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PMID:3162 | Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight. | The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme. | Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight. The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme. | [
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PMID:3163 | Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland. | 1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate. | Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland. 1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate. | [
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PMID:3164 | Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase. | 1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C10 chain but is relatively moderate compared with C10 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPAD-Sepharose. 7. These results are discussed in terms of a 'repulsion-controlled' model or hydrophobic chromatography. | Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase. 1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C10 chain but is relatively moderate compared with C10 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPAD-Sepharose. 7. These results are discussed in terms of a 'repulsion-controlled' model or hydrophobic chromatography. | [
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PMID:3165 | The effect of acid proteinase inhibitors on chicken pepsin. | 1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases. | The effect of acid proteinase inhibitors on chicken pepsin. 1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases. | [
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PMID:3166 | The purification and properties of pig spleen phosphofructokinase. | Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors. | The purification and properties of pig spleen phosphofructokinase. Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors. | [
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PMID:3167 | The effect of disulfiram on the aldehyde dehydrogenases of sheep liver. | 1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram. | The effect of disulfiram on the aldehyde dehydrogenases of sheep liver. 1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram. | [
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PMID:3168 | A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. | 1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media. | A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. 1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media. | [
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PMID:3169 | Differential effects of temperature on a membrane adenosine triphosphatase and associated phosphatase. | Arrhenius plots of a membrane (Na+ + K+)-dependent ATPase (adenosine triphosphatase) activity showed characteristic discontinuities, whereas those of the associated K+-dependent phosphatase activity did not. These findings support the contention that the phosphatase activity does not depend on phospholipid in the same way as does the ATPase activity. | Differential effects of temperature on a membrane adenosine triphosphatase and associated phosphatase. Arrhenius plots of a membrane (Na+ + K+)-dependent ATPase (adenosine triphosphatase) activity showed characteristic discontinuities, whereas those of the associated K+-dependent phosphatase activity did not. These findings support the contention that the phosphatase activity does not depend on phospholipid in the same way as does the ATPase activity. | [
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PMID:3170 | The action of chelating agents on human liver aldehyde dehydrogenase. | Human liver aldehyde dehydrogenase was inhibited by aromatic chelating agents. However, structurally related compounds with much lower metal-complexing ability displayed affinities for enzyme essentially equal to those of their respective chelating analogues. Inhibition was competitive with respect to the coenzyme. It is suggested that hydrophobic interactions between the inhibitors and the coenzyme-binding site of the enzyme are responsible for the observed effects on activity. | The action of chelating agents on human liver aldehyde dehydrogenase. Human liver aldehyde dehydrogenase was inhibited by aromatic chelating agents. However, structurally related compounds with much lower metal-complexing ability displayed affinities for enzyme essentially equal to those of their respective chelating analogues. Inhibition was competitive with respect to the coenzyme. It is suggested that hydrophobic interactions between the inhibitors and the coenzyme-binding site of the enzyme are responsible for the observed effects on activity. | [
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PMID:3171 | An improved assay for bacterial methane mono-oxygenase: some properties of the enzyme from Methylomonas methanica. | Extracts of Methylomonas methanica catalyse the O2-and NAD(P)H-dependent disappearance of bromomethane. The activity is unstable at 2 degrees C but is stable at --70 degrees C for several weeks. Bromomethane mono-oxygenase is particulate and is inhibited by metal-binding reagents, by compounds SKF 525A and Lilly 53325, by some metal ions and by acetylene. Evidence is presented that indicates that bromomethane mono-oxygenase is the enzyme responsible for methane oxidation in vivo. | An improved assay for bacterial methane mono-oxygenase: some properties of the enzyme from Methylomonas methanica. Extracts of Methylomonas methanica catalyse the O2-and NAD(P)H-dependent disappearance of bromomethane. The activity is unstable at 2 degrees C but is stable at --70 degrees C for several weeks. Bromomethane mono-oxygenase is particulate and is inhibited by metal-binding reagents, by compounds SKF 525A and Lilly 53325, by some metal ions and by acetylene. Evidence is presented that indicates that bromomethane mono-oxygenase is the enzyme responsible for methane oxidation in vivo. | [
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PMID:3172 | Iron oxidation and transferrin formation by phosvitin. | The catalytic activity of phosvitin in Fe(II) oxidation and the addition of iron to transferrin were studied under various conditions. It was concluded that the Fe(II) oxidized by phosvitin would bind to apotransferrin, although an appreciable fraction of Fe(III) remained bound to phosvitin. Fe(III) also migrated from phosvitin to apotransferrin. This reaction was first-order with respect to Fe(III)-phosvitin concentration with a half-time (t1/2) of 10 min, and a first-order rate constant, k=0.069min-1, in 700 muM-phosphate buffer, pH 7.2, at 30 degrees C. The catalysis of the oxidation of Fe(III) by phosvitin was proportional to O2 concentration, and is quite different from the relative O2 independence of Fe(II) oxidation as catalysed by ferroxidase. A scheme for the mobilization and transfer of iron in the chicken, including the role of ferroxidase, phosyitin and transferrin, is presented. | Iron oxidation and transferrin formation by phosvitin. The catalytic activity of phosvitin in Fe(II) oxidation and the addition of iron to transferrin were studied under various conditions. It was concluded that the Fe(II) oxidized by phosvitin would bind to apotransferrin, although an appreciable fraction of Fe(III) remained bound to phosvitin. Fe(III) also migrated from phosvitin to apotransferrin. This reaction was first-order with respect to Fe(III)-phosvitin concentration with a half-time (t1/2) of 10 min, and a first-order rate constant, k=0.069min-1, in 700 muM-phosphate buffer, pH 7.2, at 30 degrees C. The catalysis of the oxidation of Fe(III) by phosvitin was proportional to O2 concentration, and is quite different from the relative O2 independence of Fe(II) oxidation as catalysed by ferroxidase. A scheme for the mobilization and transfer of iron in the chicken, including the role of ferroxidase, phosyitin and transferrin, is presented. | [
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PMID:3173 | The substrate specificity of thermomycolase, an extracellular serine proteinase from the thermophilic fungus Malbranchea pulchella var. sulfurea. | The specificity of thermomycolase toward glucagon and the oxidized A and B chains of insulin was investigated. Extensive digestion of glucagon occurred when conducted at pH 7.0 and 45 degrees C for 40 min, whereas hydrolysis of only three peptide bonds occurred at pH 7.0 and 28 degrees C for 5 min. A similar situation was observed for the oxidized B chain of insulin, which exhibited only a single major cleavage after 5 min at 25 degrees C. No well-defined specificity for particular amino acid residues was evident, but ready hydrolysis of peptide bonds occurred within sequences containing non-polar residues. This endoproteinase must therefore possess an extended hydrophobic binding site for polypeptides. Thermomycolase hydrolysed acetylalanylalanylalanine methyl ester and elastin-Congo Red at 22 and 8.5 times the rate of porcine elastase respectively. A limited degradation of native collagen and significant hydrolysis of benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro were suggestive of some collagenase-like activity. No keratinase activity was apparent. | The substrate specificity of thermomycolase, an extracellular serine proteinase from the thermophilic fungus Malbranchea pulchella var. sulfurea. The specificity of thermomycolase toward glucagon and the oxidized A and B chains of insulin was investigated. Extensive digestion of glucagon occurred when conducted at pH 7.0 and 45 degrees C for 40 min, whereas hydrolysis of only three peptide bonds occurred at pH 7.0 and 28 degrees C for 5 min. A similar situation was observed for the oxidized B chain of insulin, which exhibited only a single major cleavage after 5 min at 25 degrees C. No well-defined specificity for particular amino acid residues was evident, but ready hydrolysis of peptide bonds occurred within sequences containing non-polar residues. This endoproteinase must therefore possess an extended hydrophobic binding site for polypeptides. Thermomycolase hydrolysed acetylalanylalanylalanine methyl ester and elastin-Congo Red at 22 and 8.5 times the rate of porcine elastase respectively. A limited degradation of native collagen and significant hydrolysis of benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro were suggestive of some collagenase-like activity. No keratinase activity was apparent. | [
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PMID:3174 | Stimulation of rat liver beta-galactosidase activity by ions. | 1. The p-nitrophenyl beta-D-galactosidase asctivity in rat liver homogenates of lysosome-rich fractions was shown to be markedly affected by the ionic composition of the medium. A stimulation of the reaction rate at pH 5 was produced by most of the salts tested, which contained anions such as acetate, SO4(2-) and Cl-, and cations such as Na+, K= and Mg2+. The most pronounced effect was observed with MgCl2. Only potassium glutamate was inhibitory. 2. Five peaks of beta-galactosidase activity obtained by DEAE-cellulose chromatography were equally sensitive to changes in the ionic composition of the medium. In the presence of added NaC1, the whole rate-pH curve was displaced towards higher pH values, the optimum being shifted from 2.0-2.5 to 3.5. The stimulation at pH 5.0 appeared to be mainly due to changes in Vmax., whereas the apparent Km was slightly modified. 3. Unlike the total, the free beta-galactosidase activity remained unchanged or even declined when KC1 was added to the reaction medium. | Stimulation of rat liver beta-galactosidase activity by ions. 1. The p-nitrophenyl beta-D-galactosidase asctivity in rat liver homogenates of lysosome-rich fractions was shown to be markedly affected by the ionic composition of the medium. A stimulation of the reaction rate at pH 5 was produced by most of the salts tested, which contained anions such as acetate, SO4(2-) and Cl-, and cations such as Na+, K= and Mg2+. The most pronounced effect was observed with MgCl2. Only potassium glutamate was inhibitory. 2. Five peaks of beta-galactosidase activity obtained by DEAE-cellulose chromatography were equally sensitive to changes in the ionic composition of the medium. In the presence of added NaC1, the whole rate-pH curve was displaced towards higher pH values, the optimum being shifted from 2.0-2.5 to 3.5. The stimulation at pH 5.0 appeared to be mainly due to changes in Vmax., whereas the apparent Km was slightly modified. 3. Unlike the total, the free beta-galactosidase activity remained unchanged or even declined when KC1 was added to the reaction medium. | [
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PMID:3175 | Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes. | 1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results. | Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes. 1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results. | [
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PMID:3183 | [Analytical profile of purified hexetidine (author's transl)]. | Physico-chemical, spectroscopic (UV, IR, NMR, mass), chromatographic (GLC, TLC) properties and synthesis of 1,3-bis(2-ethylhexyl)-5-amino-5-methyl-hexahydropyrimidine (hexetidine) are reported and discussed. Moreover, the difference between commercial and purified hexetidine is demonstrated. | [Analytical profile of purified hexetidine (author's transl)]. Physico-chemical, spectroscopic (UV, IR, NMR, mass), chromatographic (GLC, TLC) properties and synthesis of 1,3-bis(2-ethylhexyl)-5-amino-5-methyl-hexahydropyrimidine (hexetidine) are reported and discussed. Moreover, the difference between commercial and purified hexetidine is demonstrated. | [
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PMID:3184 | [Ditertiary diamine compounds (author's transl)]. | The preparation of ditertiary aliphatic diamines 3 designed as drugs protecting CNS acetylcholinesterase against organophosphate inhibition, is described. Owing to the radicals at the basic nitrogen atoms, these compounds should exist in appreciable amounts both as base and as diammonium ion at biological pH's. | [Ditertiary diamine compounds (author's transl)]. The preparation of ditertiary aliphatic diamines 3 designed as drugs protecting CNS acetylcholinesterase against organophosphate inhibition, is described. Owing to the radicals at the basic nitrogen atoms, these compounds should exist in appreciable amounts both as base and as diammonium ion at biological pH's. | [
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PMID:3185 | [Intracellular pH of cardiac muscle after administration of potassium-magnesium-aspartate (author's transl)]. | 27 Sprague-Dawley rats were anesthetized with pentobarbital. Artificial ventilation was given by a Starling pump respirator via a tracheal tube. Intracellular pH of cardiac muscle was determined by means of the indirect procedure of measuring the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) in intra- and extracellular spaces (DMO-method). 12 rats were injected i.p. with 0.33 mval potassium-magnesium-aspartate/100 g body weight and 15 rats served as controls. Using a regression analysis, the following relationships were obtained: 1. animals injected with potassium-magnesium-aspartate pHa = --0.75 log PaCO2 + 8.535, PHi = --0.30 log PaCO2 + 7.509, pHi = 0.41 pHa + 4.036; 2. control animals pHa = --0.59 log PaCO2 + 8.308, pHi = --0.27 log PaCO2 + 7.381, pHi = 0.47 pHa + 3.503. At a pCO2 of 40 torr a pHa of 7.33 (7.36) and a pHi of 7.03 (6.95) was obtained. At an arterial pH of 7.40 the pHi was 7.07 (6.98). The results of the control group are written in brackets. -- The experiments demonstrate an increase in the intracellular buffer bases after administration of potassium-magnesium-aspartate. This effect can be recognized by a concomitant increase in the intracellular pH of 0.09 compared to the control group. | [Intracellular pH of cardiac muscle after administration of potassium-magnesium-aspartate (author's transl)]. 27 Sprague-Dawley rats were anesthetized with pentobarbital. Artificial ventilation was given by a Starling pump respirator via a tracheal tube. Intracellular pH of cardiac muscle was determined by means of the indirect procedure of measuring the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) in intra- and extracellular spaces (DMO-method). 12 rats were injected i.p. with 0.33 mval potassium-magnesium-aspartate/100 g body weight and 15 rats served as controls. Using a regression analysis, the following relationships were obtained: 1. animals injected with potassium-magnesium-aspartate pHa = --0.75 log PaCO2 + 8.535, PHi = --0.30 log PaCO2 + 7.509, pHi = 0.41 pHa + 4.036; 2. control animals pHa = --0.59 log PaCO2 + 8.308, pHi = --0.27 log PaCO2 + 7.381, pHi = 0.47 pHa + 3.503. At a pCO2 of 40 torr a pHa of 7.33 (7.36) and a pHi of 7.03 (6.95) was obtained. At an arterial pH of 7.40 the pHi was 7.07 (6.98). The results of the control group are written in brackets. -- The experiments demonstrate an increase in the intracellular buffer bases after administration of potassium-magnesium-aspartate. This effect can be recognized by a concomitant increase in the intracellular pH of 0.09 compared to the control group. | [
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PMID:3186 | Pharmacological properties of 2-(2-chloro-p-toluidino)-2-imidazoline-nitrate (tolonidine), a new antihypertensive agent. III. Action on the secretions of the digestive tract and on the central nervous system, acute toxicity. | The pharmacological properties of 2-(2-chloro-p-toluidino)-2-imidazoline-nitrate (tolonidine) a new synthetic derivative of imidazoline are reported in a series of three successive articles. This compound has been shown to possess hypotensive and antihypertensive properties. After i.v. administration, the hypotensive phase was preceded by hypertension related to the potent direct alpha-sympatheticomimetic properties of the product. This pressor response, which was not seen after oral administration, was accompanied by a marked decrease in cardiac output and a significant increase in peripheral vascular resistance. The hypotensive action of the product was due to a drop in cardiac output probably reinforced by a decrease in vasoconstrictor sympathetic tone due to a central action. Whatever the route of administration, tolonidine slowed heart rate independently of blood pressure variations, due essentially to an increase in vagal tone. In studies of diuresis, liquid and salt loss were observed in the cat, not in the dog. At doses which induce a drop in blood pressure tolonidine did not produce a reduction in pilocarpine-induced salivary secretion and only partially inhibited gastric secretion. In the central nervous system, tolonidine produced a sedation which first appeared at doses having an antihypertensive effect but which was only fully apparent with increased doses. A decrease in the release of cerebral amines, serotonin and noradrenaline by tolonidine is proposed. Tolonidine was compared with three other antihypertensive agents: clonidine, which is structurally related, and guanethidine and mecamylamine, which are structurally unrelated and have a different mode of action. A close resemblance of the pharmacological properties of tolonidine and clonidine was established due to the chemical relationship between the two substances. | Pharmacological properties of 2-(2-chloro-p-toluidino)-2-imidazoline-nitrate (tolonidine), a new antihypertensive agent. III. Action on the secretions of the digestive tract and on the central nervous system, acute toxicity. The pharmacological properties of 2-(2-chloro-p-toluidino)-2-imidazoline-nitrate (tolonidine) a new synthetic derivative of imidazoline are reported in a series of three successive articles. This compound has been shown to possess hypotensive and antihypertensive properties. After i.v. administration, the hypotensive phase was preceded by hypertension related to the potent direct alpha-sympatheticomimetic properties of the product. This pressor response, which was not seen after oral administration, was accompanied by a marked decrease in cardiac output and a significant increase in peripheral vascular resistance. The hypotensive action of the product was due to a drop in cardiac output probably reinforced by a decrease in vasoconstrictor sympathetic tone due to a central action. Whatever the route of administration, tolonidine slowed heart rate independently of blood pressure variations, due essentially to an increase in vagal tone. In studies of diuresis, liquid and salt loss were observed in the cat, not in the dog. At doses which induce a drop in blood pressure tolonidine did not produce a reduction in pilocarpine-induced salivary secretion and only partially inhibited gastric secretion. In the central nervous system, tolonidine produced a sedation which first appeared at doses having an antihypertensive effect but which was only fully apparent with increased doses. A decrease in the release of cerebral amines, serotonin and noradrenaline by tolonidine is proposed. Tolonidine was compared with three other antihypertensive agents: clonidine, which is structurally related, and guanethidine and mecamylamine, which are structurally unrelated and have a different mode of action. A close resemblance of the pharmacological properties of tolonidine and clonidine was established due to the chemical relationship between the two substances. | [
