled drug and metabolites after iv and oral administration of 14C-bumetanide was estimated in dogs given 0.5 mg/kg and in rats given 5 mg/kg. In the dog, the primary excretion product was unchanged drug, although evidence was obtained that the acyl glucuronide of bumetanide was secreted in dog bile. The major metabolite excreted by the rat was I, and negligible quantities of intact drug were excreted in the urine after oral or iv administration. Optical activity was found for the two metabolites that contained a chiral center (III and IV), indicating that they were formed by a stereoselective hydroxylation. | Identification of the urinary metabolites of 14C-bumetanide in the rat and their excretion by rats and dogs. Unchanged bumetanide, 3-(n-butylamino)-4-phenoxy-5-sulfamoylbenzoic acid, and five metabolites were excreted in the urine of rats given 50 mg of 14C-labeled drug per kg intravenously. The metabolites, which were identified by mass spectrometry and/or nuclear magnetic resonance spectroscopy, arose by metabolic alteration of the n-butyl sidechain. In metabolites I-V, the butylamino group was converted to -NHCH2CH2CH2COOH, -NHCH2CH2CH2CH2OH,, -NHCH2CH2CHOHCH3, -NHCH2CH2CHOHCH2OH and -NH2, respectively. The total urinary and fecal excretion of labeled drug and metabolites after iv and oral administration of 14C-bumetanide was estimated in dogs given 0.5 mg/kg and in rats given 5 mg/kg. In the dog, the primary excretion product was unchanged drug, although evidence was obtained that the acyl glucuronide of bumetanide was secreted in dog bile. The major metabolite excreted by the rat was I, and negligible quantities of intact drug were excreted in the urine after oral or iv administration. Optical activity was found for the two metabolites that contained a chiral center (III and IV), indicating that they were formed by a stereoselective hydroxylation. | [
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PMID:5264 | Studies of the metabolism of parathion with an apparently homogeneous preparation of rabbit liver cytochrome P-450. | The metabolism of parathion has been examined by use of a reconstituted mixed-function oxidase enzyme system isolated from the livers of phenobarbital-pretreated rabbits. The cytochrome P-450 used in these studies was apparently homogeneous as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by assay of the preparation for contaminating microsomal enzymes and enzyme activity. These studies revealed that the apparently homogeneous preparation of cytochrome P-450, in the presence of the appropriate cofactors, can catalyze the conversions of parathion to both paraoxon and diethyl phosphorothioic acid. The present studies have also shown that parathion is metabolized by the reconstituted mixed-function oxidase enzyme system to diethyl phosphoric acid, a product which, in previous studies with intact liver microsomes, had been thought to arise exclusively from the hydrolysis of paraoxon by a microsomal esterase(s). The available data suggest that all three of the products of the metabolism of parathion by the reconstituted mixed-function oxidase enzyme system, namely, paraoxon, diethyl phosphorothioic acid, and diethyl phosphoric acid, are formed nonenzymatically by breakdown by different pathways of a common enzymatically formed intermediate. This common intermediate is thought to be the sulfine derivative of parathion formed in the mixed-function oxidase-catalyzed addition of an oxygen atom to one of the unshared electron pairs of the thiono-sulfur atom. | Studies of the metabolism of parathion with an apparently homogeneous preparation of rabbit liver cytochrome P-450. The metabolism of parathion has been examined by use of a reconstituted mixed-function oxidase enzyme system isolated from the livers of phenobarbital-pretreated rabbits. The cytochrome P-450 used in these studies was apparently homogeneous as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by assay of the preparation for contaminating microsomal enzymes and enzyme activity. These studies revealed that the apparently homogeneous preparation of cytochrome P-450, in the presence of the appropriate cofactors, can catalyze the conversions of parathion to both paraoxon and diethyl phosphorothioic acid. The present studies have also shown that parathion is metabolized by the reconstituted mixed-function oxidase enzyme system to diethyl phosphoric acid, a product which, in previous studies with intact liver microsomes, had been thought to arise exclusively from the hydrolysis of paraoxon by a microsomal esterase(s). The available data suggest that all three of the products of the metabolism of parathion by the reconstituted mixed-function oxidase enzyme system, namely, paraoxon, diethyl phosphorothioic acid, and diethyl phosphoric acid, are formed nonenzymatically by breakdown by different pathways of a common enzymatically formed intermediate. This common intermediate is thought to be the sulfine derivative of parathion formed in the mixed-function oxidase-catalyzed addition of an oxygen atom to one of the unshared electron pairs of the thiono-sulfur atom. | [
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PMID:5265 | Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. IX. The formation of a 455-nm metabolite-cytochrome P-450 complex. | The reconstituted liver microsomal hydroxylation system was used to study the formation of a metabolite-cytochrome P-450 complex absorbing maximally at 455 nm, with benzphetamine and N-hydroxyamphetamine as substrates. Complex formation required the presence of NADPH, substrate, NADPH-cytochrome c reductase, lipid, and cytochrome P-450, indicating that metabolism of the substrate is essential. In the presence of fixed amounts of lipid and NADPH-cytochrome c reductase, the rate of complex formation with cytochrome P-450 isolated from phenobarbital-treated rats was much greater than that observed with cytochrome P-48 from 3-methylcholanthrene-treated rats or rabbits. These results are consistent with recent studies indicating that different forms of cytochrome P-450 with distinct spectral, catalytic, and immunological properties exist in liver microsomes. | Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. IX. The formation of a 455-nm metabolite-cytochrome P-450 complex. The reconstituted liver microsomal hydroxylation system was used to study the formation of a metabolite-cytochrome P-450 complex absorbing maximally at 455 nm, with benzphetamine and N-hydroxyamphetamine as substrates. Complex formation required the presence of NADPH, substrate, NADPH-cytochrome c reductase, lipid, and cytochrome P-450, indicating that metabolism of the substrate is essential. In the presence of fixed amounts of lipid and NADPH-cytochrome c reductase, the rate of complex formation with cytochrome P-450 isolated from phenobarbital-treated rats was much greater than that observed with cytochrome P-48 from 3-methylcholanthrene-treated rats or rabbits. These results are consistent with recent studies indicating that different forms of cytochrome P-450 with distinct spectral, catalytic, and immunological properties exist in liver microsomes. | [
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PMID:5279 | The response of Ca-mediated action potentials and contractile activity in mammalian ventricular myocardium towards alkalosis. | Alkalosis (pH 7.8) produced by reduction of CO2 concentration augmented both upstroke velocity of Ca action potentials and isometric contractile force of mammalian heart muscle. If the increase of pH to 7.8 was achieved by a raise of HCO3 concentration (with simultaneous reduction of CO2 concentration), the positive inotropic response was not accompanied by an augmented Ca current. Obviously, the well-known positive inotropic effect of alkalosis does not only depend upon the enhancement of transmembrane Ca influx during excitation, but can be mediated alone by affecting intracellular Ca movements as well. | The response of Ca-mediated action potentials and contractile activity in mammalian ventricular myocardium towards alkalosis. Alkalosis (pH 7.8) produced by reduction of CO2 concentration augmented both upstroke velocity of Ca action potentials and isometric contractile force of mammalian heart muscle. If the increase of pH to 7.8 was achieved by a raise of HCO3 concentration (with simultaneous reduction of CO2 concentration), the positive inotropic response was not accompanied by an augmented Ca current. Obviously, the well-known positive inotropic effect of alkalosis does not only depend upon the enhancement of transmembrane Ca influx during excitation, but can be mediated alone by affecting intracellular Ca movements as well. | [
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PMID:5282 | The in vitro effect of adrenergic agents and related compounds on triglyceride levels of guinea-pig lymphoid cells. | Isoproterenol, epinephrine and norepinephrine were highly potent in decreasing the triglyceride levels of the splenic lymphoid cells from guinea-pigs in vitro, while they were quite inert upon the inguinal lymph node cells. | The in vitro effect of adrenergic agents and related compounds on triglyceride levels of guinea-pig lymphoid cells. Isoproterenol, epinephrine and norepinephrine were highly potent in decreasing the triglyceride levels of the splenic lymphoid cells from guinea-pigs in vitro, while they were quite inert upon the inguinal lymph node cells. | [
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PMID:5283 | Assimilation of ammonia and growth of biotin deficient Aspergillus nidulans. | Biotin deficiency in Aspergillus nidulans has been found to increase the uptake of ammonium ions, associated with a marked increase in the activity of NADP-linked glutamate dehydrogenase, which is found to be the major route of ammonia assimilation in this culture. The results obtained are discussed with respect to the growth of Aspergillus nidulans during biotin deficiency. | Assimilation of ammonia and growth of biotin deficient Aspergillus nidulans. Biotin deficiency in Aspergillus nidulans has been found to increase the uptake of ammonium ions, associated with a marked increase in the activity of NADP-linked glutamate dehydrogenase, which is found to be the major route of ammonia assimilation in this culture. The results obtained are discussed with respect to the growth of Aspergillus nidulans during biotin deficiency. | [
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PMID:5284 | [Differential properties of two molecular forms of 4-aminobutyrate-2-ketoglutarate transaminase (GABAT) from pig brain (author's transl)]. | The two forms isolated exhibit some differences concerning their physicochemical and functional properties. They are identical with the previously purified molecular fomrs, GABAT I and GABAT II, separated by DEAE cellulose chromatography. | [Differential properties of two molecular forms of 4-aminobutyrate-2-ketoglutarate transaminase (GABAT) from pig brain (author's transl)]. The two forms isolated exhibit some differences concerning their physicochemical and functional properties. They are identical with the previously purified molecular fomrs, GABAT I and GABAT II, separated by DEAE cellulose chromatography. | [
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PMID:5285 | Acrosomal hydrolases in buffalo spermatozoa. | Buffalo sperm acrosome resembles its counterpart in other species, being rich in hydrolytic enzymes. Of the enzyme activities estimated, acid phosphatase, beta-N-acetylglucosaminidase and hyaluronidase were low compared to those of ram semen. However, the aryl sulphatase activity was high. GOT activity estimated in sperm preparation may not be of acrosomal origin. | Acrosomal hydrolases in buffalo spermatozoa. Buffalo sperm acrosome resembles its counterpart in other species, being rich in hydrolytic enzymes. Of the enzyme activities estimated, acid phosphatase, beta-N-acetylglucosaminidase and hyaluronidase were low compared to those of ram semen. However, the aryl sulphatase activity was high. GOT activity estimated in sperm preparation may not be of acrosomal origin. | [
