Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299. Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.


Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen. Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.


Purification, crystallization, and some properties of creatine amidinohydrolase from Pseudomonas putida. A method was developed for purification and crystallization of creatinase [creatine amidinohydrolase, EC 3.5.3.3] from Pseudomonas putida var. naraensis C-83. The purified preparation appeared homogeneous on disc electrophoresis and ultracentrifugation and had a molecular weight of 94,000. It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37 degrees. SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47,000. Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme.


Charge transfer mediated by nigericin in black lipid membranes. Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.


Influence of surface potentials on the mitochondrial H+ pump and on lipid-phase transitions. It has been shown that the surface potential of lipid membranes, as well as of mitochondria, can be shifted more positive by absorption of alkylbiguanides. Both phospholipid vesicles and natural membranes respond in an analogous way to this shift. Ion activities at the immediate membrane surface are influenced by sign and magnitude of the surface charge. Corresponding effects on ion transport and on fluorescence-probe binding can be observed. The mitochondrial H+ pump is inhibited when the surface charge is shifted more positive. In contrast,the absolute charge density determines the temperature of the ordered-fluid transition. The latter is increased by biguanides, suggesting that the membrane is rendered more rigid. The experiments make obvious that physical relations derived from model systems apply equally well to lipid-containing natural membranes.


Evidence for multiple sites of ferricyanide reduction in chloroplasts. Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.


Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase. Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.


5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate. Synthesis, chemical properties, and effect on Escherichia coli thymidine kinase activity. 5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate (AIdUTP), a phosphoramidate analog of 5-iodo-2',5'-dideoxyuridine 5'-triphosphate (IdUTP), was synthesized and some of its chemical and biological properties were investigated. Although AIdUTP is stable in alkaline solutions, below pH 8 it undergoes degradation by a novel phosphorylysis reaction which exhibits first order kinetics. Inclusion of magnesium ion in the reaction mixture decreased the rate of degradation. Protonation of a group on AIdUTP which has a pKa of 6.10, presumably the secondary ionized oxygen on the gamma phosphate, precedes phosphorylysis. The only detectable reaction products are the nucleoside, 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), and trimetaphosphate. A mechanism for the acid catalyzed phosphorylysis of AIdUTP is proposed. AIdUTP, like TTP, converts Escherichia coli thymidine kinase into an inactive dimer with a sedimentation coefficient of 5.78 S. AIdUTP is, however, 60-fold more potent as an allosteric inhibitor than is TTP at pH 7.8. Although the inhibitory effect of TTP is markedly reduced at high pH, the activity of AIdUTP is lowered only slightly. The allosteric effects of AIdUTP also differ from those of IdUTP, which is an inhibitor at low pH but a strong activator above pH 7.4. 5-Iodo-2'-deoxycytidine 5'-triphosphate, a potent enzyme activator, cannot completely reverse the AIdUTP inhibition, even when present at a 150-fold molar excess.


Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver. The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.