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PMID:3187 | [On the influence of a special preparation of oxytetracycline and sodiumbituminosulfonates on amount and composition of skin surface lipids in acne vulgaris (author's transl)]. | Two groups of 27 and 23 patients with acne vulgaris were first treated for a period of one week with 1 g oxytetracycline a day p.o. In a second treatment period of 6 weeks the first group received 100 mg oxytetracycline a day p.o. and the second group a combination of 100 mg oxytetracycline and 1.2 g sodiumbituminosulfonates a day p.o. In the third treatment period, similarly continued for 6 weeks, the method was reversed. Gastric juice-insoluble preparations were used for the investigation. All criteria for a double-blind study were considered. Amount and composition of the skin surface lipids were analysed before beginning the treatment, at the end of the 2nd and at the end of the 3rd treatment period. The combination of both agents in gastric juice-insoluble preparations suppresses to a great extent the known effects brought about by the substances separately, namely the reduction in free fatty acids and the decrease in the skin surface lipids. The findings also show that the reduction of the free fatty acids was in a limited time observed only in patients treated with 100 mg oxytetracycline a day p.o. if they had been treated in the beginning of this therapy with a higher dosage of tetracycline. | [On the influence of a special preparation of oxytetracycline and sodiumbituminosulfonates on amount and composition of skin surface lipids in acne vulgaris (author's transl)]. Two groups of 27 and 23 patients with acne vulgaris were first treated for a period of one week with 1 g oxytetracycline a day p.o. In a second treatment period of 6 weeks the first group received 100 mg oxytetracycline a day p.o. and the second group a combination of 100 mg oxytetracycline and 1.2 g sodiumbituminosulfonates a day p.o. In the third treatment period, similarly continued for 6 weeks, the method was reversed. Gastric juice-insoluble preparations were used for the investigation. All criteria for a double-blind study were considered. Amount and composition of the skin surface lipids were analysed before beginning the treatment, at the end of the 2nd and at the end of the 3rd treatment period. The combination of both agents in gastric juice-insoluble preparations suppresses to a great extent the known effects brought about by the substances separately, namely the reduction in free fatty acids and the decrease in the skin surface lipids. The findings also show that the reduction of the free fatty acids was in a limited time observed only in patients treated with 100 mg oxytetracycline a day p.o. if they had been treated in the beginning of this therapy with a higher dosage of tetracycline. | [
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PMID:3188 | [Influence of ethanol on the in vitro and in vivo drug release from some sustained release tablets (author's transl)]. | The in vitro and in vivo liberation of acetylsalicylic acid from sustained release tablets in presence of ethanol is described. Simultaneous uptake of 120 ml commercial brandy resulted in a faster release of the active substance from the tablets prepared with Eudragit ret-l (PM), as has been proved by urinary excretion data. These results were supported by experiments with a pH-endoaradio transmitter and by radiography. | [Influence of ethanol on the in vitro and in vivo drug release from some sustained release tablets (author's transl)]. The in vitro and in vivo liberation of acetylsalicylic acid from sustained release tablets in presence of ethanol is described. Simultaneous uptake of 120 ml commercial brandy resulted in a faster release of the active substance from the tablets prepared with Eudragit ret-l (PM), as has been proved by urinary excretion data. These results were supported by experiments with a pH-endoaradio transmitter and by radiography. | [
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PMID:3192 | Changes in liver function after different types of surgery. | Liver function tests carried out after minor surgical procedures, under anaesthesia lasting for 1 hr, showed no abnormalities. Tests after body surface operations under the same anaesthetic techniques showed transient derangements. After intra-abdominal procedures, liver dysfunction was more marked, although no patients with evidence of preoperative liver dysfunction or postoperative surgical complications were studied and none received blood transfusions. Measurements of the serum bilirubin concentration showed the most frequent abnormalities, but the pseudocholinesterase concentration decreased progressively after intra-abdominal surgery and b.s.p. retention increased significantly. Serum concentration of intracellular enzymes (LDH, s.g.o.t. and s.g.p.t.) increased within an hour of starting surgery, changes which were probably not related to liver function. | Changes in liver function after different types of surgery. Liver function tests carried out after minor surgical procedures, under anaesthesia lasting for 1 hr, showed no abnormalities. Tests after body surface operations under the same anaesthetic techniques showed transient derangements. After intra-abdominal procedures, liver dysfunction was more marked, although no patients with evidence of preoperative liver dysfunction or postoperative surgical complications were studied and none received blood transfusions. Measurements of the serum bilirubin concentration showed the most frequent abnormalities, but the pseudocholinesterase concentration decreased progressively after intra-abdominal surgery and b.s.p. retention increased significantly. Serum concentration of intracellular enzymes (LDH, s.g.o.t. and s.g.p.t.) increased within an hour of starting surgery, changes which were probably not related to liver function. | [
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PMID:3193 | Studies on the energy metabolism in lichen planus. | Various epidermal enzymes and cofactors were measured in patients with lichen planus and in healthy controls with the aid of Lowry's microtechniques, including enzymatic cycling. The steady-state levels of the nicotinamide adenine dinucleotides NAD and NADP were decreased and this was evident even in areas still free from lesions. The oxidized and reduced portions of NAD were altered indicating changed equilibria of NAD dependent dehydrogenases. Reduced NADP was more tightly controlled at the normal level which is regarded as evidence of an unaltered biosynthetic potential in this disease. In conjunction with earlier data the results indicate a preserved glycolytic and pentose shunt activity while the mitochondria display signs of dysfunction. | Studies on the energy metabolism in lichen planus. Various epidermal enzymes and cofactors were measured in patients with lichen planus and in healthy controls with the aid of Lowry's microtechniques, including enzymatic cycling. The steady-state levels of the nicotinamide adenine dinucleotides NAD and NADP were decreased and this was evident even in areas still free from lesions. The oxidized and reduced portions of NAD were altered indicating changed equilibria of NAD dependent dehydrogenases. Reduced NADP was more tightly controlled at the normal level which is regarded as evidence of an unaltered biosynthetic potential in this disease. In conjunction with earlier data the results indicate a preserved glycolytic and pentose shunt activity while the mitochondria display signs of dysfunction. | [
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PMID:3194 | The lichen planus-like eruption after bone marrow transplantation. | A lichen planus-like eruption was seen in four patients after bone marrow transplantation. The skin and mucous membrane appearance closely mimicked lichen planus. The histopathology was also very similar to lichen planus. The occurrence of a lichen planus-like eruption (LPLE) after an immune basal cell damage related to the graft-versus-host reaction raised the question of the immune nature of this eruption. The correlation found between biological signs of graft-versus-host reaction and the out-break or relapse of the lichen planus-like eruption supports the hypothesis that the skin changes could be a sign of a chronic immune response against recipient epidermis. | The lichen planus-like eruption after bone marrow transplantation. A lichen planus-like eruption was seen in four patients after bone marrow transplantation. The skin and mucous membrane appearance closely mimicked lichen planus. The histopathology was also very similar to lichen planus. The occurrence of a lichen planus-like eruption (LPLE) after an immune basal cell damage related to the graft-versus-host reaction raised the question of the immune nature of this eruption. The correlation found between biological signs of graft-versus-host reaction and the out-break or relapse of the lichen planus-like eruption supports the hypothesis that the skin changes could be a sign of a chronic immune response against recipient epidermis. | [
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PMID:3195 | The effect of pH upon human transferrin: selective labelling of the two iron-binding sites. | The influence of pH changes upon the iron-binding properties of transferrin was investigated in the absence of chelating agents. The effects were demonstrated by spectrophotometry, gel filtration, and by studies of the intermolecular transfer of 59Fe from transferrin to conalbumin. At pH values below 6.7, diferric transferrin readily loses iron. The monoferric molecule, which is relatively resistant to acid dissociation, is preferentially formed. A temporary reduction of pH provides a simple method for selectively attaching iron to one metal-binding site, and allows double isotopic labelling of the transferrin molecule. This technique may permit further investigation of the physiological properties of the two iron-binding sites. | The effect of pH upon human transferrin: selective labelling of the two iron-binding sites. The influence of pH changes upon the iron-binding properties of transferrin was investigated in the absence of chelating agents. The effects were demonstrated by spectrophotometry, gel filtration, and by studies of the intermolecular transfer of 59Fe from transferrin to conalbumin. At pH values below 6.7, diferric transferrin readily loses iron. The monoferric molecule, which is relatively resistant to acid dissociation, is preferentially formed. A temporary reduction of pH provides a simple method for selectively attaching iron to one metal-binding site, and allows double isotopic labelling of the transferrin molecule. This technique may permit further investigation of the physiological properties of the two iron-binding sites. | [
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PMID:3196 | The effect of parturition on amniotic fluid lecithin concentration. | Amniotic fluid lecithin has been measured during the antenatal period and at comparable periods of gestation at the onset of spontaneous labour. Lecithin values were higher in labour, the difference being statistically significant in two of the three groups studied. Lecithin values were also measured serially during induced labour in 14 normal women at term. A significant fall was observed throughout labour. Creatinine levels were measured in the amniotic fluid in five of these patients and showed no significant change. | The effect of parturition on amniotic fluid lecithin concentration. Amniotic fluid lecithin has been measured during the antenatal period and at comparable periods of gestation at the onset of spontaneous labour. Lecithin values were higher in labour, the difference being statistically significant in two of the three groups studied. Lecithin values were also measured serially during induced labour in 14 normal women at term. A significant fall was observed throughout labour. Creatinine levels were measured in the amniotic fluid in five of these patients and showed no significant change. | [
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PMID:3197 | The effects of narcotics on fetal acid base status. | This paper reports two randomized control trials on the effects of nalorphine, pethidine, morphine and heroin on fetal and maternal acid base status. The drugs decreased pH and increased pCO2 in the mother, and decreased pH and base excess in the fetus. The changes in the fetus were independent of the changes in the mother. In equivalent dosages, nalorphine increased maternal pCO2 more than pethidine and morphine. The effects of heroin were found to be greater than that of other drugs, and we suggest that heroin should be avoided where the fetus is already at risk. | The effects of narcotics on fetal acid base status. This paper reports two randomized control trials on the effects of nalorphine, pethidine, morphine and heroin on fetal and maternal acid base status. The drugs decreased pH and increased pCO2 in the mother, and decreased pH and base excess in the fetus. The changes in the fetus were independent of the changes in the mother. In equivalent dosages, nalorphine increased maternal pCO2 more than pethidine and morphine. The effects of heroin were found to be greater than that of other drugs, and we suggest that heroin should be avoided where the fetus is already at risk. | [
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PMID:3198 | On the solution conformation of bradykinin and certain fragments. | A circular dichroism (CD) study of [D-Pro2]- and [D-Pro3]-bradykinin, selected peptide fragments, and the model compound. N-acetyl-L-phenylalaninamide, support our previous conclusion (Biochemistry 12, 3780, 1973) that the positive 221-nm CD band of bradykinin is a composite of bands due to two chromophores, the 217-nm band characteristic of the Phe residues overlying the 223-nm band of the N-terminal sequence, Arg-Pro-Pro. The results also indicate that the 223-nm band of Arg-Pro-Pro is associated with the configuration of the Pro-Pro sequence, Arg-D-Pro-Pro and Arg-Pro-D-Pro virtually being diastereoisomers. Accordingly, the conformation of Arg-Pro-Pro was probed in further detail. Upon increasing the temperature from about 27 to 65 degrees C, Arg-Pro-Pro undergoes a conformational transition characterized by large positive values of deltaHdegrees and deltaSdegrees, which is interpreted to mean that the structure of water and, thus, solute-solvent interactions play a dominant role in determining the conformation of the peptide 13C nuclear magnetic resonance spectroscopy indicates that the effect of lowering the pH on the CD of Arg-Pro-Pro is explicable in terms of hydrogen-bond formation between the carboxyl group and Pro2 carbonyl oxygen at acid pH with concomitant cis to trans isomerization. | On the solution conformation of bradykinin and certain fragments. A circular dichroism (CD) study of [D-Pro2]- and [D-Pro3]-bradykinin, selected peptide fragments, and the model compound. N-acetyl-L-phenylalaninamide, support our previous conclusion (Biochemistry 12, 3780, 1973) that the positive 221-nm CD band of bradykinin is a composite of bands due to two chromophores, the 217-nm band characteristic of the Phe residues overlying the 223-nm band of the N-terminal sequence, Arg-Pro-Pro. The results also indicate that the 223-nm band of Arg-Pro-Pro is associated with the configuration of the Pro-Pro sequence, Arg-D-Pro-Pro and Arg-Pro-D-Pro virtually being diastereoisomers. Accordingly, the conformation of Arg-Pro-Pro was probed in further detail. Upon increasing the temperature from about 27 to 65 degrees C, Arg-Pro-Pro undergoes a conformational transition characterized by large positive values of deltaHdegrees and deltaSdegrees, which is interpreted to mean that the structure of water and, thus, solute-solvent interactions play a dominant role in determining the conformation of the peptide 13C nuclear magnetic resonance spectroscopy indicates that the effect of lowering the pH on the CD of Arg-Pro-Pro is explicable in terms of hydrogen-bond formation between the carboxyl group and Pro2 carbonyl oxygen at acid pH with concomitant cis to trans isomerization. | [
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PMID:3199 | The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. | Ribulose-1,5-bisphosphate carboxylase was activated by incubation with CO2 and Mg2++, and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activation process involved CO2 rather than HCO3-. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH, indicating that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The initial rate of activation was linearly dependent upon the CO2 concentration but independent of the Mg2+ concentration. Kinetic analyses indicated that the enzyme reacted first with CO2 in a rate-determining and reversible step, followed by a rapid reaction with Mg2+ to form an active ternary complex (see eq 1 in text). The pseudo-first order rate constant, kobsd, for the activation process at constant pH was derived: kobsd=k1[CO2] + (k2k4/k3[Mg2+]). Experimentally, kobsd was shown to be linearly dependent upon the CO2 concentration and inversely dependent upon the Mg2+ concentration. The activity of the enzyme after preincubation to equilibrium at constant concentrations of CO2 and Mg2+ increased as the preincubation pH was raised, indicating that CO2 reacted with an enzyme group whose pK was distinctly alkaline. It is proposed that the activation of ribulose-1, 5-biphosphate carboxylane involves the formation of a carbamate. | The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. Ribulose-1,5-bisphosphate carboxylase was activated by incubation with CO2 and Mg2++, and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activation process involved CO2 rather than HCO3-. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH, indicating that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The initial rate of activation was linearly dependent upon the CO2 concentration but independent of the Mg2+ concentration. Kinetic analyses indicated that the enzyme reacted first with CO2 in a rate-determining and reversible step, followed by a rapid reaction with Mg2+ to form an active ternary complex (see eq 1 in text). The pseudo-first order rate constant, kobsd, for the activation process at constant pH was derived: kobsd=k1[CO2] + (k2k4/k3[Mg2+]). Experimentally, kobsd was shown to be linearly dependent upon the CO2 concentration and inversely dependent upon the Mg2+ concentration. The activity of the enzyme after preincubation to equilibrium at constant concentrations of CO2 and Mg2+ increased as the preincubation pH was raised, indicating that CO2 reacted with an enzyme group whose pK was distinctly alkaline. It is proposed that the activation of ribulose-1, 5-biphosphate carboxylane involves the formation of a carbamate. | [
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PMID:3200 | Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine. | The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25 degrees C. The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 muM with epsilont, the enhancement for the ternary complex, equal to 3.0. Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP. The dissociation constants were 9, 5, and 0.8 muM, respectively. The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled. The number of rapidly exchanging water molecules drops from 2 to approximately 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present. The kinetically determined KI value of approximately 4 muM for the sulfoximine is about three orders of magnitude tighter than thee Km' value of approximately 3 mM for L-glutamate. The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km'. These data suggest that L-methionine (SR)-sulfoximine is a "transition-state" analogue for the glutamine synthetase reaction. ... | Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine. The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25 degrees C. The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 muM with epsilont, the enhancement for the ternary complex, equal to 3.0. Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP. The dissociation constants were 9, 5, and 0.8 muM, respectively. The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled. The number of rapidly exchanging water molecules drops from 2 to approximately 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present. The kinetically determined KI value of approximately 4 muM for the sulfoximine is about three orders of magnitude tighter than thee Km' value of approximately 3 mM for L-glutamate. The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km'. These data suggest that L-methionine (SR)-sulfoximine is a "transition-state" analogue for the glutamine synthetase reaction. ... | [
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PMID:3201 | The interaction of borate and sulfite with pyridine nucleotides. | The kinetics and equilibria of the borate interaction at ribose with NAD+ and NMN+ have been measured using as a chromophoric probe the perturbation effect borate has on the addition of sulfite to the 4 position of the nicotinamide ring. NAD+ and NMN+ have more favorable borate association constants than do their corresponding sulfite addition complexes. The rate of interaction of the ribose moiety with borate at low borate buffer concentration is dependent on the concentration of both borate and boric acid. At high borate concentration the rate becomes independent of borate concentration, indicating the existence of a two-step process for the interaction of NAD-sulfite with borate with a change of rate-determining step from the interaction of the ribose hydroxyl group with borate at low borate to an elimination of sulfite at high borate concentration. A linear free energy relationship with a slope of 0.94 describes an increased reactivity of the nucleotide for sulfite as the affinity of the nucleotide for sulfite increases. | The interaction of borate and sulfite with pyridine nucleotides. The kinetics and equilibria of the borate interaction at ribose with NAD+ and NMN+ have been measured using as a chromophoric probe the perturbation effect borate has on the addition of sulfite to the 4 position of the nicotinamide ring. NAD+ and NMN+ have more favorable borate association constants than do their corresponding sulfite addition complexes. The rate of interaction of the ribose moiety with borate at low borate buffer concentration is dependent on the concentration of both borate and boric acid. At high borate concentration the rate becomes independent of borate concentration, indicating the existence of a two-step process for the interaction of NAD-sulfite with borate with a change of rate-determining step from the interaction of the ribose hydroxyl group with borate at low borate to an elimination of sulfite at high borate concentration. A linear free energy relationship with a slope of 0.94 describes an increased reactivity of the nucleotide for sulfite as the affinity of the nucleotide for sulfite increases. | [