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PMID:5286 | Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.). | An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue. | Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.). An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue. | [
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PMID:5287 | On the regulatory properties of a halophilic malic enzyme from Halobacterium cutirubrum. | The NADP-linked malic enzyme from Halobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3 M), considered physiological for the extremely halophilic bacteria. | On the regulatory properties of a halophilic malic enzyme from Halobacterium cutirubrum. The NADP-linked malic enzyme from Halobacterium cutirubrum is strongly inhibited by acetyl-CoA and NADH, and rather weakly inhibited by oxaloacetate and glyoxylate, in the presence of very high KCl concentrations (3 M), considered physiological for the extremely halophilic bacteria. | [
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PMID:5288 | Fermentative digestion of food in the colobus monkey, Colobus, polykomos. | Fermentation of leafy food occurs in the enlarged saccus gastricus of the colobus monkey with the formation of volatile fatty acid, as in the rumen of ruminant animals. About half of the digestible organic matter and cellulose of the diet is digested in this way. | Fermentative digestion of food in the colobus monkey, Colobus, polykomos. Fermentation of leafy food occurs in the enlarged saccus gastricus of the colobus monkey with the formation of volatile fatty acid, as in the rumen of ruminant animals. About half of the digestible organic matter and cellulose of the diet is digested in this way. | [
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] |
PMID:5303 | Isolation, purification and properties of aortic elastase. | An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16]. | Isolation, purification and properties of aortic elastase. An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16]. | [
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PMID:5312 | Some chemical aspects of histamine H2-receptor antagonists. | Certain chemical properties, which may determine the biological actions of the recently discovered histamine H2-receptor antagonists burimamide and metiamide, are identified, partly by considering the derivation of these antagonists. Examples are given of attempts to design antagonists using histamine as starting point. A partial agonist was eventually obtained through modifying the side chain of histamine but retaining the imidazole ring. Further developments led to the synthesis of uncharged thioureido analogues and to the discovery of the antagonist, burimamide. Consideration of the relative concentration of imidazole tautomers led to the replacement of a methylene group (-CH2-) with an isosteric thioether (-S-) link in the side chain, and incorporation of a methyl group in the imidazole ring; these changes afforded metiamide, an orally active antagonist. These developments emphasize that the imidazole ring appears to have a special importance at H2 receptors. Burimamide and metiamide are hydrophilic molecules that resemble histamine in having an imidazole ring but differ in the side chain which, though polar, is uncharged. By contrast, the H1-receptor antihistaminic drugs are lipophilic molecules; their resemblance to histamine is in having a positively charged ammonium side chain. These substantial chemical differences between the respective antagonists probably determine their selectivity in distinguishing between the two types of histamine receptor. Furthermore, the very low lipophilicities of these H2-receptor antagonists probably account for the lack of central nervous system and local anesthetic effects normally associated with the use of antihistaminic drugs. | Some chemical aspects of histamine H2-receptor antagonists. Certain chemical properties, which may determine the biological actions of the recently discovered histamine H2-receptor antagonists burimamide and metiamide, are identified, partly by considering the derivation of these antagonists. Examples are given of attempts to design antagonists using histamine as starting point. A partial agonist was eventually obtained through modifying the side chain of histamine but retaining the imidazole ring. Further developments led to the synthesis of uncharged thioureido analogues and to the discovery of the antagonist, burimamide. Consideration of the relative concentration of imidazole tautomers led to the replacement of a methylene group (-CH2-) with an isosteric thioether (-S-) link in the side chain, and incorporation of a methyl group in the imidazole ring; these changes afforded metiamide, an orally active antagonist. These developments emphasize that the imidazole ring appears to have a special importance at H2 receptors. Burimamide and metiamide are hydrophilic molecules that resemble histamine in having an imidazole ring but differ in the side chain which, though polar, is uncharged. By contrast, the H1-receptor antihistaminic drugs are lipophilic molecules; their resemblance to histamine is in having a positively charged ammonium side chain. These substantial chemical differences between the respective antagonists probably determine their selectivity in distinguishing between the two types of histamine receptor. Furthermore, the very low lipophilicities of these H2-receptor antagonists probably account for the lack of central nervous system and local anesthetic effects normally associated with the use of antihistaminic drugs. | [
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PMID:5313 | The pharmacology of burimamide and metiamide, two histamine H2-receptor antagonists. | Burimamide and metiamide are two histamine H2-receptor antagonists. Evidence is presented that indicates the competitive nature and the specificity of the antagonism. Metiamide is about ten times more potent than burimamide and is also more effective than burimamide when given orally. Both compounds inhibit gastric secretion and the evidence is consistent with this inhibition being due to competitive antagonism of H2 receptors in the gastric mucosa. Burimamide, unlike metiamide, causes release of catecholamines even at dose levels that are just sufficient to produce H2-receptor antagonism. Burimamide, but not metiamide, has alpha-adrenoceptor blocking activity. In certain models for inflammation, particularly rat paw edema induced by compound 48/80, burimamide in combination with the H1-receptor antagonist mepyramine shows anti-inflammatory activity. This may, in part, be associated with the catecholamine-releasing properties of the compound. Metiamide is less active in this respect. | The pharmacology of burimamide and metiamide, two histamine H2-receptor antagonists. Burimamide and metiamide are two histamine H2-receptor antagonists. Evidence is presented that indicates the competitive nature and the specificity of the antagonism. Metiamide is about ten times more potent than burimamide and is also more effective than burimamide when given orally. Both compounds inhibit gastric secretion and the evidence is consistent with this inhibition being due to competitive antagonism of H2 receptors in the gastric mucosa. Burimamide, unlike metiamide, causes release of catecholamines even at dose levels that are just sufficient to produce H2-receptor antagonism. Burimamide, but not metiamide, has alpha-adrenoceptor blocking activity. In certain models for inflammation, particularly rat paw edema induced by compound 48/80, burimamide in combination with the H1-receptor antagonist mepyramine shows anti-inflammatory activity. This may, in part, be associated with the catecholamine-releasing properties of the compound. Metiamide is less active in this respect. | [
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PMID:5314 | Identification and blockade of vascular H2 receptors. | Experiments were conducted in anesthetized dogs to determine the nature of receptors mediating vascular actions of histamine. In the perfused gracilis muscle histamine caused vasodilatation that was attenuated in part by mepyramine, an H1-receptor blocker. Metiamide, an H2 blocker, given alone had no effect on dilatation. However, the combination of mepyramine and metiamide resulted in a large attenuation of dilatation. Histamine caused constriction of the perfused saphenous vein that was totally blocked by mepyramine suggesting that venoconstriction by histamine involves only H1 receptors. Histamine infusion caused a fall in arterial pressure and a large reduction in peripheral resistance. Mepyramine attenuated the fall in pressure but not the reduction in resistance. Combined H1- and H2-receptor blockade largely eliminated the effects of histamine infusion further documenting the existence of H1 and H2 receptors. The effects of H1 and H2 antihistamines on a variety of physiological vasodilator responses were examined. Evidence was obtained to indicate that H1- and H2-histamine receptors are involved in the active component of baroreceptor-mediated reflex vasodilatation, poststimulation vasodilatation, sympathetic vasodilatation in the guanethidine-treated dog, and vasodilator responses following compound 48/80. No evidence for the participation of either H1- or H2-histamine receptors in reactive hyperemia or the dilatation accompanying exercise was found. It is concluded that in the dog both endogenously-released and exogenous histamine exert vascular effects by activation of both H1 and H2 receptors. | Identification and blockade of vascular H2 receptors. Experiments were conducted in anesthetized dogs to determine the nature of receptors mediating vascular actions of histamine. In the perfused gracilis muscle histamine caused vasodilatation that was attenuated in part by mepyramine, an H1-receptor blocker. Metiamide, an H2 blocker, given alone had no effect on dilatation. However, the combination of mepyramine and metiamide resulted in a large attenuation of dilatation. Histamine caused constriction of the perfused saphenous vein that was totally blocked by mepyramine suggesting that venoconstriction by histamine involves only H1 receptors. Histamine infusion caused a fall in arterial pressure and a large reduction in peripheral resistance. Mepyramine attenuated the fall in pressure but not the reduction in resistance. Combined H1- and H2-receptor blockade largely eliminated the effects of histamine infusion further documenting the existence of H1 and H2 receptors. The effects of H1 and H2 antihistamines on a variety of physiological vasodilator responses were examined. Evidence was obtained to indicate that H1- and H2-histamine receptors are involved in the active component of baroreceptor-mediated reflex vasodilatation, poststimulation vasodilatation, sympathetic vasodilatation in the guanethidine-treated dog, and vasodilator responses following compound 48/80. No evidence for the participation of either H1- or H2-histamine receptors in reactive hyperemia or the dilatation accompanying exercise was found. It is concluded that in the dog both endogenously-released and exogenous histamine exert vascular effects by activation of both H1 and H2 receptors. | [