Nucleophilic addition reactions of free and enzyme-bound deazaflavin. DeazaFMN-containing glycolate oxidase has been prepared and shown to catalyze the stereospecific transfer of the alpha-hydrogen from substrate to enzyme-bound deazaFMN. The reaction of sulfite, cyanide, and hydroxylamine with several deazaflavin-containing enzymes (glycolate oxidase, D-amino acid oxidase, glucose oxidase, N-methylglutamate synthetase) and free deazaFMN has been examined. All the deazaflavin systems tested form reversible 1:1 complexes with sulfite and cyanide. The pH dependence of the reaction of free deazaFMN with cyanide indicates that cyanide anion is the reacting nucleophile. Hydroxylamine complexes are formed with deazaFMN glycolate oxidase and deazaFAD glucose oxidase. The effectiveness of the various nucleophilic reagents in complex formation decreases in the following order: sulfite greater than cyanide greater than hydroxylamine. The relative stability observed for the sulfite and cyanide complexes formed with various deazaflavin systems (glycolate oxidase greater than D-amino acid oxidase greater than free deazaFMN) follows the same trend observed for the stability of the sulfite complexes formed with the corresponding flavin system. A correlation is also observed between the reduction potential (E'o) of the deazaflavin system (glycolate oxidase (- 170 mV) greater than D-amino acid oxidase (-240 mV) greater than free deazaFMN (-178 mV) and the stability of the deazaflavin-nucleophile complexes. The following evidence indicates that deazaflavin systems are generally more susceptible toward nucleophilic attack than corresponding flavin system: (a) with the exception of glucose oxidase, the dissociation constants for the deazaflavin-sulfite complexes are at least 1 order of magnitude less than the corresponding flavin sulfite complexes; (b) the least reactive nucleophile, hydroxylamine, does not form a complex with any of the flavin systems. In the case of cyanide, a complex is formed only with native glycolate oxidase, which is the flavin-containing system most susceptible to attack by the more reactive sulfite. Formation of the various (deaza)flavin-nucleophile complexes is characterized by a bleaching of the longer wavelength absorption band of the chromophore and increases in absorption below the isosbestic point of the reaction in the near-ultraviolet region of the spectrum. These results are consistent with the formation of covalent adducts via attack of the various nucleophiles at position 5 of (deaza)flavin. The reaction with cyanide provides the first example of a reversible addition of carbanion to enzyme-bound (deaza)flavin.


Formation and properties of retinylphosphate galactose. Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.


The allosteric regulation of hexokinase C from amphibian liver. A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.


Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid. The three purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase, cytochrome b5, and desaturase, have been combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the three proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12 to 19 carbon fatty acyl chains are required for desaturase activity while derivatives containing 9 to 20 carbons are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA, and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed ("gauche") conformation at carbon atoms 9,10 in the enzyme-substrate complex. Furthermore, isotope rate effects obtained with deuterated stearyl-CoA derivatives indicate that hydrogen removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5 degrees, was linear between 15 and 35 degrees, while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24 degrees coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the deuterium isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than hydrogen abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.


Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6). Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.


Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo. The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.


Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells. Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...


Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes. The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.


Reactivity of the phosphopyridoxal groups of cystathionase. Aminooxyacetate and alpha-amino-gamma-aminooxybutyrate (canaline) react specifically with the P-pyridoxal groups of cystathionase to produce characteristic changes in the absorption and fluorescence properties of the bound cofactor. The increase in fluorescence at 450 nm was used to monitor the reaction. Aminooxyacetate attacks the Schiff base linkage of the enzyme several times faster (k1 = 3700 M-1 min-1 and k2 = 1000 M-1 min-1) than it attacks the aldehydic carbon of free P-pyridoxal (k = 290 M-1 min-1). Similar results were obtained with canaline. The kinetic studies indicate that a Schiff base linkage in the enzyme cystathionase should offer direct kinetic advantage during the reaction between the substrate and the cofactor. It is also shown that the inhibitor L-alpha-gamma-aminobutyrate reacts with bound P-pyridoxal to form free P-pyridoxamine. The rate of formation of P-pyridoxamine parallels the rate of enzyme inactivation.


Digestion of native collagen, denatured collagen, and collagen fragments by extracts of rat liver lysosomes. Extracts of highly purified lysosomes from rat liver were examined for their ability to degrade native collagen and thermally denatured collagen at pH values between 3.5 and 7.0. After a 24-h digestion at 36 degrees with the lysosomal extract at a pH of 5.5 or lower (collagen/lysosomal protein; 2/1 or 8/1), both native and denatured collagen were degraded to an extent equivalent to 60 to 70% of that observed upon total acid hydrolysis in 6 N HCl as measured by the ninhydrin reaction (570 nm). At a pH of 6.0, native collagen and denatured collagen were degraded by the mixture of lysosomal proteinases to 11% and 40% of total acid hydrolysis, respectively. At pH 6.5 AND 7.0, the corresponding values were 3% versus 33% and 0.3% versus 11%, respectively. Fragments of collagen (TCA and TCB) are produced when mammalian collagenase degrades native collagen at 25 degrees. These fragments were degraded by the lysosomal extract at 36 degrees to an extent equivalent to 28% and 8% of total acid hydrolysis at pH 6.5 and 7.0, respectively. The experiments at pH 6.5 and 7.0 were done using a collagen/lysosomal protein ratio of 2/1. At pH 5.0 (a pH which is found within secondary lysosomes), the lysosomal extracts degraded collagen to a mixture of free amino acids and small peptides. Amino acid analysis established that approximately 30% of the amino acid residues of the collagen appeared in the lysosomal hydrolysate as free amino acids. Hydroxyproline and perhaps hydroxylysine were the only amino acids found in collagen which did not appear at least to some extent as the free amino acid in this hydrolysate.


Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli. Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated. Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide. Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation. ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase. Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium. Valinomycin plus nigericin completely inhibited ATP synthesis. The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined. NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect. These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.


Interaction of phenols with old yellow enzyme. Physical evidence for charge-transfer complexes. Evidence is presented that the changes in absorption spectrum obtained on complex formation between Old Yellow Enzyme and phenolic compounds are due to charge-transfer interactions. The positive correlation between the energy of the long wavelength transition and the Hammett para constant with a series of para-substituted phenols indicates that the phenol is the charge-transfer donor and the oxidized flavin of the enzyme is the charge-transfer acceptor. The same conclusion is drawn from studies in which the flavin of the native enzyme, flavin mononucleotide, was replaced by a variety of artificial flavins of different oxidation-reduction potential. The effect of pH on the dissociation constant for the enzyme-ligand binding also indicates that it is the phenolate anion, rather than the conjugate acid, which is responsible for the charge-transfer interaction. The significance of these results is discussed relative to long wavelength absorbing species detected with other flavoproteins.


Characterization of calcium binding to adipocyte plasma membranes. Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.


Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase. Structural and conformational organization of chicken liver fatty acid synthetase has been probed using its fluorescent coenzyme, NADPH. Three NADPH binding sites per mole of the enzyme complex, of apparently identical dissociation constant (KD = 0.6 muM) can be titrated at temperatures above 12 degrees. These results are in disagreement with the earlier studies of Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9, 245-251) in which four such sites could be titrated. At 12 degrees, the composite sites split into two subsets: a pair of sites with a KD of 0.3 muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees or 2 degrees), the site with weak affinity disappears, leaving a pair of sites with a Kd of 0.5 muM. Similar observations were made when the enzyme was modified with phenylmethylsulfonyl fluoride, a specific and selective inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial modification with phenylmethylsulfonyl fluoride elicits a NADPH binding response similar to the binding observed at 12 degrees, i.e. two sets of binding sites with nonidentical dissociation constants. Further modification corresponding to the complete loss of deacylase function results in a set of two apparently identical binding sites, and the third site is not available for titration. The modified enzyme retains the two reductase functions as measured by the model substrates, acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the NADPH binding affinity by a factor of 3. Other observations show that the quantum yield, as measured by the ratio of fluorescence intensity of bound and free NADPH, changes with temperature and ionic strength. Lowering the temperature from 30 degrees to 2 degrees increases the enhancement ratio by 50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium phosphate lowers it to 50% of the original level. Measurement of NADPH binding in the presence of NADP+, NADH, NAD+ and adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+ show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated interactions implicating nonspecific conformational alteration of the enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose is intermediate between NADP+ and NADH. These data are discussed in terms of substrate-mediated conformational changes and the moles of each of the reductase enzymes per mole of the enzyme complex, the polarity of the NADPH binding region, and the probable structure of the nicotinamide moiety when bound to the enzyme.


Identification of the covalently bound flavin of thiamin dehydrogenase. Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.