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PMID:3202 | Oxidation of corticosteroids to steroidal-21-oic acids by human liver enzyme. | An enzyme that oxidizes corticosteroids to acidic metabolites has been purified from postmortem human liver. The most rapidly oxidized substrate was 11-deoxycorticosterone (DOC). Other corticosteroids were oxidized at rates that were 10% or less of DOC. The products of DOC oxidation were 3, 20-dioxopregn-4-en-21-oic acid and 20-hydroxy-3-oxopregn-4-en-21-oic acid. The 20-keto acid was the predominant metabolite in all enzyme preparations. Keto acid and hydroxy acid were not interconverted. Enzyme activity was assayed by measuring the transfer of tritium from [21-3H]DOC to water. The enzyme is yellow, and has spectral maxima at 278 and 405 nm. Inhibition by o-phenanthroline suggests that it may be a metalloenzyme. Molecular weight was estimated at 74 000 +/- 8 000; a pH maximum occurred at pH 8-8.5. This enzyme may participate in the in vivo conversion of corticosteroids to the acidic metabolites that we have described previously (H.L. Bradlow et al. (1973), J. Clin. Endocrinol. Metab. 37, 811). | Oxidation of corticosteroids to steroidal-21-oic acids by human liver enzyme. An enzyme that oxidizes corticosteroids to acidic metabolites has been purified from postmortem human liver. The most rapidly oxidized substrate was 11-deoxycorticosterone (DOC). Other corticosteroids were oxidized at rates that were 10% or less of DOC. The products of DOC oxidation were 3, 20-dioxopregn-4-en-21-oic acid and 20-hydroxy-3-oxopregn-4-en-21-oic acid. The 20-keto acid was the predominant metabolite in all enzyme preparations. Keto acid and hydroxy acid were not interconverted. Enzyme activity was assayed by measuring the transfer of tritium from [21-3H]DOC to water. The enzyme is yellow, and has spectral maxima at 278 and 405 nm. Inhibition by o-phenanthroline suggests that it may be a metalloenzyme. Molecular weight was estimated at 74 000 +/- 8 000; a pH maximum occurred at pH 8-8.5. This enzyme may participate in the in vivo conversion of corticosteroids to the acidic metabolites that we have described previously (H.L. Bradlow et al. (1973), J. Clin. Endocrinol. Metab. 37, 811). | [
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PMID:3203 | A spectrophotometric and fluorimetric study of alkaline transitions of Euglena cytochrome c 552. | The behavior of the photosynthetic cytochrome c552 upon titration with alkali depends on the ionic composition of the medium. In water the disappearance of the 695-nm band, indicating the displacement of the methionine ligand, as well as a remarkable tryptophan fluorescense enhancement, follow a single proton titration curve with pK of 10.0 and n=1.0. The product is a low spin type protein. In salt-containing media two successive steps are observed: in the first one, completed at about pH 10.3, a high-spin form of cytochrome c 552 is obtained and relatively small fluorescence enhancement is detected. In the second step, more profound fluorometric changes occur, while the material reverts to its low-spin form. Addition of salts to an alkaline solution of cytochrome c 552 in water results in the formation of a 600-nm high-spin band with a concomitant quenching of tryptophan fluorescence. The results imply that at high pH unfolding of the molecule is evident only when the low-spin product is obtained. In the high-spin alkaline form, the methionine ligand is probably displaced from iron coordination by hydroxyl ions, while in the low-spin alkaline form methionine may be replaced by a lysyl residue of the cytochrome c 552 protein. The results imply that the lysyl residue is available for coordination in salt solutions at a higher pH than in water. | A spectrophotometric and fluorimetric study of alkaline transitions of Euglena cytochrome c 552. The behavior of the photosynthetic cytochrome c552 upon titration with alkali depends on the ionic composition of the medium. In water the disappearance of the 695-nm band, indicating the displacement of the methionine ligand, as well as a remarkable tryptophan fluorescense enhancement, follow a single proton titration curve with pK of 10.0 and n=1.0. The product is a low spin type protein. In salt-containing media two successive steps are observed: in the first one, completed at about pH 10.3, a high-spin form of cytochrome c 552 is obtained and relatively small fluorescence enhancement is detected. In the second step, more profound fluorometric changes occur, while the material reverts to its low-spin form. Addition of salts to an alkaline solution of cytochrome c 552 in water results in the formation of a 600-nm high-spin band with a concomitant quenching of tryptophan fluorescence. The results imply that at high pH unfolding of the molecule is evident only when the low-spin product is obtained. In the high-spin alkaline form, the methionine ligand is probably displaced from iron coordination by hydroxyl ions, while in the low-spin alkaline form methionine may be replaced by a lysyl residue of the cytochrome c 552 protein. The results imply that the lysyl residue is available for coordination in salt solutions at a higher pH than in water. | [
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PMID:3204 | Synthetic flavinyl peptides related to the active site of mitochondrial monoamine oxidase II. Fluorescence properties. | The fluorescence properties of various 8alpha-sulfur-linked flavinyl peptides and related flavin analogues were investigated as the pH solvent, temperature, and flavin concentration were varied. Substitution in the 8alpha position by a thioether-linked peptide brings about a marked quenching of fluorescence (up to 98% in water), a slight bathochromic shift and broadening of the fluorescence emission spectra, and a slight decrease in the fluorescence lifetimes. Oxidation of the thioether function to a sulfone partially releases this fluorescence quenching without further changes in the fluorescence emission spectra. The primary effect on the fluorescence intensity is due to an interaction between the nonbonding electrons of the thioether, the hydrogen-bonding, polar solvent, and the isoalloxazine ring. Dissolving these flavinyl peptides in nonaqueous solvents increases the fluorescence intensity as much as 20-fold. A secondary effect on flavinyl fluorescence can be attributed to a collisional quenching by the vicinal tyrosyl residue within tyrosine-containing flavinyl peptides. The fluorescence properties provide further confirmation of the identity of the synthetic and naturally obtained flavinyl peptides and of the interaction between the free-hydroxyl functions of the ribityl side chain and the thioether. | Synthetic flavinyl peptides related to the active site of mitochondrial monoamine oxidase II. Fluorescence properties. The fluorescence properties of various 8alpha-sulfur-linked flavinyl peptides and related flavin analogues were investigated as the pH solvent, temperature, and flavin concentration were varied. Substitution in the 8alpha position by a thioether-linked peptide brings about a marked quenching of fluorescence (up to 98% in water), a slight bathochromic shift and broadening of the fluorescence emission spectra, and a slight decrease in the fluorescence lifetimes. Oxidation of the thioether function to a sulfone partially releases this fluorescence quenching without further changes in the fluorescence emission spectra. The primary effect on the fluorescence intensity is due to an interaction between the nonbonding electrons of the thioether, the hydrogen-bonding, polar solvent, and the isoalloxazine ring. Dissolving these flavinyl peptides in nonaqueous solvents increases the fluorescence intensity as much as 20-fold. A secondary effect on flavinyl fluorescence can be attributed to a collisional quenching by the vicinal tyrosyl residue within tyrosine-containing flavinyl peptides. The fluorescence properties provide further confirmation of the identity of the synthetic and naturally obtained flavinyl peptides and of the interaction between the free-hydroxyl functions of the ribityl side chain and the thioether. | [
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PMID:3205 | High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea). | A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes. | High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea). A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes. | [
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PMID:3206 | Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester. | In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given. | Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester. In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given. | [
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PMID:3207 | Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester. | The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well. | Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester. The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well. | [
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PMID:3208 | Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases. | Chicken embryos less than 15 days old contain only the K isozyme of pyruvate kinase, which appears to exist in vivo as an R,T conformational set with pI values of 7.2 and 6.6, respectively. Sets of lower pI and higher pI K-isozyme variants also are obtained. Whole embryos of 15 days or more of development show progressively increasing amounts of higher pI, lower K0.5S enzymatic variants. Tissue distribution and kinetic properties suggest that the highest pI form (pH 8.8-9.0) is an M-isozyme analogue. The intermediate forms are postulated to be hybrids. Adult liver extracts contain only the embryonic K isozyme; no evidence for an L-isozyme analogue was obtained. All major forms of the enzymes are compared with respect to saturation by phosphoenolpyruvate in the absence of effector and in the presence of fructose 1,6-diphosphate, alanine, serine, phenylalanine, tryptophan, and/or Mg-ATP. | Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases. Chicken embryos less than 15 days old contain only the K isozyme of pyruvate kinase, which appears to exist in vivo as an R,T conformational set with pI values of 7.2 and 6.6, respectively. Sets of lower pI and higher pI K-isozyme variants also are obtained. Whole embryos of 15 days or more of development show progressively increasing amounts of higher pI, lower K0.5S enzymatic variants. Tissue distribution and kinetic properties suggest that the highest pI form (pH 8.8-9.0) is an M-isozyme analogue. The intermediate forms are postulated to be hybrids. Adult liver extracts contain only the embryonic K isozyme; no evidence for an L-isozyme analogue was obtained. All major forms of the enzymes are compared with respect to saturation by phosphoenolpyruvate in the absence of effector and in the presence of fructose 1,6-diphosphate, alanine, serine, phenylalanine, tryptophan, and/or Mg-ATP. | [
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PMID:3209 | Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. | A monooxygenase isolated from 5-day old etiolated Vinca rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was inpendent upon NADPH (neither NADH nor combination of NADH, NADP+ and ATP served as substitutes) and O2. Geraniol hydroxylation was enhanced by dithiothreitol (monothiols were less effective) and inhibited by phospholipases, thiol reagents, metyrapone, and cytochrome c, as well as other inhibitors of cytochrome P-450 systems. Geraniol was hydroxylated at a faster rate than nerol, but the alcohols possessed similar apparent Km values. The membrane-bound hydroxylase was solubilized by treatment with sodium cholate, Renex-30, or Lubrol-WX. Cholate-treated enzyme was resolved by DEAE-cellulose chromatography and reconstitution of the hydroxylase was effected utilizing different fractions containing cytochrome P-450, a NADPH-cytochrome c reductase, and lipid. | Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea. A monooxygenase isolated from 5-day old etiolated Vinca rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was inpendent upon NADPH (neither NADH nor combination of NADH, NADP+ and ATP served as substitutes) and O2. Geraniol hydroxylation was enhanced by dithiothreitol (monothiols were less effective) and inhibited by phospholipases, thiol reagents, metyrapone, and cytochrome c, as well as other inhibitors of cytochrome P-450 systems. Geraniol was hydroxylated at a faster rate than nerol, but the alcohols possessed similar apparent Km values. The membrane-bound hydroxylase was solubilized by treatment with sodium cholate, Renex-30, or Lubrol-WX. Cholate-treated enzyme was resolved by DEAE-cellulose chromatography and reconstitution of the hydroxylase was effected utilizing different fractions containing cytochrome P-450, a NADPH-cytochrome c reductase, and lipid. | [
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PMID:3210 | Methionine sulfoxide cytochrome c. | Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential. | Methionine sulfoxide cytochrome c. Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential. | [
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PMID:3211 | A kinetic study of protein-protein interactions. | Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present. | A kinetic study of protein-protein interactions. Kinetic studies have been carried out of the monomer-dimer interaction of insulin, beta-lactoglobulin, and alpha-chymotrypsin using stopped-flow and temperature-jump techniques. The pH indicators bromothymol blue, bromophenol blue, and phenol red were used to monitor pH changes associated with the monomer-dimer interaction. In all three cases a kinetic process was observed which could be attributed to a simple monomer-dimer equilibrium, and association (k1) and dissociation (k-1) rate constants were determined. The results obtained are as follows: for insulin at 23 degrees C, pH 6.8, 0.125 M KNO3, k1 = 1.14 X 10(8) M-1 s-1, k-1 - 1.48 X 10(4)s(-1); for beta-lactoglobulin AB at 35 degrees C, pH 3.7, 0.025 M KNO3, d1 = 4.7 X 10(4) M-1 s-1, k-1 = 2.1 s-1; for alpha-chymotrypsin at 25 degreesC, pH 4.3, 0.05 M KNO3 k1 - 3.7 X 10(3) M-1 s-1, k-1 - 0.68 s-1. The kinetic behavior of the separated beta-lactoglobulin A and B was similar to that of the mixture. In the case of chymotrypsin, bromophenol blue was found to activate the enzyme catalyzed hydrolysis of p-nitrophenyl acetate, and a rate process was observed with the temperature jump which could be attributed to a conformational change of the indicator-protein complex. The association rate constant for dimer formation of insulin approaches the value expected for a diffusion-controlled process, while the values obtained for the other two proteins are below those expected for a diffusion-controlled reaction unless unusally large steric and electrostatic effects are present. | [
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PMID:3212 | Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187. | The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A. | Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187. The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A. | [
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PMID:3213 | Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus. | The biochemical properties of the electrically excitable sodium channels in the electroplaque of Electrophorus electricus were investigated using tritiated tetrodotoxin (TTX) as a specific membrane probe. Membrane fragments from the electroplaque were isolated essentially by differential centrifugation and characterized with respect to the plasma membrane markers acetylcholine receptors, acetylcholinesterase, (Na+ + K+)ATPase, and [3H]TTX binding. Equilibrium binding studies showed that [3H]TTX bound to a single population of noninteracting receptor sites with an apparent dissociation constant of 6 +/- 1 X 10(-9) M. The toxin-membrane complex dissociated with a first-order rate constant of 0.012 sec-1. Studies on the pH dependence of complex formation demonstrated the requirement for an ionizable, functional group with a pK of 5.3 and this group has been shown to be a carboxyl. Treatment of the membranes with trimethyloxonium tetrafluoroborate, a carboxyl group modifying reagent, resulted in an irreversible loss in the binding of [3H]TTX, which could be prevented by low concentrations of TTX or saxitoxin. This decrease was due to a reduction in the total number of binding sites and not to a decrease in toxin binding affinities. The relative binding affinities of various monovalent alkali metal and polyatomic cations for the TTX-receptor site showed that this site displayed cation discrimination properties which were similar to those reported previously for the electrically excitable sodium channel in intact nerve fibers. A possible role for this site in the ion selectivity of the sodium channel is proposed. | Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus. The biochemical properties of the electrically excitable sodium channels in the electroplaque of Electrophorus electricus were investigated using tritiated tetrodotoxin (TTX) as a specific membrane probe. Membrane fragments from the electroplaque were isolated essentially by differential centrifugation and characterized with respect to the plasma membrane markers acetylcholine receptors, acetylcholinesterase, (Na+ + K+)ATPase, and [3H]TTX binding. Equilibrium binding studies showed that [3H]TTX bound to a single population of noninteracting receptor sites with an apparent dissociation constant of 6 +/- 1 X 10(-9) M. The toxin-membrane complex dissociated with a first-order rate constant of 0.012 sec-1. Studies on the pH dependence of complex formation demonstrated the requirement for an ionizable, functional group with a pK of 5.3 and this group has been shown to be a carboxyl. Treatment of the membranes with trimethyloxonium tetrafluoroborate, a carboxyl group modifying reagent, resulted in an irreversible loss in the binding of [3H]TTX, which could be prevented by low concentrations of TTX or saxitoxin. This decrease was due to a reduction in the total number of binding sites and not to a decrease in toxin binding affinities. The relative binding affinities of various monovalent alkali metal and polyatomic cations for the TTX-receptor site showed that this site displayed cation discrimination properties which were similar to those reported previously for the electrically excitable sodium channel in intact nerve fibers. A possible role for this site in the ion selectivity of the sodium channel is proposed. | [
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PMID:3214 | The arrangement of subunits in cholera toxin. | Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion. | The arrangement of subunits in cholera toxin. Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion. | [
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PMID:3215 | Kinetics of reaction of anions with methemerythrin derivatives. | The kinetics of anation of methemerythrin over a wide range of pH and concentration of anions have been studied at 25 degrees C. The azide and thiocyanate ions have been most intensively investigated but experiments with fluoride and chloride are also reported. The replacement of anion in methemerythrin-anionic adducts by other anions has also been studied. Except for replacement of met-fluoride by azide, all replacements can be explained by a dissociative mechanism via the aquated species. Anations are second-order and an associative mechanism is preferred. The second-order rate constant decreases with increasing anion concentrations (from 20 muM to 20 mM). This is attributed to the effect of a secondary anion binding site. The behavior of octameric and monomeric forms of the protein toward thiocyanate is identical. A comparison of results with simple Fe(III) complexes and certain metalloproteins is made. | Kinetics of reaction of anions with methemerythrin derivatives. The kinetics of anation of methemerythrin over a wide range of pH and concentration of anions have been studied at 25 degrees C. The azide and thiocyanate ions have been most intensively investigated but experiments with fluoride and chloride are also reported. The replacement of anion in methemerythrin-anionic adducts by other anions has also been studied. Except for replacement of met-fluoride by azide, all replacements can be explained by a dissociative mechanism via the aquated species. Anations are second-order and an associative mechanism is preferred. The second-order rate constant decreases with increasing anion concentrations (from 20 muM to 20 mM). This is attributed to the effect of a secondary anion binding site. The behavior of octameric and monomeric forms of the protein toward thiocyanate is identical. A comparison of results with simple Fe(III) complexes and certain metalloproteins is made. | [
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PMID:3216 | Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule. | The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored. | Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule. The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored. | [
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PMID:3217 | Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes. | Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control. | Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes. Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control. | [