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PMID:5315 | Cardiac histamine receptors. | Current evidence pertinent to the identification of cardiac histamine receptors in the guinea pig is reviewed. Pharmacological characterization has been aided by the use of selective agonists and antagonists for both types of histamine receptors. It appears that both H1 and H2 receptors mediate the cardiac effects of histamine. Histamine H2 receptors mediate the positive chronotropic and ventricular inotropic effects. H1 receptors mediate the negative dromotropic effect of histamine and possibly the atrial inotropic effect. Histamine-induced arrhythmias involve H1 receptors (arrhythmias of conduction) or H2 receptors (arrhythmias of automaticity), or both. The receptors mediating the histamine-induced increase in coronary flow are not as clearly defined: both H1 and H2 receptors might be implicated. | Cardiac histamine receptors. Current evidence pertinent to the identification of cardiac histamine receptors in the guinea pig is reviewed. Pharmacological characterization has been aided by the use of selective agonists and antagonists for both types of histamine receptors. It appears that both H1 and H2 receptors mediate the cardiac effects of histamine. Histamine H2 receptors mediate the positive chronotropic and ventricular inotropic effects. H1 receptors mediate the negative dromotropic effect of histamine and possibly the atrial inotropic effect. Histamine-induced arrhythmias involve H1 receptors (arrhythmias of conduction) or H2 receptors (arrhythmias of automaticity), or both. The receptors mediating the histamine-induced increase in coronary flow are not as clearly defined: both H1 and H2 receptors might be implicated. | [
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PMID:5322 | Lack of relationship between cyclic nucleotide levels and spermatozoal function in human semen. | Cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were determined in 103 samples of human semen and grouped according to the number of spermatozoa in the ejaculate. No correlation was found between cyclic AMP concentrations and the number, motility, and morphology of the spermatozoa or the fructose content, pH, and volume of the ejaculate. Similar findings were obtained with cyclic guanosine 3':5'-monophosphate levels in 24 samples of human semen. Therefore, cyclic nucleotide levels in human semen appear to be derived from sources other than spermatozoal adenylyl or guanylyl cyclase. | Lack of relationship between cyclic nucleotide levels and spermatozoal function in human semen. Cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were determined in 103 samples of human semen and grouped according to the number of spermatozoa in the ejaculate. No correlation was found between cyclic AMP concentrations and the number, motility, and morphology of the spermatozoa or the fructose content, pH, and volume of the ejaculate. Similar findings were obtained with cyclic guanosine 3':5'-monophosphate levels in 24 samples of human semen. Therefore, cyclic nucleotide levels in human semen appear to be derived from sources other than spermatozoal adenylyl or guanylyl cyclase. | [
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PMID:5325 | Quality, efficiency, and cost of a physician-assistant-protocol system for managment of diabetes and hypertension. | Briefly trained physicians assistants using protocols (clinical algorithms) for diabetes, hypertension, and related chronic arteriosclerotic and hypertensive heart disease abstrated information from the medical record and obtained history and physical examination data on every patient-visit to a city hospital chronic disease clinic over a 18-month period. The care rendered by the protocol system was compared with care rendered by a "traditional" system in the same clinic in which physicians delegated few clinical tasks. Increased thoroughness in collecting clinical data in the protocol system led to an increase in the recognition of new pathology. Outcome criteria reflected equivalent quality of care in both groups. Efficiency time-motion studies demonstrated a 20 per cent saving in physician time with the protocol system. Coct estimates, based on the time spent with patients by various providers and on the laboratory-test-ordering patterns, demonstrated equivalent costs of the two systems, given optimal staffing patterns. Laboratory tests were a major element of the cost of patient care,and the clinical yield per unit cost of different tests varied widely. | Quality, efficiency, and cost of a physician-assistant-protocol system for managment of diabetes and hypertension. Briefly trained physicians assistants using protocols (clinical algorithms) for diabetes, hypertension, and related chronic arteriosclerotic and hypertensive heart disease abstrated information from the medical record and obtained history and physical examination data on every patient-visit to a city hospital chronic disease clinic over a 18-month period. The care rendered by the protocol system was compared with care rendered by a "traditional" system in the same clinic in which physicians delegated few clinical tasks. Increased thoroughness in collecting clinical data in the protocol system led to an increase in the recognition of new pathology. Outcome criteria reflected equivalent quality of care in both groups. Efficiency time-motion studies demonstrated a 20 per cent saving in physician time with the protocol system. Coct estimates, based on the time spent with patients by various providers and on the laboratory-test-ordering patterns, demonstrated equivalent costs of the two systems, given optimal staffing patterns. Laboratory tests were a major element of the cost of patient care,and the clinical yield per unit cost of different tests varied widely. | [
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PMID:5326 | Suppression and stimulation mechanisms controlling glucagon secretion in a case of islet-cell tumor producing glucagon, insulin, and gastrin. | The mechanisms controlling secretion of glucagon and other pancreatic hormones were studied in a patient affected with multihormone-secreting islet-cell tumor. Fasting glucagon levels (3,000 pg./ml.) rose to 10 ng./ml. following arginine stimulation. While oral glucose load and intravenous glucose infusion did not suppress glucagon secretion, insulin administration induced a prompt depression in glucagon levels. Glucagon, insulin, and gastrin levels were suppressed by somatostatin while calcium infusion caused a paradoxical increase. It is suggested that only some of the stimulation-inhibition mechanisms were conserved in this case of glucagon-secreting pancreatic tumor. | Suppression and stimulation mechanisms controlling glucagon secretion in a case of islet-cell tumor producing glucagon, insulin, and gastrin. The mechanisms controlling secretion of glucagon and other pancreatic hormones were studied in a patient affected with multihormone-secreting islet-cell tumor. Fasting glucagon levels (3,000 pg./ml.) rose to 10 ng./ml. following arginine stimulation. While oral glucose load and intravenous glucose infusion did not suppress glucagon secretion, insulin administration induced a prompt depression in glucagon levels. Glucagon, insulin, and gastrin levels were suppressed by somatostatin while calcium infusion caused a paradoxical increase. It is suggested that only some of the stimulation-inhibition mechanisms were conserved in this case of glucagon-secreting pancreatic tumor. | [
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PMID:5335 | Histamine effects on H+ permeability by isolated gastric mucosa. | This study examines the effects of histamine on mucosal permeability to acid by isolated stomach of rabbits. Histamine (9 X 10(-5) M) decreased antral luminal acid loss which could not be accounted for by active H+ secretion or appearance of organic acid. Histamine also increased antral electrical resistance and decreased the unidirectional luminal to serosal flux (Jls) of (14)c-erythritol. Histamine, theophylline, and N(6),O(2)-dibutyryl adenosine 3',5'-monophosphate did not significantly alter secretion by fundus in the presence of salicylate (5 X 10(-3)M). However, after removal of salicylate, these agents stimulated acid secretion. Histamine decreased luminal acid loss by both antrum and fundus treated with salicylate, increased the electrical resistance of these tissues, and decreased Jls erythritol of fundic mucosa. Burimamide (1 X 10(-3) M) inhibited the permeability effects of histamine on antrum and fundus. The data indicate that histamine decreases spontaneous antral mucosal permeability as well as salicylate-induced increase in mucosal permeability of both antrum and fundus. The effects of burimamide suggest that antrum contains H2 receptor sites urelated to acid secretion and that fundus contains H2 receptor sites which also govern permeability but are independent of those related to acid secretion. | Histamine effects on H+ permeability by isolated gastric mucosa. This study examines the effects of histamine on mucosal permeability to acid by isolated stomach of rabbits. Histamine (9 X 10(-5) M) decreased antral luminal acid loss which could not be accounted for by active H+ secretion or appearance of organic acid. Histamine also increased antral electrical resistance and decreased the unidirectional luminal to serosal flux (Jls) of (14)c-erythritol. Histamine, theophylline, and N(6),O(2)-dibutyryl adenosine 3',5'-monophosphate did not significantly alter secretion by fundus in the presence of salicylate (5 X 10(-3)M). However, after removal of salicylate, these agents stimulated acid secretion. Histamine decreased luminal acid loss by both antrum and fundus treated with salicylate, increased the electrical resistance of these tissues, and decreased Jls erythritol of fundic mucosa. Burimamide (1 X 10(-3) M) inhibited the permeability effects of histamine on antrum and fundus. The data indicate that histamine decreases spontaneous antral mucosal permeability as well as salicylate-induced increase in mucosal permeability of both antrum and fundus. The effects of burimamide suggest that antrum contains H2 receptor sites urelated to acid secretion and that fundus contains H2 receptor sites which also govern permeability but are independent of those related to acid secretion. | [
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PMID:5336 | Genetics of murine liver and kidney arysulfatase b. | Mice from 12 inbred strains were surveyed for variation of kidney and liver arylsulfatase levels. Kidney variation was due to differences in the activity of arylsulfatase B. Twofold higher activities of arylsulfatase B in SWR/J kidney compared to A/HeJ kidney were determined by an autosomal gene which may be identical to the structural gene for arylsulfatase B since the SWR/J enzyme was more heat-stable than the A/HeJ enzyme. C57BL/6J mice possessed two-fold higher liver arylsulfatase levels than did A/HeJ mice. The major portion of this variation could be attributed to differences in arylsulfatase B, and appeared to be inherited in autosomal fashion. Although some evidence supports the existence of a major locus influencing liver arylsulfatase activity, this must be substantiated by further studies. Whatever the nature of the genetic factors involved, they do not appear to involve structural genes since no differences were discernible between the enzymes of the two strains relevant to Km, heat stability, electrophoretic mobility, pH optimum, activation energy, or response to several inhibitors. Furthermore, the rank ordering of strains on the basis of kidney arylsulfatase activity differed markedly from that which pertained to liver activity. Kidney arylsulfatase levels, but not brain or liver arylsulfatase activities, appear subject to androgenic influences. | Genetics of murine liver and kidney arysulfatase b. Mice from 12 inbred strains were surveyed for variation of kidney and liver arylsulfatase levels. Kidney variation was due to differences in the activity of arylsulfatase B. Twofold higher activities of arylsulfatase B in SWR/J kidney compared to A/HeJ kidney were determined by an autosomal gene which may be identical to the structural gene for arylsulfatase B since the SWR/J enzyme was more heat-stable than the A/HeJ enzyme. C57BL/6J mice possessed two-fold higher liver arylsulfatase levels than did A/HeJ mice. The major portion of this variation could be attributed to differences in arylsulfatase B, and appeared to be inherited in autosomal fashion. Although some evidence supports the existence of a major locus influencing liver arylsulfatase activity, this must be substantiated by further studies. Whatever the nature of the genetic factors involved, they do not appear to involve structural genes since no differences were discernible between the enzymes of the two strains relevant to Km, heat stability, electrophoretic mobility, pH optimum, activation energy, or response to several inhibitors. Furthermore, the rank ordering of strains on the basis of kidney arylsulfatase activity differed markedly from that which pertained to liver activity. Kidney arylsulfatase levels, but not brain or liver arylsulfatase activities, appear subject to androgenic influences. | [