Association of the Ah locus with specific changes in metyrapone and ethylisocyanide binding to mouse liver microsomes. The genetic trait of "responsiveness," which refers to the capacity for induction of cytochrome P-448 and numerous monooxygenase activities by certain aromatic hydrocarbons, is known to segregate almost exclusively as a single autosomal dominant gene among progeny of appropriate crosses originating from the responsive C57BL/6 and the nonresponsive DBA/2 inbred mouse strains. In this report the allele for responsiveness is shown to be associated with (a) increases in the apparent KS values for metyrapone bound to reduced P-450; (b) increases in the ethylisocyanide difference ratio (deltaA455-490/deltaA430-490);(c) increases in the deltaA455-490 per mg of microsomal protein but not in the deltaA430-490 per mg of protein from the reduced P-450-ethylisocyanide complex; (d) an approximately 2-nm hypsochromic shift in the spectral maximum in the 446 nm region for the reduced P-450-metyrapone complex; (e) an approximately 2-nm hypsochromic shift of the absorption maximum in the 455 nm region, but not of the maximum in the 430 nm region, for the reduced P-450-ethylisocyanide complex; and (f) larger increases in the deltaA455-490 than in the deltaA430-490 per mg of microsomal protein for the reduced P450-ethylisocyanide complex as a function of increasing pH. All of these phenomena are felt to be associated with the genetically regulated induction of liver microsomal cytochrome P-448 by polycyclic aromatic compounds. Whereas increases in the total hepatic P-450 content appear to be expressed almost exclusively as a single autosomal dominant trait, the increase in apparent KS value for metyrapone bound to reduced P-450 appears to be expressed additively. The reason for this finding is unclear. The increase in apparent KS value for metyrapone in 3-methylcholanthrene-treated rats is known to occur even when the induction process is presumably blocked by treating the rat concomitantly with cycloheximide. Several lines of evidence in this report indicate that, although total P-450 content does not increase in C57BL/6N mice treated with 3-methylcholanthrene plus cycloheximide, hepatic P-448 induction does occur; P-448 induction does not occur in DBA/2N mice under these same conditions. These results indicate that cytochrome P-448 induction is relatively resistant to the inhibition of protein synthesis and that a responsive animal treated with 3-methylcholanthrene plus cycloheximide cannot be considered experimentally the same as a genetically nonresponsive animal treated with 3-methylcholanthrene alone.


Gallium-67 citrate scintiscanning in testicular neoplasia. Gallium-67 citrate has been used to evaluate 19 patients with malignant tumors of the testes. Five patients with abnormal uptake of the radiotracer in the abdomen proved to have other evidence confirming metastatic spread to this site. Eight patients, with normal scan findings, surgery and lymphangiorgraphy, provided no conflicting evidence of disease although one of these patients has subsequently developed increased urinary gonadotrophin concentrations. Surgery caused an abnormal scan and such patients were excluded from the analysis pointing to the need for preoperative evaluation. The technique of 67-gallium scintigraphy appears of value in assessing the intra-abdominal spread of malignant tumors of the testes.


The partial replacement of the serum growth factor requirement of SV3T3 cells by lactalbumin hydrolyzate. Lactalbumin hydrolyzate (LH), a commercially prepared, enzymatic digest of a milk protein fraction, can partially replace the serum requirement of SV3T3 cells. In a low, but obligatory, background of calf serum (0.15% v/v), LH carses a large increase in the final cell density (5-10 x over the control) while modestly steimulating the actual growth rate. Lactalbumin hydrolyzate does not contain survival activity for SV3T3 cells and does not affect the growth of 3T3 cells. Since even prolonged exposure to pH 4 or 2 results in complete abolishment of the growth-rate stimulatory capability without affecting the capacity to increase the final cell density, it is possible that the LH growth activity may consist of two dissociable components. All of LH growth activity is water soluble, autoclavable, and resists proteolytic treatment. On Sephadex G15 growth activity appears as a single peak at the void volume but on G25 it is retained beyond the void volume as a broad, skewed peak. The relevant molecular weight range lies between 1,500 and 4,000. The putative low pH resistant material is strongly adsorbed to Dowex 1 x 2 and can be displaced from the column by a reduction in the pH. A comparison of the properties of the LH factors with those of known growth promoting agents isolated from serum and various enzymatic digests indicates that these new factors do not correspond to any of these agents.