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PMID:3218 | Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group. | NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl. tyrobutyricum and Cl. pasteurianum. The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH. When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell. In Cl. tyrobutyricum and Cl. pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes. In Cl. tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found. In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate. Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity. In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function. | Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group. NADH and NADPH-ferredoxin oxidoreductases have been studied in Clostridium acetobutylicum, Cl. tyrobutyricum and Cl. pasteurianum. The study of the distribution and regulation of these enzymatic activities in well-defined culture conditions, reveals that the essential function of NADPH-ferredoxin oxidoreductase is to produce NADPH, while NADH-ferredoxin oxidoreductase can, depending on cellular conditions, produce or oxidize NADH. When these Clostridia use glycolysis, regulation of the NADH-ferredoxin oxidoreductase by acetyl-CoA (obligatory activator of NADH-ferroxin reductase activity) and by NADH (competitive inhibitor of ferredoxin-NAD+ reductase activity) allow the enzymes to function correlatively with glyceraldehyde-3-phosphate dehydrogenase and thus control the levels of NAD+ and NADH in the cell. In Cl. tyrobutyricum and Cl. pasteurianum, the ferredoxin-NADP+ reductase activities are regulated by NAD+ and NADH in accordance with the intracellular concentrations of these coenzymes. In Cl. tyrobutyricum growing on pyruvate/acetate, NADH and NADPH-ferredoxin reductase activities cannot be detected; only the ferredoxin-NAD+ and ferredoxin-NADP+ reductase activities are found. In this Clostridium, regulation of the ferredoxin-NADP+ reductase activity is the same whether it is grown on glucose or pyruvate. Contrary to this, the ferredoxin-NAD+ reductase activity undergoes a drastic change, since NADH no longer controls the enzymatic activity. In this case regulation is no longer necessary, since glyceraldehyde-3-phosphate dehydrogenase does not function. | [
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PMID:3219 | Lysophospholipase activity in cell-wall fragments contaminating mitochondrial fractions of Neurospora crassa. | Crude mitochondrial preparations from Neurospora crassa contain high levels of lysophospholipase (EC 3.1.1.5) activity when assayed with lysophosphatidylcholine as a substrate. In mitochondria purified by centrifugation on a sucrose-density gradient this activity is virtually absent. The enzyme was shown to be linked to a contaminating cell fraction which mainly consists of cell-wall material as was demonstrated by electron microscopy and chemical analysis. The enzyme has no absolute Ca2+ requirement but it is slightly stimulated by 10 mM CaCl2. The pH optimum is 5.8 in presence of CaCl2 and is shifted to 4.2 when EDTA is present. In contrast to other lysophospholipases this enzyme is only slightly inhibited by deoxycholate. This detergent is able to release part of the lysophospholipase activity from the wall fragments without producing an increase in specific activity. The enzyme is possibly secreted by the cells as high lysophospholipase activities were also found in the culture medium. | Lysophospholipase activity in cell-wall fragments contaminating mitochondrial fractions of Neurospora crassa. Crude mitochondrial preparations from Neurospora crassa contain high levels of lysophospholipase (EC 3.1.1.5) activity when assayed with lysophosphatidylcholine as a substrate. In mitochondria purified by centrifugation on a sucrose-density gradient this activity is virtually absent. The enzyme was shown to be linked to a contaminating cell fraction which mainly consists of cell-wall material as was demonstrated by electron microscopy and chemical analysis. The enzyme has no absolute Ca2+ requirement but it is slightly stimulated by 10 mM CaCl2. The pH optimum is 5.8 in presence of CaCl2 and is shifted to 4.2 when EDTA is present. In contrast to other lysophospholipases this enzyme is only slightly inhibited by deoxycholate. This detergent is able to release part of the lysophospholipase activity from the wall fragments without producing an increase in specific activity. The enzyme is possibly secreted by the cells as high lysophospholipase activities were also found in the culture medium. | [
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PMID:3220 | Purification and properties of cholesterol ester hydrolase from human aortic intima and media. | 1. Cholesterol ester hydrolase of human aortic intima and media was isolated and purified about 650-fold with 10-15% recovery of the original activity by sequential precipitation with 35% acetone, gel filtration on Sephadex G-75 and DEAE-cellulose column chromatography. 2. Two pH optima of 4.5-5.0 and 7.0-7.5 were consistently observed for the partially purified cholesterol ester hydrolase of human aortic intima and media. 3. In the system used in the present study, the increasing concentration of emulsifiers, sodium taurocholate and phosphatidylcholine, inhibited the activity of the neutral enzymes but not on the acid enzymes. On the contrary, reaction products, cholesterol and oleic acid, were much more inhibitory on the acid enzymes than on the neutral ones. 4. Results of studies on the effect of presentation of substrate on the enzyme activity and on the difference between acid and neutral enzymes are also discussed. | Purification and properties of cholesterol ester hydrolase from human aortic intima and media. 1. Cholesterol ester hydrolase of human aortic intima and media was isolated and purified about 650-fold with 10-15% recovery of the original activity by sequential precipitation with 35% acetone, gel filtration on Sephadex G-75 and DEAE-cellulose column chromatography. 2. Two pH optima of 4.5-5.0 and 7.0-7.5 were consistently observed for the partially purified cholesterol ester hydrolase of human aortic intima and media. 3. In the system used in the present study, the increasing concentration of emulsifiers, sodium taurocholate and phosphatidylcholine, inhibited the activity of the neutral enzymes but not on the acid enzymes. On the contrary, reaction products, cholesterol and oleic acid, were much more inhibitory on the acid enzymes than on the neutral ones. 4. Results of studies on the effect of presentation of substrate on the enzyme activity and on the difference between acid and neutral enzymes are also discussed. | [
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PMID:3221 | Effect of maternal diet on fetal hepatic lipogenesis. | The effects of: a, maternal diet; b, cyclic-3',5'-adenosinemonophosphate (cyclic AMP) and c, clofibrate on hepatic lipogenesis in fetal rats were studied. The experimental diets contained 22% protein, 40--50% carbohydrate, adequate vitamins, and minerals. In addition, the fat-containing diets were supplemented with either 15% corn oil, 25% corn oil, or 5% cholesterol + 10% oleic acid. In the clofibrate feeding studies, 0.3% (w/v) of the ethyl ester was added to a stock ration or to fat-free diet. Lipogenesis was measured in liver slices incubated with [2-14C]pyruvate, [1-14C]acetate, or 3H2O. In addition, activities of lipogenic enzymes were measured in cytosol fractions from liver homogenates. The effec-s of the experimental diets on liver composition were also examined. Lipogenic activity was higher in fetal than in maternal liver. When 15% corn oil was added to the maternal diet, fatty acid synthesis in fetal liver did not decrease as it did in maternal liver. Maternal fasting decreased fetal fatty acid synthesys by 50% when measured with 14C and less than 10% when measured with 3H2O. Although the addition of cholesterol to the maternal diet decreased cholesterol synthesis in maternal liver, no such decrease was observed in fetal liver. Changes in enzyme activities paralleled alterations in lipogenesis in maternal but not in fetal liver. Corn oil feeding or fasting increased the rate of transfer of linoleate from the dam to the fetus. However, accumulation of linoleate in fetal liver did not correlate with a decreased rate of fatty acid synthesis as it did in maternal liver. Maternal hepatic glycogen stores were depleted by fasting, but glycogen levels in fetal liver remained high under these conditions. | Effect of maternal diet on fetal hepatic lipogenesis. The effects of: a, maternal diet; b, cyclic-3',5'-adenosinemonophosphate (cyclic AMP) and c, clofibrate on hepatic lipogenesis in fetal rats were studied. The experimental diets contained 22% protein, 40--50% carbohydrate, adequate vitamins, and minerals. In addition, the fat-containing diets were supplemented with either 15% corn oil, 25% corn oil, or 5% cholesterol + 10% oleic acid. In the clofibrate feeding studies, 0.3% (w/v) of the ethyl ester was added to a stock ration or to fat-free diet. Lipogenesis was measured in liver slices incubated with [2-14C]pyruvate, [1-14C]acetate, or 3H2O. In addition, activities of lipogenic enzymes were measured in cytosol fractions from liver homogenates. The effec-s of the experimental diets on liver composition were also examined. Lipogenic activity was higher in fetal than in maternal liver. When 15% corn oil was added to the maternal diet, fatty acid synthesis in fetal liver did not decrease as it did in maternal liver. Maternal fasting decreased fetal fatty acid synthesys by 50% when measured with 14C and less than 10% when measured with 3H2O. Although the addition of cholesterol to the maternal diet decreased cholesterol synthesis in maternal liver, no such decrease was observed in fetal liver. Changes in enzyme activities paralleled alterations in lipogenesis in maternal but not in fetal liver. Corn oil feeding or fasting increased the rate of transfer of linoleate from the dam to the fetus. However, accumulation of linoleate in fetal liver did not correlate with a decreased rate of fatty acid synthesis as it did in maternal liver. Maternal hepatic glycogen stores were depleted by fasting, but glycogen levels in fetal liver remained high under these conditions. | [
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PMID:3222 | Translational control of protein synthesis in stimulated WI-38 fibroblasts. | A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function. | Translational control of protein synthesis in stimulated WI-38 fibroblasts. A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function. | [
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PMID:3223 | A non-equilibrium thermodynamics analysis of active transport within the framework of the chemiosotic theory. | The proton circuit devised by Mitchell in the chemiosmotic theory was subjected to analysis using the formalism of irreversible thermodynamics. The phenomenological coefficients and the degree of coupling relating co-permeant flows were derived from anion/H+, substrate/H+, cation/H+ and anion/anion biporter models. Linearity and equality of the cross-coefficients in Onsager relations were always satisfied. Macroscopic flows leading to charges splitting, such as oxido-reduction, hydro-dehydratation and transhydrogenase, are driven by a composite thermodynamic force which includes the proton-motive component. Multiple coupling occurs in the circuit when it is assumed that the net inward flux of protons becomes zero, i.e. when the circulation of protons reaches a stationary state. Under these conditions, oxidative phosphorylation, ATPase- or respiration-linked transhydrogenase and uptake of anion or cation against their electrochemical gradient may be predicted, in agreement with known experimental evidence. | A non-equilibrium thermodynamics analysis of active transport within the framework of the chemiosotic theory. The proton circuit devised by Mitchell in the chemiosmotic theory was subjected to analysis using the formalism of irreversible thermodynamics. The phenomenological coefficients and the degree of coupling relating co-permeant flows were derived from anion/H+, substrate/H+, cation/H+ and anion/anion biporter models. Linearity and equality of the cross-coefficients in Onsager relations were always satisfied. Macroscopic flows leading to charges splitting, such as oxido-reduction, hydro-dehydratation and transhydrogenase, are driven by a composite thermodynamic force which includes the proton-motive component. Multiple coupling occurs in the circuit when it is assumed that the net inward flux of protons becomes zero, i.e. when the circulation of protons reaches a stationary state. Under these conditions, oxidative phosphorylation, ATPase- or respiration-linked transhydrogenase and uptake of anion or cation against their electrochemical gradient may be predicted, in agreement with known experimental evidence. | [
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PMID:3224 | Kinetics of the slow variation of peak sodium current in the membrane of myelinated nerve following changes of holding potential or extracellular pH. | (1) Changes of the holding potential applied to the membrane of myelinated nerve fibres induced slow variations of the peak sodium current, which are super-imposed on the effect of sodium inactivation. (2) These slow variations are transitions between various steady levels of available sodium conductance. Their time course can be described by the function erfc (square root t/tau) where tau is the time and erfc the error function complement. The characteristic time tau lies in the range 2-4 min and depends on the membrane potential. (3) Changes of extracellular pH cause a rapid change of the peak sodium current followed by a slow variation as observed after changes of the holding potential. This slow variation can be prevented by applying simultaneously an appropriate change of the holding potential, e.g. the effect of changing pH from 7.3 to 5.3 is balanced by changing the potential from --70 to --55 mV. (4) The results are interpreted by postulating charged components diffusion slowly within the nodal membrane. Their transverse distribution controls the number of sodium channels available at a given membrane potential. The equivalence between change of pH and voltage is explained by assuming negative fixed charges at the outer surface of the membrane, which are protonated at low pH and thus affect the intrinsic membrane potential. (5) It is concluded that effects which are ascribed to the action of agents on individual sodium channels have to be corrected for variations in the number of available channels if these agents influence the intrinsic membrane potential, e.g. changes of extracellular pH. | Kinetics of the slow variation of peak sodium current in the membrane of myelinated nerve following changes of holding potential or extracellular pH. (1) Changes of the holding potential applied to the membrane of myelinated nerve fibres induced slow variations of the peak sodium current, which are super-imposed on the effect of sodium inactivation. (2) These slow variations are transitions between various steady levels of available sodium conductance. Their time course can be described by the function erfc (square root t/tau) where tau is the time and erfc the error function complement. The characteristic time tau lies in the range 2-4 min and depends on the membrane potential. (3) Changes of extracellular pH cause a rapid change of the peak sodium current followed by a slow variation as observed after changes of the holding potential. This slow variation can be prevented by applying simultaneously an appropriate change of the holding potential, e.g. the effect of changing pH from 7.3 to 5.3 is balanced by changing the potential from --70 to --55 mV. (4) The results are interpreted by postulating charged components diffusion slowly within the nodal membrane. Their transverse distribution controls the number of sodium channels available at a given membrane potential. The equivalence between change of pH and voltage is explained by assuming negative fixed charges at the outer surface of the membrane, which are protonated at low pH and thus affect the intrinsic membrane potential. (5) It is concluded that effects which are ascribed to the action of agents on individual sodium channels have to be corrected for variations in the number of available channels if these agents influence the intrinsic membrane potential, e.g. changes of extracellular pH. | [
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PMID:3225 | Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid. | The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH2-12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the "major glycoprotein" into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fracture membrane faces. The necessity of working at low pH values from pH 2-4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-choloroethanol technique is not adequate for recombination studies tending to membrane reconsitution. | Effects of pH during recombination of human erythrocyte membrane apoprotein and lipid. The recombinates from human red cell membrane proteins and lipids resulting from dialysis of the components in 2-chloroethanol against aqueous buffers from pH2-12 have been studied by density gradient centrifugation, polyacrylamide gel electrophoresis and freeze-fracture electron microscopy. Between pH 4 and 10 most of the proteins were found in the recombinates whereas below pH 4 and above pH 10 only part of them were recovered in the lipoprotein band after density gradient centrifugation. At low pH, increasing incorporation of the "major glycoprotein" into the recombinates was detected by gel electrophoresis and in parallel increasing amounts of particles were found in the freeze-fracture membrane faces. The necessity of working at low pH values from pH 2-4, however, and a critical evaluation of all the data presently available leads to the conclusion that the 2-choloroethanol technique is not adequate for recombination studies tending to membrane reconsitution. | [
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PMID:3226 | [pH dependence and EPR spectra of Fe-NO complexes with purines and pyrimidines]. | Equilibria between different types of Fe(I)-dinitrosyl complexes with nucleobases in solution were studied by means of EPR spectroscopy. Computer simulation and 15NO isotopic substitution were used in order to make easier the interpretation of complicated EPR patterns. The pH dependence of the purine and pyrimidine complexes was investigated. Several EPR signals, under slow exchange conditions, were present in the range of pH values of biological significance. Four types of complexes were identified on the basis of the nuclear hyperfine structure: B' = where two purine molecules were bound to iron via N-7 in the imidazole ring; B'' = where two mercapto-base molecules were bound to iron via S-; B''' = where one mercapto-base molecule was bound to iron via S- and another via pyrimidine-nitrogen; B* = where two pyrimidine molecules were bound to iron via pyrimidine-nitrogen. | [pH dependence and EPR spectra of Fe-NO complexes with purines and pyrimidines]. Equilibria between different types of Fe(I)-dinitrosyl complexes with nucleobases in solution were studied by means of EPR spectroscopy. Computer simulation and 15NO isotopic substitution were used in order to make easier the interpretation of complicated EPR patterns. The pH dependence of the purine and pyrimidine complexes was investigated. Several EPR signals, under slow exchange conditions, were present in the range of pH values of biological significance. Four types of complexes were identified on the basis of the nuclear hyperfine structure: B' = where two purine molecules were bound to iron via N-7 in the imidazole ring; B'' = where two mercapto-base molecules were bound to iron via S-; B''' = where one mercapto-base molecule was bound to iron via S- and another via pyrimidine-nitrogen; B* = where two pyrimidine molecules were bound to iron via pyrimidine-nitrogen. | [
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PMID:3237 | Maximization of steady-state bacterial production in a chemostat with pH and substrate control. | This analytical study deals with the steady-state behavior and control of microbial growth in continuous cultures. A second order Haldane-Monod model of continuous cultures is used as a basis for study of the effects of the adjustment of pH by the addition of acidic (or basic) materials. The treatment of a hydrogen ion concentration, in addition to substrate and microbial concentrations as state variables, results in a third order system of equations describing the process. The analysis of the system in equilibrium yields several admissible steady states, that is, steady states which satisfy all constraints. An optimal control problem is formulated and subsequently solved to maximize steady-state microbial production. | Maximization of steady-state bacterial production in a chemostat with pH and substrate control. This analytical study deals with the steady-state behavior and control of microbial growth in continuous cultures. A second order Haldane-Monod model of continuous cultures is used as a basis for study of the effects of the adjustment of pH by the addition of acidic (or basic) materials. The treatment of a hydrogen ion concentration, in addition to substrate and microbial concentrations as state variables, results in a third order system of equations describing the process. The analysis of the system in equilibrium yields several admissible steady states, that is, steady states which satisfy all constraints. An optimal control problem is formulated and subsequently solved to maximize steady-state microbial production. | [
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PMID:3238 | Preparation and properties of soluble-insoluble nicotinamide coenzymes. | A soluble-insoluble form of nicotinamide adenine dinucleotide (NAD+), which can be rendered either soluble or insoluble by simply adjusting the pH, has been prepared by covalently coupling NAD to alginic acid using 1,2,7,8-diepoxyoctane. The NAD bound to the alginic acid showed the coenzymic function in the soluble state and could be collected for further use as precipitate by lowering the pH to below 3. Coupling soluble-insoluble coenzymes with insolubilized apoenzymes is possible in fluidized and fixed-bed reactors. | Preparation and properties of soluble-insoluble nicotinamide coenzymes. A soluble-insoluble form of nicotinamide adenine dinucleotide (NAD+), which can be rendered either soluble or insoluble by simply adjusting the pH, has been prepared by covalently coupling NAD to alginic acid using 1,2,7,8-diepoxyoctane. The NAD bound to the alginic acid showed the coenzymic function in the soluble state and could be collected for further use as precipitate by lowering the pH to below 3. Coupling soluble-insoluble coenzymes with insolubilized apoenzymes is possible in fluidized and fixed-bed reactors. | [