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PMID:5338 | [A study of H-2 mutations in mice. V. Detection of mutations M505, Hzl and M506 in the host vs transplant reaction]. | H-2 mutant/normal pairs of congenic mouse strains C57BL/6equilibriumB6.M505 (H-2Kbd), C57BL/6equilibriumB6.C(Hz1) (H-2Kba) and A.CAequilibriumA.CA(M506) (H-2fa) were studied in the graft versus host assay. All the three mutations were expressed in these tests as gain and loss of H-2 antigen. The data obtained confirm an earlier suggestion that the H-2K antigenic molecule bears antigenic determinants which could be detected by a variety of immunological techniques. | [A study of H-2 mutations in mice. V. Detection of mutations M505, Hzl and M506 in the host vs transplant reaction]. H-2 mutant/normal pairs of congenic mouse strains C57BL/6equilibriumB6.M505 (H-2Kbd), C57BL/6equilibriumB6.C(Hz1) (H-2Kba) and A.CAequilibriumA.CA(M506) (H-2fa) were studied in the graft versus host assay. All the three mutations were expressed in these tests as gain and loss of H-2 antigen. The data obtained confirm an earlier suggestion that the H-2K antigenic molecule bears antigenic determinants which could be detected by a variety of immunological techniques. | [
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PMID:5339 | [Description of new mutant forms of erythrocyte glucose-6-phosphate dehydrogenase in man]. | 9 variants of human erythrocyte glucose-6-phosphate dehydrogenase (G6PD) were isolated from erythrocytes of patients with G6PD deficiency and partially purified according to WHO program for stanartization of methods for studying G6PD. The results of physico-chemical study of these enzymes (determination of electrophoretic mobility, kappaM for G6P and NADP, pH optimum and thermostability) permit tu consider 5 of them to be new mutations of G6PD previously not described in literature. The observed high geterogeneity of variants of G6PD in Azerbaijan is discussed. | [Description of new mutant forms of erythrocyte glucose-6-phosphate dehydrogenase in man]. 9 variants of human erythrocyte glucose-6-phosphate dehydrogenase (G6PD) were isolated from erythrocytes of patients with G6PD deficiency and partially purified according to WHO program for stanartization of methods for studying G6PD. The results of physico-chemical study of these enzymes (determination of electrophoretic mobility, kappaM for G6P and NADP, pH optimum and thermostability) permit tu consider 5 of them to be new mutations of G6PD previously not described in literature. The observed high geterogeneity of variants of G6PD in Azerbaijan is discussed. | [
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PMID:5340 | [The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis]. | In the course of the investigation of piebald (black-white) cattle it is found that 17,62% animals produce the AA type beta-lactoglobulin, 49,52%--the AB type and 32,86%--the BB type. The higher inhibitory activity of milk with the BB type beta-lactoglobulin was found which retained in dilution 1 : 32. The total flora of teat, cyst and parenchyma milk of animals with the BB type beta-lactoglobulin as well as Enterococcus bacteria were much lower than in milk of cows with the AA type homogenous form. The animals with the first type of milk protein had mastitis more rarely as compared to those with the second type. The animals with heterogenous form of beta-lactoglobulin had intermediate values in most of their characteristics. | [The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis]. In the course of the investigation of piebald (black-white) cattle it is found that 17,62% animals produce the AA type beta-lactoglobulin, 49,52%--the AB type and 32,86%--the BB type. The higher inhibitory activity of milk with the BB type beta-lactoglobulin was found which retained in dilution 1 : 32. The total flora of teat, cyst and parenchyma milk of animals with the BB type beta-lactoglobulin as well as Enterococcus bacteria were much lower than in milk of cows with the AA type homogenous form. The animals with the first type of milk protein had mastitis more rarely as compared to those with the second type. The animals with heterogenous form of beta-lactoglobulin had intermediate values in most of their characteristics. | [
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PMID:5341 | [Genetic control of allogenic inhibition of hematopoietic stem cells]. | Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele. | [Genetic control of allogenic inhibition of hematopoietic stem cells]. Adult mice of C57BL/6, CBA (CBA X C57BL/6) F1, (CBA X C57BL/6) F2, F1 X CBA and F1 X C57BL/6 strains were lethally irradiated and reconstituted with a constant dose of 3-10(5) C57BL/6 bone marrow cells. At the 9th day after the bone marrow transplantation the colony count was performed in spleen of irradiated recipients. In the spleen of F1, CBA and C57BL/6 mice were registered low (0--8, intermediate (6--18) and high (22-40) numbers of colonies respectively. The segregation ratios in F2 progeny were close to 2 (low): 1(intermediate): 1(high). The segregation ratios in backcross (F1 X CBA) were close to 1(low): 1(intermediate)numbers of colonies. Backcrosses (F1 X C57BL/6) were distributed to low and high numbers of colonies with the ratio 1:1. The number of spleen colonies of males and females was the same in all segregating progeny. The results of hybrid analysis suggest that a single pair of allelic genes is involved in genetic control of allogenic inhibition, and that the resistance (manifestation of inhibition) to C57BL/6 stem cells is conferred by the dominant allele. | [
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PMID:5342 | Inhibition of food-stimulated gastric acid secretion by cimetidine. | The effect of cimetidine, a new histamine H2-receptor antagonist, on gastric acid secretion stimulated by a homogenised meal was studied in six normal volunteers using an in vivo intragastric titration technique. The subjects were studied twice, no more than 48 h apart, receiving either cimetidine 200 mg or placebo in random order. Cimetidine administered either 32 men before (three subjects) or with the meal (three subjects) significantly inhibited gastric acid secretion in all the subjects throughout the period of study; 96 min after food, total acid secretion decreased by 67 and 57% respectively. When the drug was taken with the meal absorption was slower (mean peak blood level 2-34 mumol/l, 80-128 min after dosing) than when administered on an empty stomach (mean peak blood level 5-08 mumol/l, 48-64 min after dosing). Blood cimetidine concentration correlated significantly (P less than 0-01) with percentage inhibition of acid output and the calculated concentration resulting in 50% inhibition of gastric acid secretion (IC50) was 1-6 mumol/l. Secretion of gastrin in response to food was unaffected by cimetidine. The results suggest that 200 mg cimetidine effectively inhibits food-stimulated acid secretion and that the bioavailability of the drug may be affected by the timing of dosage in relation to meals. No unwanted effect were observed. | Inhibition of food-stimulated gastric acid secretion by cimetidine. The effect of cimetidine, a new histamine H2-receptor antagonist, on gastric acid secretion stimulated by a homogenised meal was studied in six normal volunteers using an in vivo intragastric titration technique. The subjects were studied twice, no more than 48 h apart, receiving either cimetidine 200 mg or placebo in random order. Cimetidine administered either 32 men before (three subjects) or with the meal (three subjects) significantly inhibited gastric acid secretion in all the subjects throughout the period of study; 96 min after food, total acid secretion decreased by 67 and 57% respectively. When the drug was taken with the meal absorption was slower (mean peak blood level 2-34 mumol/l, 80-128 min after dosing) than when administered on an empty stomach (mean peak blood level 5-08 mumol/l, 48-64 min after dosing). Blood cimetidine concentration correlated significantly (P less than 0-01) with percentage inhibition of acid output and the calculated concentration resulting in 50% inhibition of gastric acid secretion (IC50) was 1-6 mumol/l. Secretion of gastrin in response to food was unaffected by cimetidine. The results suggest that 200 mg cimetidine effectively inhibits food-stimulated acid secretion and that the bioavailability of the drug may be affected by the timing of dosage in relation to meals. No unwanted effect were observed. | [
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PMID:5346 | [Morbus Crohn (enteritis regionalis)]. | Crohn's disease (regional enteritis) is a chronic non-specific inflammatory intestinal disorder of unknown etiology. Most commonly the terminal ileum in involved, a segmentary involvement of the bowel wall is rather characteristic. Main symptoms are recurrent abdominal pain, fever, diarrhea and weight loss. Radiological and endoscopic examination confirms the diagnosis, granulomas in the biopsy specimen are pathognomonic. In differential diagnosis ulcerative and ischaemic colitis have to be ruled out. Conservative therapy with prednisolone and salazopyrin is the method of choice, however, complications like small bowel obstruction, toxic megacolon and fistulae ask for surgical intervention. | [Morbus Crohn (enteritis regionalis)]. Crohn's disease (regional enteritis) is a chronic non-specific inflammatory intestinal disorder of unknown etiology. Most commonly the terminal ileum in involved, a segmentary involvement of the bowel wall is rather characteristic. Main symptoms are recurrent abdominal pain, fever, diarrhea and weight loss. Radiological and endoscopic examination confirms the diagnosis, granulomas in the biopsy specimen are pathognomonic. In differential diagnosis ulcerative and ischaemic colitis have to be ruled out. Conservative therapy with prednisolone and salazopyrin is the method of choice, however, complications like small bowel obstruction, toxic megacolon and fistulae ask for surgical intervention. | [