Energy metabolism in respiration-deficient and wild type Chinese hamster fibroblasts in culture. This paper presents a comparison of energy metabolism in wild type and respiration-deficient Chinese hamster cells. From previous work (DeFrancesco et. al., '75) it was concluded that the mutant satisfies essentially all of its energy requirements from glycolysis and in this study we measure precisely the amount of glucose consumed and lactate produced per milligram increment of protein in exponentially growing cultures. From these measurements we calculate the amount of ATP derived from glycolysis (and hence the total energy requirement for normal proliferation) to be 105 +/- 15 mumoles ATP/delta mg protein in the mutant. It is 63 +/- 10 mumoles ATP/delta mg protein derived from glycolysis in wild type cells. We present evidence that the total energy requirement of wild type cells is similar to that of the mutant suggesting that approximately 40% of the energy requirement is derived from respiration. The oxidation of glutamine appears to be more significant than the complete oxidation of glucose to CO2 in these Chinese hamster fibroblasts. The amount of ATP required by the mutant cells per milligram increment of protein is relatively independent of pH.


Central nervous system pH in uremia and the effects of hemodialysis. Rapid hemodialysis of uremic animals may induce a syndrome characterized by increased cerebrospinal fluid (CSF) pressure, grand mal seizures, and electroencephalographic abnormalities. There is a fall in pH and bicarbonate concentration in CSF, and brain osmolality exceeds that of plasma, resulting in a net movement of water into the brain. This syndrome has been called experimental dialysis disequilibrium syndrome. The fall in pH of CSF may be secondary to a fall of intracellular pH (pHi) in brain. Since changes in pHi can alter intracellular osmolality in other tissues, it was decided to investigate brain pHi in uremia, and the effects of hemodialysis. Brain pHi was measured by evaluating the distribution of 14C-labeled dimethadione in brain relative to CSF, while extracellular space was calculated as the 35504=/4 space relative to CSF. In animals with acute renal failure, brain (cerebral cortex) pHi was 7.06+/-0.02 (+/-SE) while that in CSF was 7.31+/-0.02, both values not different from normal. After rapid hemodialysis (100 min) of uremic animals, plasma creatinine fell from 11.8 to 5.9 mg/dl. Brain pHi was 6.89+/-0.02 and CSF pH and 7.19+/-0.02, both values significantly lower than in uremic animals (P less than 0.01), and there was a 12% increase in brain water content. After slow hemodialysis (210 min), brain pHi (7.01+/-0.02) and pH in CSF (7.27+/-0.02) were both significantly greater than values observed after rapid hemodialysis (P less than 0.01), and brain water content was normal. None of the above maneuvers had any effect on pHi of skeletal muscle or subcortical white matter. The data show that rapid hemodialysis of uremic dogs is accompanied by a significant fall in pH of CSF and pHi in cerebral cortex. Accompanying the fall in brain pHi is cerebral edema.


Activation-induced restoration of sensorimotor functions in rats with dopamine-depleting brain lesions. Bilateral electrolytic lesions of the lateral hypothalamus or intraventricular 6-hydroxydopamine injections produced substantial depletions of striatal dopamine in rates. All animals with brain damage showed marked sensorimotor impairments. However, they began to move and respond appropriately to environmental stimuli when placed in a sink of water, in a shallow ice bath, or among a colony of cats or rats. A reversal of the sensorimotor dysfunctions was still apparent shortly after the animals were removed from each activating situation. However, the terapeutic effects dissipated rapidly, and by 4 hr after an exposure the rats responded as poorly as they had prior to activation. These findings are strikingly similar to the "paradoxical kinesia" seen in parkinsonism, a clinical disorder attributed to degeneration of central dopamine-containing neurons. Collectively, they suggest the importance of activation in maintaining responsiveness to senory stimuli in rats following dopamine-depleting brain lesions.


Homogenized milk: is it really the culprit in dietary-induced atherosclerosis? Xanthine oxidase activity was assayed in commercial samples of homogenized milk subjected to pH ranging from 6.7 to 2.0 and held at room temperature for 5 min. Activity decreased sharply between pH 5.5 and 3.2. Below pH 3.2 no activity was detected. Also, rabbit anti-bovine xanthine oxidase failed to crossreact immunologically with xanthine oxidase of mouse milk. These results cast doubt on the hypothesis that dietary xanthine oxidase participates in the formation of atherosclerotic plaques.