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PMID:3240 | Studies on some lipogenic enzymes of cultured myeloid leukemic cells. | The microsomal fraction of M1 cells (an established cell line of myeloid leukemia) was capable of catalyzing acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters. The principal lipid product formed was identified as phosphatidic acid. Palmityl-CoA, stearyl-CoA, and oleyl-CoA were more effective acyl donors than linoleyl-CoA and arachidonyl-CoA. M1 cells and macrophages differentiated from them exhibited similar levels of sn-glycerol 3-phosphate-acylating activity, which were approximately one-half that in mouse liver and approximately four times that in peritoneal macrophages. The levels of acetyl-CoA carboxylase activity in M1 cells and macrophages differentiated from them were not significantly different from each other and were comparable to those in mouse liver, whereas no activity was detected in peritoneal macrophages. These results indicated that differentiation of the myeloid leukemic cells, which results in loss of leukemogenicity and mitotic activity, is not associated with changes in the activities of these lipogenic enzymes, although the cultured cells exhibited remarkably higher activities than freshly harvested peritoneal macrophages. Furthermore, the present study supports the view that the glycerophosphate pathway makes an essential contribution to the de novo synthesis of phospholipids in M1 cells, as well as in both types of macrophages. | Studies on some lipogenic enzymes of cultured myeloid leukemic cells. The microsomal fraction of M1 cells (an established cell line of myeloid leukemia) was capable of catalyzing acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters. The principal lipid product formed was identified as phosphatidic acid. Palmityl-CoA, stearyl-CoA, and oleyl-CoA were more effective acyl donors than linoleyl-CoA and arachidonyl-CoA. M1 cells and macrophages differentiated from them exhibited similar levels of sn-glycerol 3-phosphate-acylating activity, which were approximately one-half that in mouse liver and approximately four times that in peritoneal macrophages. The levels of acetyl-CoA carboxylase activity in M1 cells and macrophages differentiated from them were not significantly different from each other and were comparable to those in mouse liver, whereas no activity was detected in peritoneal macrophages. These results indicated that differentiation of the myeloid leukemic cells, which results in loss of leukemogenicity and mitotic activity, is not associated with changes in the activities of these lipogenic enzymes, although the cultured cells exhibited remarkably higher activities than freshly harvested peritoneal macrophages. Furthermore, the present study supports the view that the glycerophosphate pathway makes an essential contribution to the de novo synthesis of phospholipids in M1 cells, as well as in both types of macrophages. | [
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PMID:3245 | Impairment in the hepatic clearance of (35S)-bromosulphophthalein in paracetamol-intoxicated rats. | 1 The overall functional capacity of the liver was evaluated using [35S]-bromosulphophthalein (BSP, 100 mg/kg, i.v.) in biliary fistulated adult rats pretreated orally with different doses of paracetamol (APAP) for varying time intervals. 2 The maximal hepatic damage occurred between 12-18 h after single doses of APAP (0.5 or 1 g/kg); hepatic excretory function returned to control levels by 48-72 hours. 3 Administration of either 0.5 or 1 g/kg APAP 18 h before BSP caused a dose-dependent inhibition of the choleretic effect of BSP and of the 60 min cumulative excretion of the dye, but conversely, produced a significant increase in the liver and plasma concentrations of 35S. 4 Following acute (0.25 g/kg), or subacute (0.5 g/kg, twice daily for 7 days) treatment with APAP, the total excretion of 35S in bile and the retention of 35S in the liver or plasma remained essentially the same as that for the controls. 5 In rats given single doses of 1 g/kg APAP, the hepatic uptake of the dye was significantly increased during the early stages of intoxication, while the opposite effect was observed at late periods. 6 The bile flow appeared to be inversely related to the excretion of unchanged BSP, and directly related to the excretion of the major BSP conjugate in bile. 7 The hepatic clearance of BSP was more rapid in rats treated subacutely with 0.5 or 1 g/kg APAP, than in those treated acutely with equal doses, suggesting that the intensity of APAP-induced hepatotoxicity became less severe after the repeated administration of this drug. 8 It is concluded that the hepatic uptake, metabolism and excretion of BSP are reversibly impaired following APAP-induced liver injury. | Impairment in the hepatic clearance of (35S)-bromosulphophthalein in paracetamol-intoxicated rats. 1 The overall functional capacity of the liver was evaluated using [35S]-bromosulphophthalein (BSP, 100 mg/kg, i.v.) in biliary fistulated adult rats pretreated orally with different doses of paracetamol (APAP) for varying time intervals. 2 The maximal hepatic damage occurred between 12-18 h after single doses of APAP (0.5 or 1 g/kg); hepatic excretory function returned to control levels by 48-72 hours. 3 Administration of either 0.5 or 1 g/kg APAP 18 h before BSP caused a dose-dependent inhibition of the choleretic effect of BSP and of the 60 min cumulative excretion of the dye, but conversely, produced a significant increase in the liver and plasma concentrations of 35S. 4 Following acute (0.25 g/kg), or subacute (0.5 g/kg, twice daily for 7 days) treatment with APAP, the total excretion of 35S in bile and the retention of 35S in the liver or plasma remained essentially the same as that for the controls. 5 In rats given single doses of 1 g/kg APAP, the hepatic uptake of the dye was significantly increased during the early stages of intoxication, while the opposite effect was observed at late periods. 6 The bile flow appeared to be inversely related to the excretion of unchanged BSP, and directly related to the excretion of the major BSP conjugate in bile. 7 The hepatic clearance of BSP was more rapid in rats treated subacutely with 0.5 or 1 g/kg APAP, than in those treated acutely with equal doses, suggesting that the intensity of APAP-induced hepatotoxicity became less severe after the repeated administration of this drug. 8 It is concluded that the hepatic uptake, metabolism and excretion of BSP are reversibly impaired following APAP-induced liver injury. | [
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PMID:3246 | Dual effect of alpha-adrenoceptor antagonists in rat isolated vas deferens. | 1 In rat isolated vas deferens, the isotonic contractile responses to low doses of noradrenaline or adrenaline were antagonized, and those to high doses were potentiated, by yohimbine, piperoxan, phentolamine and tolazoline. Effects due to intermediate doses were not affected, or were potentiated within about 30 min, following an initial inhibition. 2 The alpha-adrenoceptor blockers thus caused a shift to the right and an increase of the maximum height of log dose-response curves of alpha-adrenoceptor stimulants. For a given dose of antagonist, the onset was slower for the potentiating than for the blocking effect. 3 The shift to the right induced by piperoxan and yohimbine on dose-response curves of noradrenaline and adrenaline was analysed with the Schild plot, and the slopes obtained, around 0.3, were lower than expected from receptor theory. When cocaine was used to block neuronal uptake, the slopes were close to 1.0. 4 The increase in maximum response to noradrenaline and adrenaline induced by alpha-adrenoceptor blockers was dependent on the time of incubation, on the dose of antagonist, and on the initial height of responses to the agonist. A less pronounced potentiation was also obtained when acetylcholine was used as agonist. 5 The findings are explained in terms of receptor theory as being due to a dual effect of alpha-adrenoceptor antagonists; competitive antagonism proper, which may be disclosed after blockade of neuronal uptake, and an interaction at a different locus, which results in potentiation of the effects of noradrenaline and adrenaline. | Dual effect of alpha-adrenoceptor antagonists in rat isolated vas deferens. 1 In rat isolated vas deferens, the isotonic contractile responses to low doses of noradrenaline or adrenaline were antagonized, and those to high doses were potentiated, by yohimbine, piperoxan, phentolamine and tolazoline. Effects due to intermediate doses were not affected, or were potentiated within about 30 min, following an initial inhibition. 2 The alpha-adrenoceptor blockers thus caused a shift to the right and an increase of the maximum height of log dose-response curves of alpha-adrenoceptor stimulants. For a given dose of antagonist, the onset was slower for the potentiating than for the blocking effect. 3 The shift to the right induced by piperoxan and yohimbine on dose-response curves of noradrenaline and adrenaline was analysed with the Schild plot, and the slopes obtained, around 0.3, were lower than expected from receptor theory. When cocaine was used to block neuronal uptake, the slopes were close to 1.0. 4 The increase in maximum response to noradrenaline and adrenaline induced by alpha-adrenoceptor blockers was dependent on the time of incubation, on the dose of antagonist, and on the initial height of responses to the agonist. A less pronounced potentiation was also obtained when acetylcholine was used as agonist. 5 The findings are explained in terms of receptor theory as being due to a dual effect of alpha-adrenoceptor antagonists; competitive antagonism proper, which may be disclosed after blockade of neuronal uptake, and an interaction at a different locus, which results in potentiation of the effects of noradrenaline and adrenaline. | [
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PMID:3242 | [Isotopic study of fluid and electrolyte disturbances in decompensated chronic respiratory insufficiency (author's transl)]. | The study of fluid and electrolyte disturbances by isotope radiodilution method is carried out in 22 patients with chronic respiratory insufficiency and cardiac failure. The simultaneous measurements of hydro-ionic compartments have been carried out with tritiated water (HTO), labelled sodium (22Na), labelled potassium (42K) and labelled bromine (82Br). From these measurements, the various water spaces are calculated: total water (ET) and extracellular fluids (LEC), also exchangeable electrolytes: sodium (NaE), potassium (KE), chlorine (ClE) and derived values. Results are compared to corresponding values in controls with the same obesity index. Patients with respiratory insufficiency show a fluid and sodium rise, similar to that found in cardiac failure and denutrition. The (NaE + KE)/ET ratio is not significantly decreased and the natremia is only slightly lower. There is no real potassium depletion in most patients. | [Isotopic study of fluid and electrolyte disturbances in decompensated chronic respiratory insufficiency (author's transl)]. The study of fluid and electrolyte disturbances by isotope radiodilution method is carried out in 22 patients with chronic respiratory insufficiency and cardiac failure. The simultaneous measurements of hydro-ionic compartments have been carried out with tritiated water (HTO), labelled sodium (22Na), labelled potassium (42K) and labelled bromine (82Br). From these measurements, the various water spaces are calculated: total water (ET) and extracellular fluids (LEC), also exchangeable electrolytes: sodium (NaE), potassium (KE), chlorine (ClE) and derived values. Results are compared to corresponding values in controls with the same obesity index. Patients with respiratory insufficiency show a fluid and sodium rise, similar to that found in cardiac failure and denutrition. The (NaE + KE)/ET ratio is not significantly decreased and the natremia is only slightly lower. There is no real potassium depletion in most patients. | [
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PMID:3241 | [Role of P50 in resuscitation (author's transl)]. | The amount of oxygen made available to the tissues of the body depends essentially upon pulmonary gas exchanges, cardiac output and its regional distribution, haemoglobin concentration and also upon the oxygen affinity of the haemoglobin molecule. That a standard oxyhaemoglobin dissociation curve faithfully describes oxygen loading and unloading both in healthy subjects and in those suffering from pathological process has come under attack. Beside the effect of pH, PCO2 and temperature, the oxyhaemoglobin dissociation curve can be modified by alterations of other factors (concentration of 2,3-diphosphoglycerate, hormones, drugs). Although the shifts of the oxyhaemoglobin dissociation curve, expressed by variations of P50 may seem minute, the effect of these shifts, expressed in terms of the "functional value of haemoglobin" are very large. Assessment of the intensive care patient must take into account the effect of alterations of the oxyhaemoglobin dissociation curve which can either increase or diminish tissue oxygenation. | [Role of P50 in resuscitation (author's transl)]. The amount of oxygen made available to the tissues of the body depends essentially upon pulmonary gas exchanges, cardiac output and its regional distribution, haemoglobin concentration and also upon the oxygen affinity of the haemoglobin molecule. That a standard oxyhaemoglobin dissociation curve faithfully describes oxygen loading and unloading both in healthy subjects and in those suffering from pathological process has come under attack. Beside the effect of pH, PCO2 and temperature, the oxyhaemoglobin dissociation curve can be modified by alterations of other factors (concentration of 2,3-diphosphoglycerate, hormones, drugs). Although the shifts of the oxyhaemoglobin dissociation curve, expressed by variations of P50 may seem minute, the effect of these shifts, expressed in terms of the "functional value of haemoglobin" are very large. Assessment of the intensive care patient must take into account the effect of alterations of the oxyhaemoglobin dissociation curve which can either increase or diminish tissue oxygenation. | [
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PMID:3243 | [Acid-base disorders in status asthmaticus (author's transl)]. | In 85 patinets withstatus asthmaticus, the authors have studied the acid-base balance, the blood gas tensions and various humoral parameters. The values were classified into two groups according to the PaCO2 level: below or equal to 44 torr (Group I), higher than 44 torr (Group II). In the 58 cases of Group II, there was a very close positive correlation between PaCO2 and H + ions, practically the same as that established by BRACKETT et al. [3] in experimental acute hypercapnia in man. On the contrary, the correlation derived from cases of status asthmaticus in the literature showed, in some cases, a metabolic component in acidosis. In the present work, the mean value of lactates was close to normal; there was a slow increase in protein content and hematocrit, in the two groups. The prognosis of the status asthmaticus depends on the degrees of hypercapnia: when it reaches 70 torr, mechanical ventilation is urgently needed and is the main part of the treatment; the use of additional drugs remains a matter of specific case. | [Acid-base disorders in status asthmaticus (author's transl)]. In 85 patinets withstatus asthmaticus, the authors have studied the acid-base balance, the blood gas tensions and various humoral parameters. The values were classified into two groups according to the PaCO2 level: below or equal to 44 torr (Group I), higher than 44 torr (Group II). In the 58 cases of Group II, there was a very close positive correlation between PaCO2 and H + ions, practically the same as that established by BRACKETT et al. [3] in experimental acute hypercapnia in man. On the contrary, the correlation derived from cases of status asthmaticus in the literature showed, in some cases, a metabolic component in acidosis. In the present work, the mean value of lactates was close to normal; there was a slow increase in protein content and hematocrit, in the two groups. The prognosis of the status asthmaticus depends on the degrees of hypercapnia: when it reaches 70 torr, mechanical ventilation is urgently needed and is the main part of the treatment; the use of additional drugs remains a matter of specific case. | [
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PMID:3247 | The evaluation of the novel pressor activity of gamma-piperidinobutyramide (WY 20051, DF480). | 1 gamma-Piperidinobutyramide (Wy 20051, DF480) injected intravenously evoked pressor responses in the anaesthetized ganglion blocked rat preparation over the dose range 2.4 x 10(-6)-3.0 x 10(-4) mol/kg. 2 High doses (greater than 3.8 x 10(-5) mol/kg) or even repeated submaximal doses (1.9 x 10(-5) mol/kg) of Wy 20051 caused tachyphylaxis of this pressor response. 3 The noradrenaline pressor-response curve was shifted significantly to the right of the control curve following a dose of Wy 20051 (1.5 x 10(-4) mol/kg cumulative). 4 The dose-response curve for the pressor action of Wy 20051 was potentiated in reserpine-treated anaesthetized rats. In contrast, tyramine-induced pressor responses were abolished. 5 Wy 20051 contracted the guinea-pig isolated aortic spiral preparation (3.8 x 10(-5)-6.0 x 10(-4) mol) and evoked constrictor responses in the perfused mesenteric vasculature preparation of the rat (5.9 x 10(-7)-1.2 x 10(-5) mol). At higher doses the responses were reduced. 6 Wy 20051-induced constrictor responses of the perfused mesentery were unaffected by blockade of alpha-adrenoceptors or by tachyphylaxis of 5-hydroxytryptamine receptors. 7 The time for abolition of Wy 20051-induced constrictor responses of the mesentery in a calcium-free medium was not significantly different from that required for noradrenaline, but was significantly greater than that for KCl (P less than 0.001). 8 Wy 20051 and noradrenaline, but not KCl, evoked constrictor responses in the depolarized rat mesenteric vasculature. 9 The results indicate that Wy 20051 evokes pressor responses which have some of the characteristics of those of noradrenaline. However, the responses are not elicited by an alpha-adrenoceptor mechanism. | The evaluation of the novel pressor activity of gamma-piperidinobutyramide (WY 20051, DF480). 1 gamma-Piperidinobutyramide (Wy 20051, DF480) injected intravenously evoked pressor responses in the anaesthetized ganglion blocked rat preparation over the dose range 2.4 x 10(-6)-3.0 x 10(-4) mol/kg. 2 High doses (greater than 3.8 x 10(-5) mol/kg) or even repeated submaximal doses (1.9 x 10(-5) mol/kg) of Wy 20051 caused tachyphylaxis of this pressor response. 3 The noradrenaline pressor-response curve was shifted significantly to the right of the control curve following a dose of Wy 20051 (1.5 x 10(-4) mol/kg cumulative). 4 The dose-response curve for the pressor action of Wy 20051 was potentiated in reserpine-treated anaesthetized rats. In contrast, tyramine-induced pressor responses were abolished. 5 Wy 20051 contracted the guinea-pig isolated aortic spiral preparation (3.8 x 10(-5)-6.0 x 10(-4) mol) and evoked constrictor responses in the perfused mesenteric vasculature preparation of the rat (5.9 x 10(-7)-1.2 x 10(-5) mol). At higher doses the responses were reduced. 6 Wy 20051-induced constrictor responses of the perfused mesentery were unaffected by blockade of alpha-adrenoceptors or by tachyphylaxis of 5-hydroxytryptamine receptors. 7 The time for abolition of Wy 20051-induced constrictor responses of the mesentery in a calcium-free medium was not significantly different from that required for noradrenaline, but was significantly greater than that for KCl (P less than 0.001). 8 Wy 20051 and noradrenaline, but not KCl, evoked constrictor responses in the depolarized rat mesenteric vasculature. 9 The results indicate that Wy 20051 evokes pressor responses which have some of the characteristics of those of noradrenaline. However, the responses are not elicited by an alpha-adrenoceptor mechanism. | [
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PMID:3248 | Electroconvulsive shock increases the behavioural responses of rats to brain 5-hydroxytryptamine accumulation and central nervous system stimulant drugs. | 1 A single electroconvulsive shock (ECS) of 150 V for 1 s increased the concentration of rat brain 5-hydroxyindoleacetic acid (5-HIAA) but did not alter brain 5-hydroxytryptamine (5-HT) or tryptophan concentrations 3 h later. 2 A single ECS decreased 5-HT synthesis 3 h and 6 h later. Synthesis was back to normal after 24 hours. The ECS-treated rats did not show greater hyperactivity produced by the increased brain 5-HT accumulation following administration of L-tryptophan and tranylcypromine at any time up to 24 h later. This suggests that a single electroshock does not alter 5-HT functional activity. 3 Twenty-four hours after the final ECS of a series of 10 shocks given once daily, the rats were given tranylcypromine and L-tryptophan. They displayed greater hyperactivity than control rats not treated with ECS, suggesting that ECS increases 5-HT functional activity. Brain concentrations of 5-HT, 5-HIAA and tryptophan were then unchanged by ECS. 5-HT synthesis and accumulation of 5-HT following tranylcypromine and L-tryptophan were not altered by ECS. 4 The hyperactivity following administration of the 5-HT agonist 5-methoxy N,N-dimethyltryptamine was enhanced by repeated (10 day) ECS, suggesting altered post-synaptic responses to 5-HT receptor stimulation. 5 Repeated ECS enhanced locomotor activity following tranylcypromine and L-DOPA. It did not alter brain noradrenaline or dopamine concentrations. 6 The latent period before a pentylenetetrazol-induced convulsion was shortened by repeated ECS. 7 Following repeated ECS there appears to be increased neuronal sensitivity to certain stimuli producing centrally mediated behavioural stimulation. This is discussed in relation to the mechanism by which electroconvulsive therapy (ECT) produces its therapeutic effect. | Electroconvulsive shock increases the behavioural responses of rats to brain 5-hydroxytryptamine accumulation and central nervous system stimulant drugs. 1 A single electroconvulsive shock (ECS) of 150 V for 1 s increased the concentration of rat brain 5-hydroxyindoleacetic acid (5-HIAA) but did not alter brain 5-hydroxytryptamine (5-HT) or tryptophan concentrations 3 h later. 2 A single ECS decreased 5-HT synthesis 3 h and 6 h later. Synthesis was back to normal after 24 hours. The ECS-treated rats did not show greater hyperactivity produced by the increased brain 5-HT accumulation following administration of L-tryptophan and tranylcypromine at any time up to 24 h later. This suggests that a single electroshock does not alter 5-HT functional activity. 3 Twenty-four hours after the final ECS of a series of 10 shocks given once daily, the rats were given tranylcypromine and L-tryptophan. They displayed greater hyperactivity than control rats not treated with ECS, suggesting that ECS increases 5-HT functional activity. Brain concentrations of 5-HT, 5-HIAA and tryptophan were then unchanged by ECS. 5-HT synthesis and accumulation of 5-HT following tranylcypromine and L-tryptophan were not altered by ECS. 4 The hyperactivity following administration of the 5-HT agonist 5-methoxy N,N-dimethyltryptamine was enhanced by repeated (10 day) ECS, suggesting altered post-synaptic responses to 5-HT receptor stimulation. 5 Repeated ECS enhanced locomotor activity following tranylcypromine and L-DOPA. It did not alter brain noradrenaline or dopamine concentrations. 6 The latent period before a pentylenetetrazol-induced convulsion was shortened by repeated ECS. 7 Following repeated ECS there appears to be increased neuronal sensitivity to certain stimuli producing centrally mediated behavioural stimulation. This is discussed in relation to the mechanism by which electroconvulsive therapy (ECT) produces its therapeutic effect. | [