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PMID:5347 | [Levamisole, a chemically defined substance with immunostimulating potential--a review]. | In contrast to immunosuppression, immunostimulation is still at an early stage of development. Levamisole is the first chemically defined substance with immunostimulating potential which can be applied systemically. After a short consideration of the chemistry and pharmacology of Levamisole, its effect in different animal experimental models of humoral and cell-mediated immunity are discussed in detail. Moreover, the following review deals with the influence of Levamisole on several immunological parameters in man. Finally, the therapeutical results achieved up to now by different clinical indications are reported. | [Levamisole, a chemically defined substance with immunostimulating potential--a review]. In contrast to immunosuppression, immunostimulation is still at an early stage of development. Levamisole is the first chemically defined substance with immunostimulating potential which can be applied systemically. After a short consideration of the chemistry and pharmacology of Levamisole, its effect in different animal experimental models of humoral and cell-mediated immunity are discussed in detail. Moreover, the following review deals with the influence of Levamisole on several immunological parameters in man. Finally, the therapeutical results achieved up to now by different clinical indications are reported. | [
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PMID:5345 | Oral lesions caused by habits. | Oral lesions caused by habits can be of teeth only, of teeth and the soft tissues of the mouth or may be only of the soft tissues. Lesions of teeth are permanent and may remain even when there has been total destruction of soft tissues. Recognition of lesions due to habits such as betel chewing, snuff dipping, pipe smoking and certain sexual practices may help towards establishing the sex, the ethnic grouping or even the place of origin of a person or their remains. Certain dental and gingival changes may indicate a person's working circumstances and even the type of treatment received. | Oral lesions caused by habits. Oral lesions caused by habits can be of teeth only, of teeth and the soft tissues of the mouth or may be only of the soft tissues. Lesions of teeth are permanent and may remain even when there has been total destruction of soft tissues. Recognition of lesions due to habits such as betel chewing, snuff dipping, pipe smoking and certain sexual practices may help towards establishing the sex, the ethnic grouping or even the place of origin of a person or their remains. Certain dental and gingival changes may indicate a person's working circumstances and even the type of treatment received. | [
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PMID:5372 | Adherence of bacterial to vaginal epithelial cells. | Vaginal epithelial cells from healthy women were washed and incubated in tissue culture medium with freshly isolated bacteria of the indigenous vaginal flora and with bacteria of species that have been discussed in conjunction with genital infections. After incubation and washing, the number of bacteria that adhered per cell was determined. The influence on the attachment rate of such factors as variations in the washing procedure, bacterial density, and incubation time was assessed. Lactobacillus acidophilus and other bacterial species that occur in the lower genital tract of healthy women, e.g., some strictly anaerobic species, adhered by significantly lower numbers per cell than Neisseria gonorrhoeae, group B streptococci, and Corynebacterium vaginale. Significantly more freshly isolated gonococci adhered per cell than gonococci that had been passaged on artificial medium. The adherence of gonococci increased with increasing acidity of the test medium. | Adherence of bacterial to vaginal epithelial cells. Vaginal epithelial cells from healthy women were washed and incubated in tissue culture medium with freshly isolated bacteria of the indigenous vaginal flora and with bacteria of species that have been discussed in conjunction with genital infections. After incubation and washing, the number of bacteria that adhered per cell was determined. The influence on the attachment rate of such factors as variations in the washing procedure, bacterial density, and incubation time was assessed. Lactobacillus acidophilus and other bacterial species that occur in the lower genital tract of healthy women, e.g., some strictly anaerobic species, adhered by significantly lower numbers per cell than Neisseria gonorrhoeae, group B streptococci, and Corynebacterium vaginale. Significantly more freshly isolated gonococci adhered per cell than gonococci that had been passaged on artificial medium. The adherence of gonococci increased with increasing acidity of the test medium. | [
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PMID:5373 | Biological properties of an immunogenic pneumococcal subcellular preparation. | A subcellular fraction, designated PSP-3R, prepared from rough, type 3R Streptococcus pneumoniae is described, which affords excellent protection to mice against challenge with smooth organisms of the homologous serotype 3S and significant protection against heterologous challenge with serotypes 1S and 2S. Adjuvant enhances the protective capacity of the vaccine but is not necessary for immunogenicity. Protection induced by PSP-3R can be passively transferred tp normal mice with serum from actively immunized animals. The protective capacity can be completely absorbed out with rough or smooth type 3 organisms but not with rough type 1R or 2R cells. PSP-3R immune serum was tested in a passive hemagglutination assay against type 3 capsular polysaccharide-coated erythrocytes and found to have no detectable anticapsular antibody. The possible identity of the immunogen (s) in the vaccine is discussed. | Biological properties of an immunogenic pneumococcal subcellular preparation. A subcellular fraction, designated PSP-3R, prepared from rough, type 3R Streptococcus pneumoniae is described, which affords excellent protection to mice against challenge with smooth organisms of the homologous serotype 3S and significant protection against heterologous challenge with serotypes 1S and 2S. Adjuvant enhances the protective capacity of the vaccine but is not necessary for immunogenicity. Protection induced by PSP-3R can be passively transferred tp normal mice with serum from actively immunized animals. The protective capacity can be completely absorbed out with rough or smooth type 3 organisms but not with rough type 1R or 2R cells. PSP-3R immune serum was tested in a passive hemagglutination assay against type 3 capsular polysaccharide-coated erythrocytes and found to have no detectable anticapsular antibody. The possible identity of the immunogen (s) in the vaccine is discussed. | [
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PMID:5374 | Nonspecific bactericidal activity of the lactoperoxidases-thiocyanate-hydrogen peroxide system of milk against Escherichia coli and some gram-negative pathogens. | Two strains of Escherichia coli and one strain each of Salmonella typhimurium and Pseudomonas aeruginosa were killed by the bactericidal activity of the lactoperoxidase-thiocyanate-hydrogen peroxide system in milk and in a synthetic medium. H2O2 was supplied exogenously by glucose oxidase, and glucose was produced at a level which was itself noninhibitory. Two phases were distinguished: the first phase was dependent on the oxidation of SCN(-) by lactoperoxidase and H2O2, which was reversed by reducing agent, and the second phase was dependent on the presence of accumulated H2O2, which was reversed by catalase. The latter enzyme could also reverse the first phase, but only when present in excessive and unphysiological levels. The bactericidal activity was greatest at pH 5 and below, and it depended on the SCN(-)concentration and on the number of organisms. Since raw or heated milk neutralizes the acid barrier against infection in the stomach, the bactericidal system discussed may contribute to the prevention of enteric infections in neonates. | Nonspecific bactericidal activity of the lactoperoxidases-thiocyanate-hydrogen peroxide system of milk against Escherichia coli and some gram-negative pathogens. Two strains of Escherichia coli and one strain each of Salmonella typhimurium and Pseudomonas aeruginosa were killed by the bactericidal activity of the lactoperoxidase-thiocyanate-hydrogen peroxide system in milk and in a synthetic medium. H2O2 was supplied exogenously by glucose oxidase, and glucose was produced at a level which was itself noninhibitory. Two phases were distinguished: the first phase was dependent on the oxidation of SCN(-) by lactoperoxidase and H2O2, which was reversed by reducing agent, and the second phase was dependent on the presence of accumulated H2O2, which was reversed by catalase. The latter enzyme could also reverse the first phase, but only when present in excessive and unphysiological levels. The bactericidal activity was greatest at pH 5 and below, and it depended on the SCN(-)concentration and on the number of organisms. Since raw or heated milk neutralizes the acid barrier against infection in the stomach, the bactericidal system discussed may contribute to the prevention of enteric infections in neonates. | [
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PMID:5375 | Germination of Candida albicans induced by proline. | Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts. | Germination of Candida albicans induced by proline. Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts. | [
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PMID:5376 | Partial purification and properties of Enterobacter cloacae heat-stable enterotoxin. | Cell free preparations of the whole-cell lysate and ultrafiltration (UF) fractions of broth cultures of a strain of Enterobacter cloacae, isolated from a Puerto Rican with tropical sprue, were assayed for their ability to induce in vivo net water secretion in the rat jejunum. The whole-cell lysate and UM-10 retentate of broth cultures were inactive. The UM-2 retentate and filtrate were active at a concentration of 100 mug/ml or more; the toxigenic activity was entirely retained, and increased to 1 mug/ml, by a UM-05 membrane; washing this retentate yielded a fraction with an activity of 10 ng/ml. Stationary aerobic culture conditions yielded the most active UF fractions when ammonium sulfate was used as the precipitating agent, whereas anaerobic culture conditions produced the most active fractions in broth cultures precipitated by acetone. Passage of the active acetone-precipitated UF fractions through a Sephadex G-25 column yielded eluate pools with enhanced toxigenic activity in, or adjacent to, the void volume, but maximum activity of the ammonium sulfate-precipitated UM-05 retentate eluated at a Kav of 0.38 to 0.52. Neither of the most active gel filtration elution fractions of the UM-05 retentates contained detectable carbohydrate, suggesting that the toxin is not associated with endotoxin. Toxigenic activity was unaltered by exposure to a temperature of 100C for 30 min, lowering the pH to 1, or incubation with either Pronase or trypsin. These observations indicate that the strain of E. cloacae under study elaborates a heat-stable enterotoxin htat has approximately the same molecular weight and shares many of the characteristics of the heat-stable enterotoxin produced by some strains of Escherichia coli and Klebsiella pneumoniae. | Partial purification and properties of Enterobacter cloacae heat-stable enterotoxin. Cell free preparations of the whole-cell lysate and ultrafiltration (UF) fractions of broth cultures of a strain of Enterobacter cloacae, isolated from a Puerto Rican with tropical sprue, were assayed for their ability to induce in vivo net water secretion in the rat jejunum. The whole-cell lysate and UM-10 retentate of broth cultures were inactive. The UM-2 retentate and filtrate were active at a concentration of 100 mug/ml or more; the toxigenic activity was entirely retained, and increased to 1 mug/ml, by a UM-05 membrane; washing this retentate yielded a fraction with an activity of 10 ng/ml. Stationary aerobic culture conditions yielded the most active UF fractions when ammonium sulfate was used as the precipitating agent, whereas anaerobic culture conditions produced the most active fractions in broth cultures precipitated by acetone. Passage of the active acetone-precipitated UF fractions through a Sephadex G-25 column yielded eluate pools with enhanced toxigenic activity in, or adjacent to, the void volume, but maximum activity of the ammonium sulfate-precipitated UM-05 retentate eluated at a Kav of 0.38 to 0.52. Neither of the most active gel filtration elution fractions of the UM-05 retentates contained detectable carbohydrate, suggesting that the toxin is not associated with endotoxin. Toxigenic activity was unaltered by exposure to a temperature of 100C for 30 min, lowering the pH to 1, or incubation with either Pronase or trypsin. These observations indicate that the strain of E. cloacae under study elaborates a heat-stable enterotoxin htat has approximately the same molecular weight and shares many of the characteristics of the heat-stable enterotoxin produced by some strains of Escherichia coli and Klebsiella pneumoniae. | [