Biological aspects of leukotactic factors. Quantitation of leukotaxis involves direct visualization of migrated cells either as total cell numbers or according to depth of penetration of cells into filters. Newer approaches utilize radioactively labeled cells or movement in gel. Considerable controversy exists regarding the structural attributes of chemotactic activity, although synthetic peptides emphasize the importance of primary structure. Leukotactic factors, both complement-derived and lymphocyte-generated, have been found in many conditions both in vitro and in vivo. There is increasing evidence that the leukotactic system plays an important role in the pathogenesis of inflammation.


Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy. The monoamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and tryptophan hydroxylase (TrH) were immunocytochemical localized in dopaminergic, noradrenergic and serotonergic neurons of rat brain by light and electron microscopy. In dopaminergic and serotonergic neurons, the respective synthesizing enzymes. TH and TrH, were distributed throughout the cytoplasm of the neuronal perikarya, dendrites, axons and terminals. The most selective accumulation of reaction product for the specific enzyme was associated: (a) in perikarya with endoplasmic reticulum, Golgi apparatus and microtubules, (b) in processes with microtubules, and (c) in terminals with dense granules or clear vesicles. The labeled terminals were characterized by their content of labeled organelles and the absence of synaptic junctions. In noradrenergic neurons, both TH and DBH were localized in the perikarya, similar to TH in dopamine neurons. TH and DBH differed in their localization within proximal axons and dendrites in that TH was associated with microtubules but DBH was not. These results provide ultrastructural evidence to suggest that monoamines may be: (a) synthesized by enzymes which are associated with different organelles depending on the portion of the neuron and the type of enzyme; (b) synthesized in both axons and dendrites and (c) released from terminals without postsynaptic membrane specializations.


Separation of slow reacting substance of anaphylaxis (SRS-A) from human lung into four biologically active fractions. Slow reacting substance of anaphylaxis (SRS-A) was released from human lung passively sensitized with ragweed antibody and challenged with specific antigen E. After purification by ethanol extraction, incubation with alkali (0.1 M NaOH for 30 min at 37 degrees C) and chromatography on silicic acid and DEAE-cellulose, human SRS-A was separated into four biologically active fractions (Fractions I to IV). Arylsulfatase (Type H-1) in 0.1 M sodium acetate buffer, pH 4.5, destroyed the biologic activity of only Fraction I. All four fractions, like SO4=, inhibited the arylsulfatase activity at pH 4.5 but not at pH 6.0 when p-nitrocatechol sulfate was used as substrate. These results suggest that SRS-A contain a sulfur group and that human STS-A, like the prostaglandins, may be a family of compounds. The instability of the purified SRS-A to storage remains a major barrier to their further purification and chemical identification.


Ability of H-2 regions to induce graft-vs-host disease. Individual young adult F1 hybrid mice were irradiated with 500 R and 24 hr later injected with 5 X 10(7) spleen cells obtained from a sex-matched parental-strain donor. The injected animals were then followed for a period of 3 months and loss of body weight, mortality rate, and other signs of fatal graft-vs-host disease (GVHD) were recorded. The donor-recipient strain combinations were selected in such a way as to provide genetic differences in the entire H-2 complex, the K or D regions alone, the K or the D end, and the central (I) regions alone. The data obtained on only few combinations indicate that strong GVHD (100% mortality rate within the first month after the injection) occurs only in those donor-recipient combinations which differ in the entire H-2 complex or in the K end (K + I regions). Much weaker GVHD (mortality rate of only 50% or less and death of individual mice spread over the entire observation period) is observed when the donor and the host differ in either the K, I, or D region alone. The degree of GVHD induced by three regions, when taken singularly, is about the same. Surprisingly, the K-region GVHD was somewhat stronger in combinations of mutant strains in comparison with recombinant-strain combinations.


Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit. Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.