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PMID:3249 | Effects of chronic nicotine administration on the denervated rat adrenal medulla. | 1 The effects of chronic nicotine administration (1 or 10 mg/kg, s.c., twice daily) were studied in intact and denervated rat adrenal glands to determine the relative roles of central input and direct actions on catecholamines. 2 Catecholamine depletion was obtained in the intact glands from 1-7 days of treatment with 10 mg/kg, with recovery by 14 days of treatment; catecholamines were not decreased in denervated adrenal glands. 3 Catecholamine depletion was accompanied by a decline in functional storage vesicles (determined by [3H]-adrenaline uptake per gland) in the intact side, while no change was seen in the denervated side; the proportion of newly synthesized vesicles increased markedly during 1-7 days of treatment with 10 mg/kg in the intact side, while a much smaller increase of shorter duration was seen in the denervated adrenal gland. 4 Chronic nicotine administration at either dose level induced tyrosine hydroxylase in both intact and denervated glands, but the increase occurred more slowly in the denervated glands. 5 Dopamine beta-hydroxylase levels increased similarly in both sides during treatment with nicotine (10 mg/kg). 6 These studies suggest that although long-term adrenal denervation eliminates the catecholamine depletion caused by chronic administration of nicotine, the mechanisms for induction of catecholamine synthesizing enzymes are still capable of responding to the drug. | Effects of chronic nicotine administration on the denervated rat adrenal medulla. 1 The effects of chronic nicotine administration (1 or 10 mg/kg, s.c., twice daily) were studied in intact and denervated rat adrenal glands to determine the relative roles of central input and direct actions on catecholamines. 2 Catecholamine depletion was obtained in the intact glands from 1-7 days of treatment with 10 mg/kg, with recovery by 14 days of treatment; catecholamines were not decreased in denervated adrenal glands. 3 Catecholamine depletion was accompanied by a decline in functional storage vesicles (determined by [3H]-adrenaline uptake per gland) in the intact side, while no change was seen in the denervated side; the proportion of newly synthesized vesicles increased markedly during 1-7 days of treatment with 10 mg/kg in the intact side, while a much smaller increase of shorter duration was seen in the denervated adrenal gland. 4 Chronic nicotine administration at either dose level induced tyrosine hydroxylase in both intact and denervated glands, but the increase occurred more slowly in the denervated glands. 5 Dopamine beta-hydroxylase levels increased similarly in both sides during treatment with nicotine (10 mg/kg). 6 These studies suggest that although long-term adrenal denervation eliminates the catecholamine depletion caused by chronic administration of nicotine, the mechanisms for induction of catecholamine synthesizing enzymes are still capable of responding to the drug. | [
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PMID:3250 | Catechol O-methyltransferase in red blood cells of schizophrenic, depressed, and normal human subjects. | Catechol O-methyltransferase of lysed human red blood cells was assayed under optimal conditions, using saturating concentrations of the substrates, S-adenosyl-L-methionine and 3-4-dihydroxybenzoic acid. The mean enzyme activity found in 24 normal subjects was 29-2 nmol/hr/ml RBC. The mean activity in blood of 33 female unipolar depressives was not significantly different from normal. However, higher enzyme activities were observed in the blood of 11 schizophrenic patients (38-9 nmol/hr/ml RBC). Partially purified enzyme preparations from blood of normal and schizophrenic individuals were indistinguishable with respect to substrate specificities, isoelectric pH values, and ratios of the two O-methylated products. Therefore it is unlikely that any defect in O-methylation which may occur in schizophrenia can be attributed to a change in the intrinsic properties of erythrocyte catechol O-methyltransferase. | Catechol O-methyltransferase in red blood cells of schizophrenic, depressed, and normal human subjects. Catechol O-methyltransferase of lysed human red blood cells was assayed under optimal conditions, using saturating concentrations of the substrates, S-adenosyl-L-methionine and 3-4-dihydroxybenzoic acid. The mean enzyme activity found in 24 normal subjects was 29-2 nmol/hr/ml RBC. The mean activity in blood of 33 female unipolar depressives was not significantly different from normal. However, higher enzyme activities were observed in the blood of 11 schizophrenic patients (38-9 nmol/hr/ml RBC). Partially purified enzyme preparations from blood of normal and schizophrenic individuals were indistinguishable with respect to substrate specificities, isoelectric pH values, and ratios of the two O-methylated products. Therefore it is unlikely that any defect in O-methylation which may occur in schizophrenia can be attributed to a change in the intrinsic properties of erythrocyte catechol O-methyltransferase. | [
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PMID:3251 | The expectation of outcome from maintenance therapy in chronic schizophrenic patients. | The results from a prospective follow-up study of a group of schizophrenic patients suggest that a significant proportion (41 per cent) are likely to relapse during a two-year period despite the prescription of long-acting injectable neuroleptic drugs. Some will relapse because of a failure of the regime, but others (32-37 per cent) because the pharmacological protection of these drugs would appear to be less effective in certain patients. Even with the major advantages of the long-acting injectable neuroleptics over oral medication, the schizophrenic patient population remains a group with a high incidence of psychiatric and social morbidity which continues to require the full resources of both the hospital and community services. | The expectation of outcome from maintenance therapy in chronic schizophrenic patients. The results from a prospective follow-up study of a group of schizophrenic patients suggest that a significant proportion (41 per cent) are likely to relapse during a two-year period despite the prescription of long-acting injectable neuroleptic drugs. Some will relapse because of a failure of the regime, but others (32-37 per cent) because the pharmacological protection of these drugs would appear to be less effective in certain patients. Even with the major advantages of the long-acting injectable neuroleptics over oral medication, the schizophrenic patient population remains a group with a high incidence of psychiatric and social morbidity which continues to require the full resources of both the hospital and community services. | [
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PMID:3258 | Salicylates and renal function in rheumatoid arthritis. | The effect of salicylate treatment on the kidney, particularly medullary function, was investigated. In a retrospective analysis patients with rheumatoid arthritis (RA) treated with high doses of salicylates were shown to have inferior urinary concentrating power and increased excretion of N-acetyl-beta-D-glucosaminidase (NAG) when compared with patients who had not received salicylate treatment. A prospective study of renal funcition in healthy people and patients with RA starting salicylate in therapeutic doses showed that while epithelial cell excretion was only transiently raised in both groups the excretion of NAG was increased in all cases at three days and this increase was sustained at 10 days, all values being much higher in the patients than in the healthy subjects. Thus salicylate treatment does cause renal tubular damage but this damage results in only minimal impairment of function and does not constitute a reason for withholding salicylate treatment. | Salicylates and renal function in rheumatoid arthritis. The effect of salicylate treatment on the kidney, particularly medullary function, was investigated. In a retrospective analysis patients with rheumatoid arthritis (RA) treated with high doses of salicylates were shown to have inferior urinary concentrating power and increased excretion of N-acetyl-beta-D-glucosaminidase (NAG) when compared with patients who had not received salicylate treatment. A prospective study of renal funcition in healthy people and patients with RA starting salicylate in therapeutic doses showed that while epithelial cell excretion was only transiently raised in both groups the excretion of NAG was increased in all cases at three days and this increase was sustained at 10 days, all values being much higher in the patients than in the healthy subjects. Thus salicylate treatment does cause renal tubular damage but this damage results in only minimal impairment of function and does not constitute a reason for withholding salicylate treatment. | [
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PMID:3259 | Benzodiazepine drugs in general medical patients. | Data from a hospital-based drug surveillance programme were used to determine how often benzodiazepine drugs were used in general medical wards. Benzodiazepines were the drugs most commonly used as hypnotics and were given to 32% of these patients. Concomitant use of more than one benzodiazepine drug or of benzodiazepines with other psychoactive drugs was common and often irrational. A series of double-blind patient-preference studies comparing various benzodiazepines and a benzodiazepine with an antihistamine showed that for short-term hypnotic effect there were no differences between three common benzodiazepines but elderly patients preferred benzodiazepines to the antihistamine, which produced more undesired effects. These results suggest that currently diazepam is the hypnotic of choice for medical ward inpatients. | Benzodiazepine drugs in general medical patients. Data from a hospital-based drug surveillance programme were used to determine how often benzodiazepine drugs were used in general medical wards. Benzodiazepines were the drugs most commonly used as hypnotics and were given to 32% of these patients. Concomitant use of more than one benzodiazepine drug or of benzodiazepines with other psychoactive drugs was common and often irrational. A series of double-blind patient-preference studies comparing various benzodiazepines and a benzodiazepine with an antihistamine showed that for short-term hypnotic effect there were no differences between three common benzodiazepines but elderly patients preferred benzodiazepines to the antihistamine, which produced more undesired effects. These results suggest that currently diazepam is the hypnotic of choice for medical ward inpatients. | [
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PMID:3261 | Decrease of uptake and exchange of neurotransmitter amino acids after depletion of their synaptosomal pools. | Synaptosomes prelabeled at 37 degrees C with radioactive amino acids (GABA, glutamate, glycine, taurine, alpha-aminoisobutyric acid, phenylalanine, leucine) and then washed at 0 degrees C on Millipore filters (DAWP 02500) lost 60-70% of the accumulated radioactivity. The loss was similar with exogenous tritiated GABA and glutamate, and with [14C]GABA and [14C]glutamate metabolically derived from [14C]glucose. In contrast, radioactive norepinephrine, dopamine and 5-hydroxytryptamine were almost totally retained by cold shocked synaptosomes. After pretreatment with reserpine and nialamide the loss of norepinephrine became significantly greater (about 25%). The uptake of radioactive GABA, glutamate and clycine after cold shock was about 50% reduced, whereas that of radioactive biogenic amines was less affected (reduction of 22% for norepinephrine, 29% for 5-hydroxytryptamine and 35% for dopamine). The loss of amino acids and the reduction of uptake could be minimized by performing the cold shock in hypertonic conditions. In synaptosomes prelabeled with [3H]GABA, a good correlation was observed among magnitude of amino acid pool depletion induced by cold shock or by 56 mM KCl, decrease of subsequent accumulation of [14C]GABA, and decrease of [14C]-GABA-stimulated [3H]GABA release (homoexchange). | Decrease of uptake and exchange of neurotransmitter amino acids after depletion of their synaptosomal pools. Synaptosomes prelabeled at 37 degrees C with radioactive amino acids (GABA, glutamate, glycine, taurine, alpha-aminoisobutyric acid, phenylalanine, leucine) and then washed at 0 degrees C on Millipore filters (DAWP 02500) lost 60-70% of the accumulated radioactivity. The loss was similar with exogenous tritiated GABA and glutamate, and with [14C]GABA and [14C]glutamate metabolically derived from [14C]glucose. In contrast, radioactive norepinephrine, dopamine and 5-hydroxytryptamine were almost totally retained by cold shocked synaptosomes. After pretreatment with reserpine and nialamide the loss of norepinephrine became significantly greater (about 25%). The uptake of radioactive GABA, glutamate and clycine after cold shock was about 50% reduced, whereas that of radioactive biogenic amines was less affected (reduction of 22% for norepinephrine, 29% for 5-hydroxytryptamine and 35% for dopamine). The loss of amino acids and the reduction of uptake could be minimized by performing the cold shock in hypertonic conditions. In synaptosomes prelabeled with [3H]GABA, a good correlation was observed among magnitude of amino acid pool depletion induced by cold shock or by 56 mM KCl, decrease of subsequent accumulation of [14C]GABA, and decrease of [14C]-GABA-stimulated [3H]GABA release (homoexchange). | [
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PMID:3263 | Ultrastructural hypoxic changes in Ammon's horn and Purkinje cells. | Guinea pigs were exposed for varying periods to different degrees of hypoxia by respiration of controlled mixtures of O2-N2 and the CNS subjected to both light and electron microscopic examination after aldehyde fixation by perfusion. The subacute anoxia experiments revealed an alteration in the rough endoplasmic reticulum of the Purkinje cells consisting of the formation of 'paired cisternae'. In the chronic anoxia experiments, small ultrastructural alterations of the same cells were found in association with the appearance of monoparticulate glycogen. The authors debate the significance of these ultrastructural alterations in relation to the damage found in other organs. | Ultrastructural hypoxic changes in Ammon's horn and Purkinje cells. Guinea pigs were exposed for varying periods to different degrees of hypoxia by respiration of controlled mixtures of O2-N2 and the CNS subjected to both light and electron microscopic examination after aldehyde fixation by perfusion. The subacute anoxia experiments revealed an alteration in the rough endoplasmic reticulum of the Purkinje cells consisting of the formation of 'paired cisternae'. In the chronic anoxia experiments, small ultrastructural alterations of the same cells were found in association with the appearance of monoparticulate glycogen. The authors debate the significance of these ultrastructural alterations in relation to the damage found in other organs. | [
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PMID:3264 | The effect of taget organ removal on the development of sympathetic neurons. | The role of target organs in the maturation of adrenergic neurons was studied in the neonatal rat. The superior cervical ganglion (SCG) and its end organs, the salivary glands and iris were employed as a model system. Unilateral sialectomy and iridectomy in 3-day-old animals prevented the normal development of ganglion tyrosine hydroxylase (T-OH) and DOPA decarboxylase activities. These enzymes are highly localized to adrenergic neurons in the SCG, and were used to monitor maturation of these cells. Enzyme activity remained depressed for at least two months, the longest time tested. In contrast, total ganglion protein, a measure of ganglion growth as a whole, initially developed normally. Six weeks after surgery, however, protein content was significantly lower in ganglia deprived of the normal field of innervation. Failure of normal enzyme maturation was apparently dependent on removal of ipsilateral end organs only, since bilateral sialectomy exerted no greater effect than unilateral sialectomy. In adults, unilateral sialectomy and iridectomy did not significantly alter ganglion T-OH activity or protein in rats followed up to one month after surgery. | The effect of taget organ removal on the development of sympathetic neurons. The role of target organs in the maturation of adrenergic neurons was studied in the neonatal rat. The superior cervical ganglion (SCG) and its end organs, the salivary glands and iris were employed as a model system. Unilateral sialectomy and iridectomy in 3-day-old animals prevented the normal development of ganglion tyrosine hydroxylase (T-OH) and DOPA decarboxylase activities. These enzymes are highly localized to adrenergic neurons in the SCG, and were used to monitor maturation of these cells. Enzyme activity remained depressed for at least two months, the longest time tested. In contrast, total ganglion protein, a measure of ganglion growth as a whole, initially developed normally. Six weeks after surgery, however, protein content was significantly lower in ganglia deprived of the normal field of innervation. Failure of normal enzyme maturation was apparently dependent on removal of ipsilateral end organs only, since bilateral sialectomy exerted no greater effect than unilateral sialectomy. In adults, unilateral sialectomy and iridectomy did not significantly alter ganglion T-OH activity or protein in rats followed up to one month after surgery. | [
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PMID:3265 | Sensitization and habituation of the plantar cushion reflex in cats. | The plantar cushion reflex in cats was examined as a model system in a mammal for the study of the effects of repeated stimulation on neural transmission. Effects of various frequencies and intensities of stimulation were similar to those seen in other reflex systems. For instance, for a fixed number of stimuli, habituation of the plantar cushion reflex was more marked at 10 Hz than at 2.0 Hz, and with 1.0 X threshold stimulation than with 5.0 X threshold stimulation. Sensitization occurred at intermediate intensities and frequencies of stimulation. Dorsal root potentials were studied; changes in dorsal root potentials during iterated stimulation did not correlate with the changes in the plantar cushion reflex. These changes in the plantar cushion reflex were also unrelated to variations in afferent transmission peripheral to the spinal cord. Sensitization and habituation in the plantar cushion reflex occurred during iterated stimulation, were produced centrally, and were unrelated to mechanisms of presynaptic inhibition. | Sensitization and habituation of the plantar cushion reflex in cats. The plantar cushion reflex in cats was examined as a model system in a mammal for the study of the effects of repeated stimulation on neural transmission. Effects of various frequencies and intensities of stimulation were similar to those seen in other reflex systems. For instance, for a fixed number of stimuli, habituation of the plantar cushion reflex was more marked at 10 Hz than at 2.0 Hz, and with 1.0 X threshold stimulation than with 5.0 X threshold stimulation. Sensitization occurred at intermediate intensities and frequencies of stimulation. Dorsal root potentials were studied; changes in dorsal root potentials during iterated stimulation did not correlate with the changes in the plantar cushion reflex. These changes in the plantar cushion reflex were also unrelated to variations in afferent transmission peripheral to the spinal cord. Sensitization and habituation in the plantar cushion reflex occurred during iterated stimulation, were produced centrally, and were unrelated to mechanisms of presynaptic inhibition. | [