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PMID:5377 | Further studies of the physical and metabolic properties of foot-and-mouth disease virus temperature-sensitive mutants. | Three temperature-sensitive (ts) mutants of foot-and-mouth disease virus were classified as ribonucleic acid negative and as belonging to the same complementation group when measured by virus yields and [3H] uridine incorporation in paired, mixed infections at the nonpermissive temperature (38.5C). Mutant ts-22, the only mutant able to produce plaques at 38.5 C, was more sensitive to acid than were the parental wild-type or other mutant viruses. Diethylaminoethyl-dextran did not enhance the plaque-forming ability of the mutant viruses at 38.5C. All of the viruses inhibited host cell protein syntehsis at both permissive (33C) and nonpermissive (38.5C) temperatures. | Further studies of the physical and metabolic properties of foot-and-mouth disease virus temperature-sensitive mutants. Three temperature-sensitive (ts) mutants of foot-and-mouth disease virus were classified as ribonucleic acid negative and as belonging to the same complementation group when measured by virus yields and [3H] uridine incorporation in paired, mixed infections at the nonpermissive temperature (38.5C). Mutant ts-22, the only mutant able to produce plaques at 38.5 C, was more sensitive to acid than were the parental wild-type or other mutant viruses. Diethylaminoethyl-dextran did not enhance the plaque-forming ability of the mutant viruses at 38.5C. All of the viruses inhibited host cell protein syntehsis at both permissive (33C) and nonpermissive (38.5C) temperatures. | [
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PMID:5378 | Interaction of cholera toxin and toxin derivatives with lymphocytes. II. Modulating effects of cholera toxin on in vivo humoral and cellular immune responses. | The in vivo effects of cholera toxin on lymphoid organ structure and function in mice were investigated. It was found that within a day following intravenous injection of 1 mug of toxin, thymus as well as spleen weight decreased but the animals remained healthy. Histological studies suggested that the involution of lymphoid organs was due to cell death. Injection of cholera toxin into adrenalectomized mice was lethal within 36 h. In these animals no decrease in lymphoid organ weight was noted. Thymus cells from toxin-treated mice were found to be much inferior to thymocytes of untreated animals in their in vitro response to Concanavalin A, whereas the response of spleen cells from toxin-treated animals to mitogens was slightly increased. 1 mug of cholera toxin increased primary antibody formation when given to mice together with antigen (sheep erythrocytes) and decreased primary antibody formation when given before or after the antigen. The toxin also increased secondary antibody formation when injected simultaneously with or after the booster antigen dose, and decreased the antibody formation when given a few days before the booster injection. Treatment of mice with toxin was found to increase the capacity of spleen cells from these animals to induce the parental effect on antibody formation and to induce graft-versus-host reactions. The mechanisms behind the observed effects are discussed. It is suggested that cholera toxin affects different types of cells involved in immune responses primarily by a direct inhibitory action on cellular proliferation but also indirectly by causing release of adrenal gland hormones. | Interaction of cholera toxin and toxin derivatives with lymphocytes. II. Modulating effects of cholera toxin on in vivo humoral and cellular immune responses. The in vivo effects of cholera toxin on lymphoid organ structure and function in mice were investigated. It was found that within a day following intravenous injection of 1 mug of toxin, thymus as well as spleen weight decreased but the animals remained healthy. Histological studies suggested that the involution of lymphoid organs was due to cell death. Injection of cholera toxin into adrenalectomized mice was lethal within 36 h. In these animals no decrease in lymphoid organ weight was noted. Thymus cells from toxin-treated mice were found to be much inferior to thymocytes of untreated animals in their in vitro response to Concanavalin A, whereas the response of spleen cells from toxin-treated animals to mitogens was slightly increased. 1 mug of cholera toxin increased primary antibody formation when given to mice together with antigen (sheep erythrocytes) and decreased primary antibody formation when given before or after the antigen. The toxin also increased secondary antibody formation when injected simultaneously with or after the booster antigen dose, and decreased the antibody formation when given a few days before the booster injection. Treatment of mice with toxin was found to increase the capacity of spleen cells from these animals to induce the parental effect on antibody formation and to induce graft-versus-host reactions. The mechanisms behind the observed effects are discussed. It is suggested that cholera toxin affects different types of cells involved in immune responses primarily by a direct inhibitory action on cellular proliferation but also indirectly by causing release of adrenal gland hormones. | [
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PMID:5380 | Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation. | Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra. | Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation. Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra. | [
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PMID:5381 | Characterization of an alkaline subtilopeptidase type Pfizer. | The physiochemical properties, amino acid composition and profile of the the tryptic peptides for an alkaline subtilopeptidase type Pfizer have been determined. The enzyme is stable in the pH range from 5 to 10, has a pH optimum of 9.5 to 10, and is relatively stable for a period of 2 h up to a temperature of 50C. Homogeneity was demonstrated by electrophoretic techniques and the mobilities indicated on isoelectric point of 8.7. The molecular weight was found to be 25,000 by gel filtration. The amino acid composition was found to be Ala32, Arg4, Aspgamma8, Glu15, Gly29, His4, Ile9, Leu13, Lys11, Met5, Phe4, Pro14, Ser31, Thr17, Tyr9, Val22, a total of 247 amino acid residues. The enzyme does not contain either disulfide bonds or cysteine, and lacks tryptophan as well. The N-terminal end-group residue is alanine: the C-terminal amino acid is arginine. Tryptic hydrolysis of the enzyme produced 15 peptides which were separated by gradient elution on Dowex 50-X2. The amino acid composition of each appropriately purified tryptic peptide was established. | Characterization of an alkaline subtilopeptidase type Pfizer. The physiochemical properties, amino acid composition and profile of the the tryptic peptides for an alkaline subtilopeptidase type Pfizer have been determined. The enzyme is stable in the pH range from 5 to 10, has a pH optimum of 9.5 to 10, and is relatively stable for a period of 2 h up to a temperature of 50C. Homogeneity was demonstrated by electrophoretic techniques and the mobilities indicated on isoelectric point of 8.7. The molecular weight was found to be 25,000 by gel filtration. The amino acid composition was found to be Ala32, Arg4, Aspgamma8, Glu15, Gly29, His4, Ile9, Leu13, Lys11, Met5, Phe4, Pro14, Ser31, Thr17, Tyr9, Val22, a total of 247 amino acid residues. The enzyme does not contain either disulfide bonds or cysteine, and lacks tryptophan as well. The N-terminal end-group residue is alanine: the C-terminal amino acid is arginine. Tryptic hydrolysis of the enzyme produced 15 peptides which were separated by gradient elution on Dowex 50-X2. The amino acid composition of each appropriately purified tryptic peptide was established. | [
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PMID:5382 | Psychosomatic studies of psychiatric disorders: schizophrenia. | This paper uses some of the data on central nervous dysfunction in schizophrenic patients as a paradigm for the integration of biologic data with psychopathologic conditions. As such, a good portion of the paper emphasizes the methodologic and interpretive problems one must consider in "psychosomatic" studies of mental illness. A possible theory to integrate these findings is presented. | Psychosomatic studies of psychiatric disorders: schizophrenia. This paper uses some of the data on central nervous dysfunction in schizophrenic patients as a paradigm for the integration of biologic data with psychopathologic conditions. As such, a good portion of the paper emphasizes the methodologic and interpretive problems one must consider in "psychosomatic" studies of mental illness. A possible theory to integrate these findings is presented. | [
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PMID:5383 | Biofeedback and self-control of physiological functions: clinical applications. | The parameters amenable to biofeedback learning are mentioned, including brainwaves, muscle tension, temperature, the cardiovascular system, and others. A discussion follows of the clinical application of biofeedback in the treatment of such disorders as tension headaches, neuromuscular re-education, epilepsy, "dysponesis," cardiac arrhythmias, blood pressure and migraines. The usefulness of biofeedback has been demonstrated also in the field of psychotherapy for purposes of desensitization, treating anxious patients, encouraging specific personality changes, and indicating stress to patients. | Biofeedback and self-control of physiological functions: clinical applications. The parameters amenable to biofeedback learning are mentioned, including brainwaves, muscle tension, temperature, the cardiovascular system, and others. A discussion follows of the clinical application of biofeedback in the treatment of such disorders as tension headaches, neuromuscular re-education, epilepsy, "dysponesis," cardiac arrhythmias, blood pressure and migraines. The usefulness of biofeedback has been demonstrated also in the field of psychotherapy for purposes of desensitization, treating anxious patients, encouraging specific personality changes, and indicating stress to patients. | [