Participation of cyclophosphamide-sensitive T cells in graft-vs-host reactions. Cyclophosphamide (CY)3 is toxic for a population of mouse T cells which is active in graft vs host (GVH) responses. This observation contrasts with previous reports which indicate that treatment of mice with CY regimens, similar to those used in this study, enhances the reactivity of T cells which mediate delayed type hypersensitivity to sheep red blood cells. The GVH-inducing capacity of T cells is not altered by removal of B cells by nylon wool columns. We suggest that CY-sensitive T cells may be "regulatory cells," and that the effect they have depends on, among other things, the nature of the stimulating antigen to which the cells are responding and/or the assay used for study.


Degradation of gastrin by gastric mucosal cells. Specific binding of radiolabeled gastrin by gastric mucosal cells prepared from guinea pigs was detected and examined. Specific cell binding of gastrin was found to be both pH and temperature dependent. Maximum binding of radiolabeled gastrin by gastric mucosal cells was demonstrated at pH 7.4 and at 4 degrees C. Substantial degradation of 125I-gastrin occurred during incubation with gastric mucosal cells. Gastrin degradation was detected both by loss of immunological reactivity with antibodies to gastrin and by abolition of subsequent specific binding after prior incubation with gastric mucosal cells. Degradation of gastrin was  most marked with incubations conducted at pH 7.4 and at low pH (pH 2.0), suggesting the activities of at least two gastrin-degrading enzyme systems in the gastric mucosal cell preparation.


Renal acidification in sickle-cell disease. Renal acidification was evaluated in patients with sickle-cell disease (HvSS) with both oral NH4CI and NaHC03 and the results were compared to those of subjects with sickle-cell trait (HbAS) and controls. The pH of arterial blood was normal in HbSS subjects but their PC02 and [HC03] were lower than those of controls. In response to NH4CI, six of 20 HbSS subjects had an abnormal minimal urine pH (greater than 5.3) and the entire HbSS group had a higher mean value than did either controls or HbAS subjects. Since none of the six HbSS subjects had evidence of proximal tubular abnormalities, it was concluded that they exhibited the syndrome of incomplete distal renal tubular acidosis. Only one of the six HbSS volunteers with an abnormal response to NH4CI and two of seven with a normal response increased their urinary PC02 normally after bicarbonate loading. PAH clearance was significantly higher and inulin clearance tended to be higher in HbSS subjects than in either controls or HbAS subjects. Maximal concentrating ability was decreased in both sickle-cell groups but more so in HbSS. No adverse effects occurred and no appearance or increase in per cent of sickled cells resulted from short-duration NH4CI acid-loading. No differences were found either in the clinical characterstics or in hematological, renal, and acid-base variables between the HbSS subjects with and without a normal response to acid-loading. The mechanism for the observed renal acidification abnormality remains unknown.


Inflammatory reaction and airway damage in cystic fibrosis. In cystic fibrosis there is chronic infection and inflammatory reaction in the airways, accompanied by destruction and shedding of airway epithelium. Leukocytes migrate into the airways and some disintegrate, liberating deoxyribonucleoprotein that is incorporated into the gel structure of the bronchial mucus. We compared the status of these processes in cystic fibrosis with that in chronic bronchitis and bronchiectasis, by examining the sputum raised from the lower airways. Measurements also were made on sputum induced in normal subjects. The results indicate that migration of leukocytes into the airways and shedding of damaged airway epithelium were minimal in the normal subjects; they were significant in the patients with chronic bronchitis, higher in those with bronchiectasis, and still higher in those with cystic fibrosis. The large increases found in the total content of DNA and solids in the cystic fibrosis sputum were due to increases in the insoluble fraction containing the whole leukocytes and particulate debris that remained when the sputum mucus gel was solubilized with mercaptoethanol. Despite the large increases in the total content of DNA and solids, the contents of mucus gel components and of deoxyribonucleoprotein from disintegrated leukocytes actually present in the mucus gel structure of the cystic fibrosis sputum were not significantly higher than in the sputum from the patients with chronic bronchitis or brochiectasis.