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PMID:3266 | Quantitative localization of tyrosine hydroxylase, dopamine-beta-hydroxylase, phenolethanolamine-N-methyl transferase, and glutamic acid decarboxylase in spinal cord. | Sensitive radiometric assays for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyl transferase (PNMT), and glutamic acid decarboxylase (GAD) have been combined with microdissection techniques for quantitative localization of these synthetic enzymes in rabbit spinal cord. TH was present uniformly in gray and white matter. DBH was higher in the lateral horns than in the other gray matter areas, and was not detectable in white matter. PNMT was detectable in gray but not white matter, and was considerably lower in activity than the other catecholamine synthetic enzymes. GAD was higher in the dorsal horns at the cervical and lumbar levels than in other gray matter areas and relatively low in white matter. GAD activity was considerably higher than the catecholamine synthetic enzyme activities. The quantitative localizations are consistent with the qualitative immunohistochemical enzymes maps and distributions of the related putative neurotransmitters. | Quantitative localization of tyrosine hydroxylase, dopamine-beta-hydroxylase, phenolethanolamine-N-methyl transferase, and glutamic acid decarboxylase in spinal cord. Sensitive radiometric assays for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyl transferase (PNMT), and glutamic acid decarboxylase (GAD) have been combined with microdissection techniques for quantitative localization of these synthetic enzymes in rabbit spinal cord. TH was present uniformly in gray and white matter. DBH was higher in the lateral horns than in the other gray matter areas, and was not detectable in white matter. PNMT was detectable in gray but not white matter, and was considerably lower in activity than the other catecholamine synthetic enzymes. GAD was higher in the dorsal horns at the cervical and lumbar levels than in other gray matter areas and relatively low in white matter. GAD activity was considerably higher than the catecholamine synthetic enzyme activities. The quantitative localizations are consistent with the qualitative immunohistochemical enzymes maps and distributions of the related putative neurotransmitters. | [
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PMID:3268 | Extraction of a calcium-phospholipid-phosphate complex from bone. | A calcium-phospholipid-phosphate complex with a constant 1:1 calcium to total phosphate molar ratio is shown to exist in rabbit and calf bone. This complex, which may be involved in the transport and deposition of bone mineral, appears to constitute a significantly greater proportion of the lipids of younger bone than of more mature bone. The comples was isolated by a modified Folch extraction employing ultrasonic disruption of cellular material. Evidence is presented to show that the complex is a natural constituent of bone rather than one created artifactually during extraction. | Extraction of a calcium-phospholipid-phosphate complex from bone. A calcium-phospholipid-phosphate complex with a constant 1:1 calcium to total phosphate molar ratio is shown to exist in rabbit and calf bone. This complex, which may be involved in the transport and deposition of bone mineral, appears to constitute a significantly greater proportion of the lipids of younger bone than of more mature bone. The comples was isolated by a modified Folch extraction employing ultrasonic disruption of cellular material. Evidence is presented to show that the complex is a natural constituent of bone rather than one created artifactually during extraction. | [
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PMID:3269 | Magill circuit and controlled ventilation. | Nine patients under anaesthesia were controlled manually with the Magill attachment. Nine other patients using the same circuit under anaesthesia breathed spontaneously. Blood gases were studied throughout the period of operation to determine the adequacy of ventilation. The results show that the Magill attachment was adequate for controlled ventilation using normal flows (9 1/min) in young healthy patients. | Magill circuit and controlled ventilation. Nine patients under anaesthesia were controlled manually with the Magill attachment. Nine other patients using the same circuit under anaesthesia breathed spontaneously. Blood gases were studied throughout the period of operation to determine the adequacy of ventilation. The results show that the Magill attachment was adequate for controlled ventilation using normal flows (9 1/min) in young healthy patients. | [
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PMID:3270 | The reduction of coma time in lipophilic drug overdose using castor oil. | A clinical trial of castor oil in overdoses of lipophilic drugs gave a strong clinical impression that it was effective in speeding up recovery. Therefore, animal experiments were undertaken to confirm that castor oil acts as a ligand in Ethchlorvynol poisoning and that its use reduces coma time. Serial serum levels of Ethchlorvynol were obtained from dogs given Ethchlorvynol 150 mg/kg alone, and the same dose dissolved in castor oil 15 ml/kg in a crossover fashion. The result was a reduction of peak serum levels and of the half-life of the drug when the castor oil solution was used. In order to mimic the clinical situation more closely, a further crossover study was undertaken using Ethchlorvynol 300 mg/kg alone and the same dose followed by castor oil 15 ml/kg repeated q12h. This showed no delay in reaching peak serum concentration and no reduction of peak levels. However, it did show a 31 per cent reduction in the half-life of the drug. This change is statistically significant, and supports the continued use of castor oil in lipophilic drug overdose. | The reduction of coma time in lipophilic drug overdose using castor oil. A clinical trial of castor oil in overdoses of lipophilic drugs gave a strong clinical impression that it was effective in speeding up recovery. Therefore, animal experiments were undertaken to confirm that castor oil acts as a ligand in Ethchlorvynol poisoning and that its use reduces coma time. Serial serum levels of Ethchlorvynol were obtained from dogs given Ethchlorvynol 150 mg/kg alone, and the same dose dissolved in castor oil 15 ml/kg in a crossover fashion. The result was a reduction of peak serum levels and of the half-life of the drug when the castor oil solution was used. In order to mimic the clinical situation more closely, a further crossover study was undertaken using Ethchlorvynol 300 mg/kg alone and the same dose followed by castor oil 15 ml/kg repeated q12h. This showed no delay in reaching peak serum concentration and no reduction of peak levels. However, it did show a 31 per cent reduction in the half-life of the drug. This change is statistically significant, and supports the continued use of castor oil in lipophilic drug overdose. | [
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PMID:3271 | An endogalactosaminidase from Streptomyces griseus. | An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules. | An endogalactosaminidase from Streptomyces griseus. An endogalactosaminidase has been purified 34-fold from the culture filtrate of Streptomyces griseus. This enzyme cleaves GalN-GalN linkages in oligogalactosaminoglycan, a galactosamine-rich oligosaccharide isolated from the culture filtrate of a Neurospora mutant. Since some or all of the GalN-GalN bonds in this molecule link positions 1 and 4, and are in the alpha-configuration, we are probably dealing with an endo-alpha-(1 leads to 4)-galactosaminidase, bu this characterization is only tentative because the few bonds cleaved by the enzyme could have a different structure. The enzyme is inactive towards N-acetyl-oligogalactosaminoglycan and chitosan. The endogalactosaminidase preparations also cleave high molecular weight galactosaminoglycan (obtained from Neurospora) into fragments greater than or equal to 10(4) daltons in molecular weight, and catalyze the release of Neurospora sporelings from the glass surfaces to which they are anchored. Galactosaminoglycan-cleaving and sporeling-releasing activities elute jointly from DEAE-cellulose columns. This observation provides further support for an earlier proposal that the sporelings are anchored to the glass by means of galactosaminoglycan molecules. | [
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PMID:3272 | Fractionation of nucleolar proteins by two-dimensional gel electrphoresis. | Isolation of nucleolar proteins was obtained by dissociation in the presence of urea-guanidine hydrochloride, followed by high-speed centrifugation to remove nucleic acids. At least 31 fractions of nucleolar proteins were detected by isoelectrofocusing gel electrophoresis in pH range 3.5-10. Following two-dimensional gel electrophoresis on sodium dodecyl sulfate-polyacrylamide slab gels, more than 100 components of nucleolar proteins were identifieid. Two-thirds of nucleolar proteins were located in the pH range 5-8 following isoelectrofocusing. The molecular weights of these classes of proteins were shown to be mostly 30000-70000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. | Fractionation of nucleolar proteins by two-dimensional gel electrphoresis. Isolation of nucleolar proteins was obtained by dissociation in the presence of urea-guanidine hydrochloride, followed by high-speed centrifugation to remove nucleic acids. At least 31 fractions of nucleolar proteins were detected by isoelectrofocusing gel electrophoresis in pH range 3.5-10. Following two-dimensional gel electrophoresis on sodium dodecyl sulfate-polyacrylamide slab gels, more than 100 components of nucleolar proteins were identifieid. Two-thirds of nucleolar proteins were located in the pH range 5-8 following isoelectrofocusing. The molecular weights of these classes of proteins were shown to be mostly 30000-70000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. | [
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PMID:3273 | Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas. | A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 degrees C and grew over the range 0 degrees C-35 degrees C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 degrees C was lower than that of cells grown at 22 degrees C. However, the consumption of oxygen after heat treatment at 35 degrees for 35 min was reduced considerably by 2 degrees C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 degrees C and 22 degrees C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 degrees C produced an alanine oxidase with a temperature optimum of 35 degrees C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 degrees C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 degrees C contained an alanine oxidase system with an optimum temperature of 45 degrees C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 degrees C. The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 degrees C and 37 degrees C had a common optimum temperature of 45 degrees C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions. | Temperature-induced alanine oxidation in a psychrotrophic Pseudomonas. A psychrotrophic pseudomonad isolated from iced fish oxidized alanine at temperatures close to 0 degrees C and grew over the range 0 degrees C-35 degrees C. The rate of oxidation of alanine, measured manometrically, by cells grown at 2 degrees C was lower than that of cells grown at 22 degrees C. However, the consumption of oxygen after heat treatment at 35 degrees for 35 min was reduced considerably by 2 degrees C grown cells. Alanine oxidase activity was tested in an extract from cells grown at 2 degrees C and 22 degrees C with alanine as the sole carbon, nitrogen, and energy source. Cells grown at 2 degrees C produced an alanine oxidase with a temperature optimum of 35 degrees C and pH optimum of 8, which lost about 80% activity by heat treatment at 40 degrees C for 30 min. There was no change in activity after dialysis at pH 7, 8, or 9. Extracts from cells grown at 22 degrees C contained an alanine oxidase system with an optimum temperature of 45 degrees C, a pH optimum above 8, and only about 30% reduction of activity after heat treatment. This enzyme activity was concentrated in the 0.5 M elution fraction from a Sephadex column, and dialysis reduced the activity at pH 7 and 8. Mesophilic enzyme synthesis apparently started around a growth temperature of 10 degrees C. The crude alanine oxidase systems of Pseudomonas aeruginosa derived from cells grown at 13 degrees C and 37 degrees C had a common optimum temperature of 45 degrees C. These data suggest that one mechanism of psychrophilic growth by psychrotrophic bacteria may be the induction of enzymes with low optimum temperatures in response to low temperature conditions. | [
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PMID:3274 | Growth characteristics of a cell line derived from the pig oviduct. | Further evidence for the establishment of the pig fallopian tube (PFT) cell line as a continuous cell line was shown by an increase in the maximum population density as the number of subcultures increased. The optimal pH and temperature-growth ranges appeared to be 7.4-7.8 and 37-41 degrees C respectively, and the population doubling time was 20-25 h under optimal growth conditions. With progressive subculture, the serum requirements dropped from 20 to 2%. A plating efficiency of 2 to 4% was found in all serial subcultures. Colonies were observed in agar suspension culture at the 146th subculture and thereafter. Chromosomal alterations were found in the 100th subculture and thereafter. | Growth characteristics of a cell line derived from the pig oviduct. Further evidence for the establishment of the pig fallopian tube (PFT) cell line as a continuous cell line was shown by an increase in the maximum population density as the number of subcultures increased. The optimal pH and temperature-growth ranges appeared to be 7.4-7.8 and 37-41 degrees C respectively, and the population doubling time was 20-25 h under optimal growth conditions. With progressive subculture, the serum requirements dropped from 20 to 2%. A plating efficiency of 2 to 4% was found in all serial subcultures. Colonies were observed in agar suspension culture at the 146th subculture and thereafter. Chromosomal alterations were found in the 100th subculture and thereafter. | [
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PMID:3275 | Characteristics of a facultatively psychrophilic Acinetobacter species isolated from river sediment. | A facultatively psychrophilic bacterium isolated from river sediment was identified as an Acinetobacter species, similar to those previously characterized as A. lwoffi. The strain was extremely lipolytic and hemolytic. Some action on crude oil was also observed. The organism was able to utilize a wide variety of carbon and energy sources when tested at both 20 and 30 degrees C. A comparison is made with the previously proposed type strain of A. lwoffi. The bacteria had a Gram-negative cell wall containing an electron-dense intermediate layer. Cell division occurred with the formation of a septum and slight constriction. | Characteristics of a facultatively psychrophilic Acinetobacter species isolated from river sediment. A facultatively psychrophilic bacterium isolated from river sediment was identified as an Acinetobacter species, similar to those previously characterized as A. lwoffi. The strain was extremely lipolytic and hemolytic. Some action on crude oil was also observed. The organism was able to utilize a wide variety of carbon and energy sources when tested at both 20 and 30 degrees C. A comparison is made with the previously proposed type strain of A. lwoffi. The bacteria had a Gram-negative cell wall containing an electron-dense intermediate layer. Cell division occurred with the formation of a septum and slight constriction. | [
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PMID:3276 | Zoospore chemotaxis in Australian isolates of Phytophthora species. | Zoospores of Australian isolates of Phytophthora drechsleri, P. cryptogea, P. cinnamomi, P. nicotianae var. parasitica, and P. citricola were examined for their chemotactic responses to asparagine, glutamine, aspartate, glutamate, and structurally related compounds. Structural requirements for attraction include the alpha-amino-acid group with a short carbon chain terminating in an amide group. The one American isolate tested gave a different result and possible reasons for this are discussed. The pH of the environment was important, a neutral-charged molecule was more attractive than a negatively charged molecule, hence glutamine and aspartate were more attractive at pH 3.0 than pH 5.0. Zoospores tended to move away from regions with a high hydrogen ion concentration. Compounds other than amino acids were slightly attractive including several sugars and ethanol. Synergistic interactions between amino acids, ethanol, and sucrose were observed and may account for the high levels of attraction of zoospores to root exudates and extracts. | Zoospore chemotaxis in Australian isolates of Phytophthora species. Zoospores of Australian isolates of Phytophthora drechsleri, P. cryptogea, P. cinnamomi, P. nicotianae var. parasitica, and P. citricola were examined for their chemotactic responses to asparagine, glutamine, aspartate, glutamate, and structurally related compounds. Structural requirements for attraction include the alpha-amino-acid group with a short carbon chain terminating in an amide group. The one American isolate tested gave a different result and possible reasons for this are discussed. The pH of the environment was important, a neutral-charged molecule was more attractive than a negatively charged molecule, hence glutamine and aspartate were more attractive at pH 3.0 than pH 5.0. Zoospores tended to move away from regions with a high hydrogen ion concentration. Compounds other than amino acids were slightly attractive including several sugars and ethanol. Synergistic interactions between amino acids, ethanol, and sucrose were observed and may account for the high levels of attraction of zoospores to root exudates and extracts. | [
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PMID:3279 | Double-blind evaluation of oral L-prolyl-Lleucyl-glycine amide in Parkinson's disease. | A 4-month double-blind study comparing the effect of increasing oral doses (up to 1.0 g daily) of synthetic L-proyl-L-leucyl-glycine amide (PLG) and placebo in 20 parkinsonian patients showed no significant improvement in objective scores of functional disability. However, important trends and some significant results were observed with the lower doses of PLG. These essentially negative results may be attributed to poor intestinal absorption of the compound, a short biologic half-life in the blood, or administration of oral doses that were much higher than required, or a combination of factors. In further studies with this peptide, which are encouraged, the intravenous route should be used until the question of intestinal absorption is resolved. | Double-blind evaluation of oral L-prolyl-Lleucyl-glycine amide in Parkinson's disease. A 4-month double-blind study comparing the effect of increasing oral doses (up to 1.0 g daily) of synthetic L-proyl-L-leucyl-glycine amide (PLG) and placebo in 20 parkinsonian patients showed no significant improvement in objective scores of functional disability. However, important trends and some significant results were observed with the lower doses of PLG. These essentially negative results may be attributed to poor intestinal absorption of the compound, a short biologic half-life in the blood, or administration of oral doses that were much higher than required, or a combination of factors. In further studies with this peptide, which are encouraged, the intravenous route should be used until the question of intestinal absorption is resolved. | [
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PMID:3280 | Carcinoid tumors of the thymus. | Three patients with carcinoid tumors of the anterior mediastinum are described. Study of these patients and an analysis of previously reported cases indicates that the thymus is the primary site of these tumors, which are probably related to the presence of Kulchitsky cells in normal thymus. These neoplasms differ clinically and anatomically from conventional thymomas. They occur predominantly in men, are not associated with myasthenia gravis or red-cell hypoplasia, and are more aggressive tumors than thymomas. Histologically, they are similar to carcinoid tumors of other organs and differ from the variable combination of epithelial cells and lymphocytes of thymomas. Although they are usually locally invasive and frequently metastasize, the clinical course is usually protracted. It is probable that the reported examples of Cushing's syndrome related to thymomas were actually associated with thymic carcinoid tumors. | Carcinoid tumors of the thymus. Three patients with carcinoid tumors of the anterior mediastinum are described. Study of these patients and an analysis of previously reported cases indicates that the thymus is the primary site of these tumors, which are probably related to the presence of Kulchitsky cells in normal thymus. These neoplasms differ clinically and anatomically from conventional thymomas. They occur predominantly in men, are not associated with myasthenia gravis or red-cell hypoplasia, and are more aggressive tumors than thymomas. Histologically, they are similar to carcinoid tumors of other organs and differ from the variable combination of epithelial cells and lymphocytes of thymomas. Although they are usually locally invasive and frequently metastasize, the clinical course is usually protracted. It is probable that the reported examples of Cushing's syndrome related to thymomas were actually associated with thymic carcinoid tumors. | [
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PMID:3281 | Effects of hypoxia on distribution of cardiac output and organ blood flow in the rabbit. Regional vascular response to hypoxia. | The hemodynamic responses of various vascular beds in the systemic circulation to prolonged moderate hypoxia were studied in the rabbit using the radioactive microsphere method. Although cardiac output remained unchanged, there was a redistribution of blood flow in which blood was mainly diverted from the kidneys to provide greater supply to heart, brain and skeletal muscle. These regional adjustments are similar to those seen after low cardiac output due to hemorrhage or endotoxic shock. | Effects of hypoxia on distribution of cardiac output and organ blood flow in the rabbit. Regional vascular response to hypoxia. The hemodynamic responses of various vascular beds in the systemic circulation to prolonged moderate hypoxia were studied in the rabbit using the radioactive microsphere method. Although cardiac output remained unchanged, there was a redistribution of blood flow in which blood was mainly diverted from the kidneys to provide greater supply to heart, brain and skeletal muscle. These regional adjustments are similar to those seen after low cardiac output due to hemorrhage or endotoxic shock. | [