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PMID:5384 | Psychophysiology of pain. | The recent literature on pain states shows: pain thresholds are relatively constant for an individual, but pain tolerance is influenced by psychological state; the expression of pain is a function partly of ethnic membership and degree of extroversion; pain complaints are determined as well by cultural and extroversive factors, and also degree of neuroticism. Studies of pain patients reveals that those with acute pain tend to show normal personality profiles, but the degree of pain experienced is related to the degree of anxiety present. Most chronic pain patients, like those with psychogenic pain, show somatic preoccupations and reactive depression. The treatment and/or rehabilitation of pain patients has developed in three areas. In cases of peripheral neuropathy and some spinal cord lesions, electrical stimulation with "neural pacemakers" can often "close the gate" to pain signals and provide significant reduction or abolition of pain. Psychotropic medications, particularly the tricyclic antidepressants, sometimes in combination with phenothiazines and antihistamines, are effective in many instances of central pain, and help increase the pain tolerance and decrease the need for narcotics in other pain states. Operant conditioning, including the use of biofeedback, extinguishes pain behavior and increases pain-incompatible behaviors, with good long-term results. | Psychophysiology of pain. The recent literature on pain states shows: pain thresholds are relatively constant for an individual, but pain tolerance is influenced by psychological state; the expression of pain is a function partly of ethnic membership and degree of extroversion; pain complaints are determined as well by cultural and extroversive factors, and also degree of neuroticism. Studies of pain patients reveals that those with acute pain tend to show normal personality profiles, but the degree of pain experienced is related to the degree of anxiety present. Most chronic pain patients, like those with psychogenic pain, show somatic preoccupations and reactive depression. The treatment and/or rehabilitation of pain patients has developed in three areas. In cases of peripheral neuropathy and some spinal cord lesions, electrical stimulation with "neural pacemakers" can often "close the gate" to pain signals and provide significant reduction or abolition of pain. Psychotropic medications, particularly the tricyclic antidepressants, sometimes in combination with phenothiazines and antihistamines, are effective in many instances of central pain, and help increase the pain tolerance and decrease the need for narcotics in other pain states. Operant conditioning, including the use of biofeedback, extinguishes pain behavior and increases pain-incompatible behaviors, with good long-term results. | [
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PMID:5388 | [Foot tendon dislocations]. | Two luxations of the tendon of the Peroneus longus and one luxation of the Tibialis posterior muscle are reported. All the three cases had a traumatical cause and none of them showed symptoms of dysplasia. That is why a simple reconstruction of the retinaculum strengthened by a flap of the periost seemed to be justified. One year after the operation good results were verified by controls, therefore the method may be recommended for similar cases. | [Foot tendon dislocations]. Two luxations of the tendon of the Peroneus longus and one luxation of the Tibialis posterior muscle are reported. All the three cases had a traumatical cause and none of them showed symptoms of dysplasia. That is why a simple reconstruction of the retinaculum strengthened by a flap of the periost seemed to be justified. One year after the operation good results were verified by controls, therefore the method may be recommended for similar cases. | [
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] |
PMID:5389 | Simultaneous pH and fluoride telemetry. A supplemental report. | Simultaneous telemetry of salivary fluoride and of interproximal plaqua pH at the entrance to an interproximal space was used to investigate the effects of sucrose-containing fluoride tablets dissolving in the vicinity of the fluoride and pH sensors. The dissolving of 1.0 mg fluoride-42 mg sucrose tablets caused only slight pH depressions ranging between 6.7 and 6.0 with concurrent increases in salivary F to as high as 190 ppm. Acid formation in interproximal plaque by fluoride-free, sucrose-containing placebo tablets decreased pH to 4.2. | Simultaneous pH and fluoride telemetry. A supplemental report. Simultaneous telemetry of salivary fluoride and of interproximal plaqua pH at the entrance to an interproximal space was used to investigate the effects of sucrose-containing fluoride tablets dissolving in the vicinity of the fluoride and pH sensors. The dissolving of 1.0 mg fluoride-42 mg sucrose tablets caused only slight pH depressions ranging between 6.7 and 6.0 with concurrent increases in salivary F to as high as 190 ppm. Acid formation in interproximal plaque by fluoride-free, sucrose-containing placebo tablets decreased pH to 4.2. | [
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PMID:5385 | Isolations in a mosquito (Aedes pseudoscutellaris) cell line (Mos. 61) of yellow fever virus strains from original field material. | A simple, rapid and inexpensive method of isolating yellow fever (YF) virus from naturally infected mosquitoes, human liver and the serum of a sentinel monkey by inoculation of a continuous line of mosquito cells is described. The mosquito cells were more sensitive than suckling mice and marginally better than Vero cells for primary isolation. This is the first time that mosquito cells have been successfully used for primary isolation of YF virus from field material. | Isolations in a mosquito (Aedes pseudoscutellaris) cell line (Mos. 61) of yellow fever virus strains from original field material. A simple, rapid and inexpensive method of isolating yellow fever (YF) virus from naturally infected mosquitoes, human liver and the serum of a sentinel monkey by inoculation of a continuous line of mosquito cells is described. The mosquito cells were more sensitive than suckling mice and marginally better than Vero cells for primary isolation. This is the first time that mosquito cells have been successfully used for primary isolation of YF virus from field material. | [
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PMID:5390 | The effect of sorbose on pH of mixed saliva and interproximal plaque. | Stimulated mixed saliva and interproximal plaque were exposed to the ketohexose sorbose. The average pH of an in vitro 1%-sorbose/saliva mixture increased with time when compared with a highly significant pH-decrease of a sucrose/saliva mixture. In contrast to sucrose rinses, the telemetrically recorded pH of interproximal plaque did not drop below pH 5.5 during and subsequent to rinsing with sorbose solutions. | The effect of sorbose on pH of mixed saliva and interproximal plaque. Stimulated mixed saliva and interproximal plaque were exposed to the ketohexose sorbose. The average pH of an in vitro 1%-sorbose/saliva mixture increased with time when compared with a highly significant pH-decrease of a sucrose/saliva mixture. In contrast to sucrose rinses, the telemetrically recorded pH of interproximal plaque did not drop below pH 5.5 during and subsequent to rinsing with sorbose solutions. | [
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PMID:5395 | Comparison of antibiotic-amended potato dextrose agar and acidified potato dextrose agar as growth substrates for fungi. | Fifteen fungal species, all isolated from food, were compared for their growth abilities on potato dextrose agar acidified to pH 3.5, and on nonacidified potato dextrose agar amended with 40 ppm chlortetracycline hydrochloride. Comparisons were made at 16, 21, 26, 32, and 37 degrees C. Of the 15 species, only Penicillium expansum exhibited better growth on the acidified medium than on the nonacidified antibiotic medium, while 9 species grew better on the nonacidified antibiotic medium. Five species grew equally well on either medium. | Comparison of antibiotic-amended potato dextrose agar and acidified potato dextrose agar as growth substrates for fungi. Fifteen fungal species, all isolated from food, were compared for their growth abilities on potato dextrose agar acidified to pH 3.5, and on nonacidified potato dextrose agar amended with 40 ppm chlortetracycline hydrochloride. Comparisons were made at 16, 21, 26, 32, and 37 degrees C. Of the 15 species, only Penicillium expansum exhibited better growth on the acidified medium than on the nonacidified antibiotic medium, while 9 species grew better on the nonacidified antibiotic medium. Five species grew equally well on either medium. | [
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PMID:5397 | Luminescence and respiratory activities of Photobacterium phosphoreum. Competition for cellular reducing power. | Changes in the in vivo luminescence, respiratory activities, contents of cytochromes, extractable luciferase and NAD(P)H-FMN reductase during growth of the wild (bright) strain of Photobacterium phosphoreum and its dim mutant were determined. The intensity of the in vivo luminescence per cell increased 10 times in the wild strain and 750 times in the dim strain during logarithmic growth, while the contents of luciferase and NAD(P)H-FMN reductase remained almost constant. It is suggested that a characteristic change in the mode of competition of the luminescence reaction system with another electron transfer chain involving cytochromes for NAD(P)H take place during the growth of this bacterium. | Luminescence and respiratory activities of Photobacterium phosphoreum. Competition for cellular reducing power. Changes in the in vivo luminescence, respiratory activities, contents of cytochromes, extractable luciferase and NAD(P)H-FMN reductase during growth of the wild (bright) strain of Photobacterium phosphoreum and its dim mutant were determined. The intensity of the in vivo luminescence per cell increased 10 times in the wild strain and 750 times in the dim strain during logarithmic growth, while the contents of luciferase and NAD(P)H-FMN reductase remained almost constant. It is suggested that a characteristic change in the mode of competition of the luminescence reaction system with another electron transfer chain involving cytochromes for NAD(P)H take place during the growth of this bacterium. | [
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PMID:5398 | Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus. | Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly. | Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus. Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly. | [
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PMID:5399 | Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes. | After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats. | Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes. After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats. | [
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PMID:5400 | Purification and characterization of pyruvate decarboxylase from sweet potato roots. | Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting. | Purification and characterization of pyruvate decarboxylase from sweet potato roots. Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting. | [