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PMID:3284 | Quenching of tryptophanyl fluorescence of human growth hormone by iodide. | Quenching of tryptophanyl fluorescence of human growth hormone by I- followed saturation kinetics and was abolished by KSCN. In the presence of 6 M guanidine hydrochloride quenching was linear between 0 to 0.2 M KI. These results suggest that I- quenched the fluorescence of the native hormone by binding at or near the single tryptophanyl residue. Quenching by 0.1 M KI decreased exponentially with increasing concentrations of human and bovine growth hormones. Acidification did not have a significant effect on quenching of the human hormone, but it markedly decreased quenching of the bovine hormone. Conformational differences at the vicinity of the lone tryptophanyl residue that could be inferred by these and other experiments may be contributing to the biological specificity of native human and bovine growth hormones. | Quenching of tryptophanyl fluorescence of human growth hormone by iodide. Quenching of tryptophanyl fluorescence of human growth hormone by I- followed saturation kinetics and was abolished by KSCN. In the presence of 6 M guanidine hydrochloride quenching was linear between 0 to 0.2 M KI. These results suggest that I- quenched the fluorescence of the native hormone by binding at or near the single tryptophanyl residue. Quenching by 0.1 M KI decreased exponentially with increasing concentrations of human and bovine growth hormones. Acidification did not have a significant effect on quenching of the human hormone, but it markedly decreased quenching of the bovine hormone. Conformational differences at the vicinity of the lone tryptophanyl residue that could be inferred by these and other experiments may be contributing to the biological specificity of native human and bovine growth hormones. | [
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PMID:3285 | Comparison of the metabolism of benzo[alpha]pyrene and binding to DNA caused by rat liver nuclei and microsomes. | Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP. | Comparison of the metabolism of benzo[alpha]pyrene and binding to DNA caused by rat liver nuclei and microsomes. Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP. | [
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PMID:3286 | Inhibition of nadph-driven microsomal lipid peroxidation by cytosol factor(s). Effect of a fat-free, high carbohydrate diet. | Male Swiss mice were given free access to a fat-free, high carbohydrate diet. The liver cytosol fraction from these mice contained a heat-sensitive factor that markedly inhibited microsomal, ferric pyrophosphate stimulated, NADPH-driven lipid peroxidation. The diet-induced factor was apparently incorporated into the microsomes after 12 days of continuous feeding, since lipid peroxidation by these microsomes was strongly diminished. The factor disappeared from the cytosol after 24 h of fasting and reappeared after refeeding the mice with the fat-free, high carbohydrate diet. | Inhibition of nadph-driven microsomal lipid peroxidation by cytosol factor(s). Effect of a fat-free, high carbohydrate diet. Male Swiss mice were given free access to a fat-free, high carbohydrate diet. The liver cytosol fraction from these mice contained a heat-sensitive factor that markedly inhibited microsomal, ferric pyrophosphate stimulated, NADPH-driven lipid peroxidation. The diet-induced factor was apparently incorporated into the microsomes after 12 days of continuous feeding, since lipid peroxidation by these microsomes was strongly diminished. The factor disappeared from the cytosol after 24 h of fasting and reappeared after refeeding the mice with the fat-free, high carbohydrate diet. | [
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PMID:3287 | The repression and derepression of hepatic tyrosine aminotransferase by carcinogens. | Like hydrocortisone, a single carcinogenic dose of dimethylnitrosamine (50 mg/kg) initiates the induction cycle for hepatic tyrosine aminotransferase in adrenalectomized rats. However, following this initial induction in the presence of dimethylnitrosamine, the enzyme becomes refractory to reinduction by known inducers. The administration of thioacetamide to either adrenalectomized or intact rats leads to an immediate and progressive loss of inducibility by hydrocortisone, dibutyrylcyclic AMP or dimethylnitrosamine. Although the thioacetamide-induced repression was not reversed even up to 10 weekds after the cessation of treatment, it was reversed after the induction of liver regeneration. Both the carcinogen-mediated induction and repression of tyrosine aminotransferase appears to occur by mechanisms which do not involve the corticosteroid-binding proteins which normally mediate the induction by glucocorticoids. | The repression and derepression of hepatic tyrosine aminotransferase by carcinogens. Like hydrocortisone, a single carcinogenic dose of dimethylnitrosamine (50 mg/kg) initiates the induction cycle for hepatic tyrosine aminotransferase in adrenalectomized rats. However, following this initial induction in the presence of dimethylnitrosamine, the enzyme becomes refractory to reinduction by known inducers. The administration of thioacetamide to either adrenalectomized or intact rats leads to an immediate and progressive loss of inducibility by hydrocortisone, dibutyrylcyclic AMP or dimethylnitrosamine. Although the thioacetamide-induced repression was not reversed even up to 10 weekds after the cessation of treatment, it was reversed after the induction of liver regeneration. Both the carcinogen-mediated induction and repression of tyrosine aminotransferase appears to occur by mechanisms which do not involve the corticosteroid-binding proteins which normally mediate the induction by glucocorticoids. | [
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PMID:3291 | [Effect of intratumoral injection of bacterial and viral neuraminidase in rats]. | We studied the effect of neuraminidase injection in rat's tumor at different doses: 5,10,50,100, 500 U and we concluded that: There was no difference between the rats treated with 5,10,50 U and the controls. The y died 3 weeks after the injection. But the rats treated by 100 at 500 U of NA died quickley, in the week, of long metastases. | [Effect of intratumoral injection of bacterial and viral neuraminidase in rats]. We studied the effect of neuraminidase injection in rat's tumor at different doses: 5,10,50,100, 500 U and we concluded that: There was no difference between the rats treated with 5,10,50 U and the controls. The y died 3 weeks after the injection. But the rats treated by 100 at 500 U of NA died quickley, in the week, of long metastases. | [
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PMID:3292 | [Changes in transmitter release at frog neuromuscular junction induced by 4-aminopyridine]. | 4-aminopyridine (4-AP) at micromolar concentrations, increases the end-plate potential amplitude in curarized preparations and the mean quantal content in every preparation tested, but the spontaneous release is not modified by 4-AP. These results can explain the anticurare activity observed in the wole animal or in vitro. 4-AP prolongs the falling phase of the muscle action potential without change in the muscle membrane potential. | [Changes in transmitter release at frog neuromuscular junction induced by 4-aminopyridine]. 4-aminopyridine (4-AP) at micromolar concentrations, increases the end-plate potential amplitude in curarized preparations and the mean quantal content in every preparation tested, but the spontaneous release is not modified by 4-AP. These results can explain the anticurare activity observed in the wole animal or in vitro. 4-AP prolongs the falling phase of the muscle action potential without change in the muscle membrane potential. | [
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PMID:3293 | Contribution of tissue acidosis to ischemic injury in the perfused rat heart. | The isolated perfused working rat heart preparation has been used to study the effects of respiratory acidosis on myocardial metabolism and contractilly. Hearts were perfused with 5 mM glucose and 10(-2) U/ml of insulin in order to enhance metabolsim of glucose relative to that of fatty acids. After perfusion with Krebs bicarbonate medium at pH 6.6, hearts rapidly ceased performing external work and peak left ventricular pressure fell by 75% after 5 minutes. Oxygen consumption, rate of ATP generation and overall glycolytic flux also declined rapidly. After about 2 minutes of perfusion, the fall of glycolytic flux showed a partial reversal, which was largely accounted for by increased lactate production, so that glucose oxidation decreased further. The reversal of glycoltic flux could be accounted for by partial release of H+ inhibition of phospho-fructokinase by increased tissue levels of adenosine 5'-diphosphate (ADP), adenosine monophosphate (AMP) and P1 and decreased levels of adenosine triphosphate (ATP) and creatine phosphate. The increased proportion of glucose uptake converted to lactate together with an increase of the tissue lactate/pyruvate ratio could be accounted for by inhibition of the malate-aspartate cycle combined with tissue hypoxia. Lactate accumulated in the tissue as a result of a decreased permeability of the plasma membrane to lactate. Decreased oxygen delivery to the myocardium was caused by secondary constriction of the coronary vessels. In further experiments, the coronary flow was regulated by an external pump which delivered fluid at a controlled rate into the aortic cannula above the coronary arteries, and the degree of tissue hypoxia was monitored by measuring changes of pyridine nucleotide reduction state by surface fluorescence techniques. The effects of acidosis uncomplicated by possible hypoxia were compared directly with those produced by ischemic hypoxia. The effects of acidosis under these conditions were similar to those described above, and to those produced by ischemia. From these and other data it is concluded that the effects of ischemia are caused by a lowering of the intracellular pH, which decreases the rate of energy production relative to the rate of energy demand. However, it is suggested that the primary cause of the decreased peak systolic pressure with either acidosis or ischemia is not a result of a defect of energy metabolism, but is due to alteration of the calcium cycle of the heart. Possible causes of irreversible heart failure after prolonged ischemia are discussed. | Contribution of tissue acidosis to ischemic injury in the perfused rat heart. The isolated perfused working rat heart preparation has been used to study the effects of respiratory acidosis on myocardial metabolism and contractilly. Hearts were perfused with 5 mM glucose and 10(-2) U/ml of insulin in order to enhance metabolsim of glucose relative to that of fatty acids. After perfusion with Krebs bicarbonate medium at pH 6.6, hearts rapidly ceased performing external work and peak left ventricular pressure fell by 75% after 5 minutes. Oxygen consumption, rate of ATP generation and overall glycolytic flux also declined rapidly. After about 2 minutes of perfusion, the fall of glycolytic flux showed a partial reversal, which was largely accounted for by increased lactate production, so that glucose oxidation decreased further. The reversal of glycoltic flux could be accounted for by partial release of H+ inhibition of phospho-fructokinase by increased tissue levels of adenosine 5'-diphosphate (ADP), adenosine monophosphate (AMP) and P1 and decreased levels of adenosine triphosphate (ATP) and creatine phosphate. The increased proportion of glucose uptake converted to lactate together with an increase of the tissue lactate/pyruvate ratio could be accounted for by inhibition of the malate-aspartate cycle combined with tissue hypoxia. Lactate accumulated in the tissue as a result of a decreased permeability of the plasma membrane to lactate. Decreased oxygen delivery to the myocardium was caused by secondary constriction of the coronary vessels. In further experiments, the coronary flow was regulated by an external pump which delivered fluid at a controlled rate into the aortic cannula above the coronary arteries, and the degree of tissue hypoxia was monitored by measuring changes of pyridine nucleotide reduction state by surface fluorescence techniques. The effects of acidosis uncomplicated by possible hypoxia were compared directly with those produced by ischemic hypoxia. The effects of acidosis under these conditions were similar to those described above, and to those produced by ischemia. From these and other data it is concluded that the effects of ischemia are caused by a lowering of the intracellular pH, which decreases the rate of energy production relative to the rate of energy demand. However, it is suggested that the primary cause of the decreased peak systolic pressure with either acidosis or ischemia is not a result of a defect of energy metabolism, but is due to alteration of the calcium cycle of the heart. Possible causes of irreversible heart failure after prolonged ischemia are discussed. | [
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PMID:3294 | Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase. | Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods. | Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase. Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods. | [
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PMID:3295 | Measurement of 25-hydroxyvitamin D3 in serum. | We describe a method for measuring 25-hydroxyvitamin D3 in serum. Extraction with dichloromethane/methanol (2/1 by vol), followed by chromatography on a column of Sephadex LH-20, resulted in an overall analytical recovery of 82% +/- 3.5% (SD). Diluted normal rat serum was used as binding protein because it contains a transport protein that has both a high affinity (Ka = 2 X 10(10) liter/mol) and a high capacity (3 X 10(-6) mol/liter) for 25-hydroxyvitamin D3. There is no advantage in using more complex binding proteins derived either from rachitic animals or from cytosol preparations. Concentrations of 25-hydroxyvitamin D3 (13.4 +/- 4 mug/liter) in the serum of apparently normal Belgian subjects are lower than those reported for North Americans, but resemble those reported for the United Kingdom. | Measurement of 25-hydroxyvitamin D3 in serum. We describe a method for measuring 25-hydroxyvitamin D3 in serum. Extraction with dichloromethane/methanol (2/1 by vol), followed by chromatography on a column of Sephadex LH-20, resulted in an overall analytical recovery of 82% +/- 3.5% (SD). Diluted normal rat serum was used as binding protein because it contains a transport protein that has both a high affinity (Ka = 2 X 10(10) liter/mol) and a high capacity (3 X 10(-6) mol/liter) for 25-hydroxyvitamin D3. There is no advantage in using more complex binding proteins derived either from rachitic animals or from cytosol preparations. Concentrations of 25-hydroxyvitamin D3 (13.4 +/- 4 mug/liter) in the serum of apparently normal Belgian subjects are lower than those reported for North Americans, but resemble those reported for the United Kingdom. | [
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PMID:3296 | Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions. | Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively. | Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions. Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively. | [
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PMID:3297 | New techniques for ion-selective measurements of ionized calcium in serum after pH adjustment of aerobically handled sera. | I report further experience in measuring ionized calcium (Ca2+) with the AMT Electron System and its serum standards and solid-state, dip, calcium-selective electrodes. With this system, serum pH can be adjusted with CO2 gas and Ca2+ and pH simultaneously measured; when 5.2% CO2 (40 mm pco2) is used for sample equilibration, the standard bicarbonate concentration is also provided. I measured serum Ca2+ as a function of pH between pH 7.0 and 9.0 and found the relationship to be reproducible, with no evidence of irreversible complexing of Ca2+. When the pH of aerobically exposed, mailed sera was restored to the original values, their values for Ca2+ were the same as for the fresh sera. Measurement of Ca2+ in routinely (aerobically) handled sera after pH restoration with CO2 gas was therefore validated, both samples from within an institution and mailed specimens. Standardization to pH 7.40 is recommended for routine measurements, is generally more accurate than use of heparin or quasianaerobic techniques, and is a practical approach. In patients with possible uncompensated acid-base disturbance (which may be indicated by an abnormal standard bicarbonate concentration if not suspected clinically), patient pH should be measured independently as part of the usual strict, anaerobic blood-gas-analysis procedures. Abnormal patient pH must be considered in the interpretation of Ca2+ results determined at pH 7.40 which are borderline or slightly abnormal; most accurately, Ca2+ may be measured in the separated sera at the previously determined patient pH value. Studies of aqueous solutions with the currently used Ca2+ electrodes showed a selectivity coefficient (the constant which relates the activity of an interfering ion to the activity of calcium that would contribute the same emf) KNa=0.0031 +/- 0.0003 (SE) and KMg=0.046 +/- 0.004 (SE). At physiological concentrations of Ca2+, physiologically encountered variation in Na+ is of no significance in resulting Ca2+, but extreme variation in Mg2+ may cause an error of approximately 1%. | New techniques for ion-selective measurements of ionized calcium in serum after pH adjustment of aerobically handled sera. I report further experience in measuring ionized calcium (Ca2+) with the AMT Electron System and its serum standards and solid-state, dip, calcium-selective electrodes. With this system, serum pH can be adjusted with CO2 gas and Ca2+ and pH simultaneously measured; when 5.2% CO2 (40 mm pco2) is used for sample equilibration, the standard bicarbonate concentration is also provided. I measured serum Ca2+ as a function of pH between pH 7.0 and 9.0 and found the relationship to be reproducible, with no evidence of irreversible complexing of Ca2+. When the pH of aerobically exposed, mailed sera was restored to the original values, their values for Ca2+ were the same as for the fresh sera. Measurement of Ca2+ in routinely (aerobically) handled sera after pH restoration with CO2 gas was therefore validated, both samples from within an institution and mailed specimens. Standardization to pH 7.40 is recommended for routine measurements, is generally more accurate than use of heparin or quasianaerobic techniques, and is a practical approach. In patients with possible uncompensated acid-base disturbance (which may be indicated by an abnormal standard bicarbonate concentration if not suspected clinically), patient pH should be measured independently as part of the usual strict, anaerobic blood-gas-analysis procedures. Abnormal patient pH must be considered in the interpretation of Ca2+ results determined at pH 7.40 which are borderline or slightly abnormal; most accurately, Ca2+ may be measured in the separated sera at the previously determined patient pH value. Studies of aqueous solutions with the currently used Ca2+ electrodes showed a selectivity coefficient (the constant which relates the activity of an interfering ion to the activity of calcium that would contribute the same emf) KNa=0.0031 +/- 0.0003 (SE) and KMg=0.046 +/- 0.004 (SE). At physiological concentrations of Ca2+, physiologically encountered variation in Na+ is of no significance in resulting Ca2+, but extreme variation in Mg2+ may cause an error of approximately 1%. | [
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PMID:3298 | Effect of tobramycin on urinary gamma-glutamyltransferase activity: Studies in a case of renal carcinoma. | Gamma-Glutamyltransferase activity was studied in a man presenting with recurrent septicemia owing to pyonephrosis and renal carcinoma. Increased activity in the urine was ascribable to administration of the aminoglycoside antibiotic, tobramycin. That the renal carcinoma did not contribute to the increased values was confirmed by homogenization and enzyme histochemistry of the tumor. Although the activity of this enzyme in serum was greater than normal, this persisted postoperatively, and thus was not related to the renal carcinoma. | Effect of tobramycin on urinary gamma-glutamyltransferase activity: Studies in a case of renal carcinoma. Gamma-Glutamyltransferase activity was studied in a man presenting with recurrent septicemia owing to pyonephrosis and renal carcinoma. Increased activity in the urine was ascribable to administration of the aminoglycoside antibiotic, tobramycin. That the renal carcinoma did not contribute to the increased values was confirmed by homogenization and enzyme histochemistry of the tumor. Although the activity of this enzyme in serum was greater than normal, this persisted postoperatively, and thus was not related to the renal carcinoma. | [
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PMID:3301 | Electrophoresis of gamma-glutamyltranspeptidase on cellogel. The appearance of the alpha2-beta band in positive LP-X sera. | Fractionations of serum gamma-glutamyltranspeptidase (gamma-GT) and determinations of the "abnormal serum lipoprotein X" (LP-X) have been carried out in sera from patients with different hepatobiliary disorders. LP-X was used to demonstrate or exclude cholestasis. One gamma-GT fraction, alpha2-beta, may be of interest to distinguish between extrahepatic obstruction and intrahepatic cholestasis as was revealed by statistical analysis. | Electrophoresis of gamma-glutamyltranspeptidase on cellogel. The appearance of the alpha2-beta band in positive LP-X sera. Fractionations of serum gamma-glutamyltranspeptidase (gamma-GT) and determinations of the "abnormal serum lipoprotein X" (LP-X) have been carried out in sera from patients with different hepatobiliary disorders. LP-X was used to demonstrate or exclude cholestasis. One gamma-GT fraction, alpha2-beta, may be of interest to distinguish between extrahepatic obstruction and intrahepatic cholestasis as was revealed by statistical analysis. | [
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