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PMID:5401 | Quantitative determination of anomeric forms of sugar produced by amylases. V. Anomeric forms of maltose produced in the hydrolytic reaction of substituted phenyl alpha-maltosides catalyzed by saccharifying alpha-amylase from B. subtilis. | 1. Hydrolyses of phenyl alpha-maltoside and its derivatives with various substituents (p-NO2, p-C1, p-CH3, p-C2H5, and p-C(CH3)3) catalyzed by saccharifying alpha-amylase from B. subtilis3 [EC 3.2.1.1] were studied under conditions such that the products were only maltose and the corresponding phenols (1), in order to determine quantitatively the anomeric form of the sugar produced from each substrate. 2. At the optimum pH of this enzyme (pH-5.4), maltose released from all the substituted substrates studied was entirely in the beta-form. These results are in remarkable contrast to the previous finding that alpha-maltose is exclusively produced from unsubstituted phenyl alpha-maltoside by this enzyme (2). 3. At pH 6.18 and 6.73, maltose produced from unsubstituted phenyl alpha-maltoside (øM) or p-tert-butylphenyl alpha-maltoside (PTBøM) was a mixture of alpha- and beta-anomers, the ratio being dependent on pH as follows: For øM, the percentage of alpha-anomer was 100% (pH 5.4), 80 (pH 6.18), and 55% (pH 6.73), whereas for PTBøM, the percentage of beta-anomer was 100% (pH 5.4), 75% (pH 6.18), and 60% (pH 6.73). | Quantitative determination of anomeric forms of sugar produced by amylases. V. Anomeric forms of maltose produced in the hydrolytic reaction of substituted phenyl alpha-maltosides catalyzed by saccharifying alpha-amylase from B. subtilis. 1. Hydrolyses of phenyl alpha-maltoside and its derivatives with various substituents (p-NO2, p-C1, p-CH3, p-C2H5, and p-C(CH3)3) catalyzed by saccharifying alpha-amylase from B. subtilis3 [EC 3.2.1.1] were studied under conditions such that the products were only maltose and the corresponding phenols (1), in order to determine quantitatively the anomeric form of the sugar produced from each substrate. 2. At the optimum pH of this enzyme (pH-5.4), maltose released from all the substituted substrates studied was entirely in the beta-form. These results are in remarkable contrast to the previous finding that alpha-maltose is exclusively produced from unsubstituted phenyl alpha-maltoside by this enzyme (2). 3. At pH 6.18 and 6.73, maltose produced from unsubstituted phenyl alpha-maltoside (øM) or p-tert-butylphenyl alpha-maltoside (PTBøM) was a mixture of alpha- and beta-anomers, the ratio being dependent on pH as follows: For øM, the percentage of alpha-anomer was 100% (pH 5.4), 80 (pH 6.18), and 55% (pH 6.73), whereas for PTBøM, the percentage of beta-anomer was 100% (pH 5.4), 75% (pH 6.18), and 60% (pH 6.73). | [
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PMID:5402 | Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action. | 1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups. | Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action. 1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups. | [
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PMID:5403 | Substrate specificity of carboxypeptidase from Watermelon. | The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0. | Substrate specificity of carboxypeptidase from Watermelon. The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0. | [
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PMID:5404 | Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue. | The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified. | Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue. The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified. | [
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PMID:5405 | Inhibition of polypeptide synthesis by tRNA fractions in rat liver cell-free systems. | The mechanism of inhibition of polypeptide synthesis by the addition of a tRNA fraction in a rat liver cell-free system was studied. The inhibition was found to occur at the step of aminoacyl-tRNA binding to ribosomes, in which aminoacyl-tRNA's were mainly responsible for the inhibition. The addition of EF-1 decreased the inhibition by the tRNA fraction. The tRNA fraction inhibited polypeptide synthesis in a polysome-S100 system under conditions in which poly U- and poly A-dependent polypeptide syntheses were not inhibited. The possibility that the aminoacyl-tRNA inhibitory activity functions through improper binding to the ribosomes in the polysome-S100 system is discussed. | Inhibition of polypeptide synthesis by tRNA fractions in rat liver cell-free systems. The mechanism of inhibition of polypeptide synthesis by the addition of a tRNA fraction in a rat liver cell-free system was studied. The inhibition was found to occur at the step of aminoacyl-tRNA binding to ribosomes, in which aminoacyl-tRNA's were mainly responsible for the inhibition. The addition of EF-1 decreased the inhibition by the tRNA fraction. The tRNA fraction inhibited polypeptide synthesis in a polysome-S100 system under conditions in which poly U- and poly A-dependent polypeptide syntheses were not inhibited. The possibility that the aminoacyl-tRNA inhibitory activity functions through improper binding to the ribosomes in the polysome-S100 system is discussed. | [
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PMID:5406 | alpha-Chymotryptic hydrolysis of derivatives of the specific substrates with substituents in the nucleus. | Steady state kinetic studies of alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of nucleus-substituted derivatives of the specific substrates were made at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily than Ac-Trp-OMe owing to its smaller Km value. The kcat values of Ac-Trp(CHO)-OMe and Ac-Tyr(3-no2)-ome were higher than those of the corresponding unmodified substrates, suggesting that derivatives with a substituent as large as a formyl or nitro group at the epsilon-position are stereochemically favorable to the catalytic process. Derivatives of Ac-Phe-OMe with a chain of four atoms at the 3 or 4-position of the phenyl nucleus and 2,3-dihydropyrrolo[2,3-b]indoles derived from Ac-Trp-OMe were not hydrolyzed at all. | alpha-Chymotryptic hydrolysis of derivatives of the specific substrates with substituents in the nucleus. Steady state kinetic studies of alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of nucleus-substituted derivatives of the specific substrates were made at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily than Ac-Trp-OMe owing to its smaller Km value. The kcat values of Ac-Trp(CHO)-OMe and Ac-Tyr(3-no2)-ome were higher than those of the corresponding unmodified substrates, suggesting that derivatives with a substituent as large as a formyl or nitro group at the epsilon-position are stereochemically favorable to the catalytic process. Derivatives of Ac-Phe-OMe with a chain of four atoms at the 3 or 4-position of the phenyl nucleus and 2,3-dihydropyrrolo[2,3-b]indoles derived from Ac-Trp-OMe were not hydrolyzed at all. | [
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PMID:5407 | Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions. | The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5,5'-dimethyloxazoldine,-2,4-dione (DMO) method. The initial pHi at 4degrees was about 7.6 and was higher than the extracellular pH (pHe) at 4degrees. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4degrees) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2,3-diphosphoglycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4degrees were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood. | Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions. The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5,5'-dimethyloxazoldine,-2,4-dione (DMO) method. The initial pHi at 4degrees was about 7.6 and was higher than the extracellular pH (pHe) at 4degrees. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4degrees) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2,3-diphosphoglycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4degrees were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood. | [
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PMID:5408 | Purification and properties of two ribonucleases in different intracellular compartments in pea root tissue. | Two RNases in bound forms associated with the microsomal membrane and with the ribosomes or unknown particles in pea root tissue were solubilized by subjecting the membrane to sonic oscillation in the presence of EDTA and KC1 and by treating the particles with EDTA, respectively. The RNases were than purified by DEAE-cellulose and Sephadex G-75 column chromatographies. The elution profiles of RNases from the columns were very similar. No significant differences were observed in their electrophoretic mobilities in polyacrylamide gels, in molecular weight, in activation by inorganic ions, urea or phospholipid micelles or in the dependence of their activities upon pH. The purified RNASES were not different from the bound enzymes as regards activation by inorganic ions and urea and the dependence of the activity upon pH. Triton X-100 stimulated the activity only if RNase was in a bound form associated with the microsomal membrane. We propose that the two RNases may be the same molecular species and differ only in the form of association with intracellular structures. | Purification and properties of two ribonucleases in different intracellular compartments in pea root tissue. Two RNases in bound forms associated with the microsomal membrane and with the ribosomes or unknown particles in pea root tissue were solubilized by subjecting the membrane to sonic oscillation in the presence of EDTA and KC1 and by treating the particles with EDTA, respectively. The RNases were than purified by DEAE-cellulose and Sephadex G-75 column chromatographies. The elution profiles of RNases from the columns were very similar. No significant differences were observed in their electrophoretic mobilities in polyacrylamide gels, in molecular weight, in activation by inorganic ions, urea or phospholipid micelles or in the dependence of their activities upon pH. The purified RNASES were not different from the bound enzymes as regards activation by inorganic ions and urea and the dependence of the activity upon pH. Triton X-100 stimulated the activity only if RNase was in a bound form associated with the microsomal membrane. We propose that the two RNases may be the same molecular species and differ only in the form of association with intracellular structures. | [
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PMID:5409 | Purification and properties of an exo-cellulase component of novel type from Trichoderma miride. | An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure. | Purification and properties of an exo-cellulase component of novel type from Trichoderma miride. An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure. | [
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PMID:5410 | Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver. | D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits. | Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver. D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits. | [
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PMID:5411 | Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C. | 1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid. | Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C. 1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid. | [
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PMID:5412 | Thermal denaturation and regeneration of japanese-radish peroxidase. | Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin. | Thermal denaturation and regeneration of japanese-radish peroxidase. Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin. | [
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