In vitro demonstration of a particular affinity of glomerular basement membrane and collagen for DNA. A possible basis for a local formation of DNA-anti-DNA complexes in systemic lupus erythematosus. In vitro, collagen and collagen-like material in GBM, were demonstrated to have a particular high affinity for any DNA tested (mammalian, bacterial, viral, and plant). GBM fixed DNA 40-80 times more than HGG and BSA and 10-40 times more than bacterial LPS. GBM has a higher affinity for SSDNA than for DSDNA. This binding was inhibited at low pH, low ionic strength, and in the presence of anionic detergents, indicating that the highly negatively charged DNA may interact with the basic site on collagen or GBM by electrostatic forces. This interaction was competitively interfered with by DNA-binding proteins such as Clq. Complexes formed of DNA and anti-DNA antibodies did not exhibit the same binding property as free DNA. However, DNA which was already bound to GBM or to collagen could very efficiently bind anti-DNA antibodies and form immune complexes which would remain on these structures. The biological significance of the binding of DNA to GBM or to collagen should be particularly considered in relation to the pathogenesis of SLE. It is possible that DNA released from disrupted or degenerating cells would bind to surrounding collagen fibers or to basement membranes and then act as an immunoabsorbant for circulating anti-DNA antibodies. Some evidence for an in vivo binding of SSDNA to renal structures was obtained in mice treated with bacterial LPS 2 days before the injection of SSDNA.


The mediator of cellular immunity. XII. Inhibition of activated T cells by Newcastle disease virus. Newcastle disease virus (NDV) can interact in at least two ways with rat T cells. By adsorbing to circulating lymphocytes, the virus can transiently deflect the cells from lymph nodes and inflammatory exudates induced in the peritoneal cavity. T cells are affected regardless of age, state of activation, or position in the mitotic cycle. The effect is reversible and is mediated not only by infectious (I)-NDV, but also by UV-NDV which cannot achieve a complete replication cycle in eggs. But I-NDV has another lasting effect on activated T cells. It is revealed in the failure of virus-treated thoracic duct lymphocytes to transfer cellular resistance to Listeria monocytogenes, delayed-type hypersensitivity to soluble antigens of the parasite, and the permanent exclusion of labeled S-phase lymphocytes from inflammatory foci. Activated T cells are inhibited by virus multiplicites which have little if any effect upon  the proliferative potential of antigen-sensitive T cells or localization of labeled small lymphocytes in lymph nodes. The underlying mechanism has not been determined; however, there are reasons for thinking that NDV has a lethal effect upon activated T cells, because the latter are permissive for virus replication.


An improved method for the determination of creatine kinase activity in serum. An improved method for the determination of creatine kinase activity (EC 2.7.3.2) is described. For the reactivation of creatine kinase serum is first preincubated in the test solution in the presence of dithioerythritol. Then the enzymatic reaction is started by adding creatine phosphate. By optimizing the concentrations in the test solution and by altering the measurement procedure using dithioerythritol as reactivator, the assay is made more sensitive, and a far higher enzyme activity in serum is measured in comparison with other recommended tests. The reaction rate is linear for ten minutes up to 700 U/l. An increase in activity by enzyme dilution was not observed. In this study a sensitivity of detection of 0.9 U/l was achieved. This would be especially advantageous in the detection of the isoenzymes of creatine kinase after chromatography. Within-run precision was 1.2% (CV), day-to-day precision was 2.0% (CV). The test solution is stable for at least 24 hours.


[The determination of cystine aminopoptidase (oxytocinase) with a ENI fast analyzer (author's transl)]. A procedure for the determination of cystine aminopeptidase activity was adapted to a fast analyzer. The various reaction parameters were checked. The precision of the method was about 5%. Cystine aminopeptidase activity was stable for long storage periods at +4 degrees C and -20 degrees C. The normal range for the terminal weeks of pregnancy was determined on samples from female patients with normal pregnancies.


Spectrophotometric studies on the pH of frog skeletal muscle. PH change during and after contractile activity. The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pHi) of sartorius muscles from Rana pipiens, both during the course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodology. Neither the fast realkalinization observed during a twitch nor the slow alkalizing phase of recovery metabolism was affected in an obvious manner when phosphocreatine (PC) hydrolysis was blocked by 1-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscle approach tonic equilibrium are proposed.


Acetyl-CoA production and utilization during growth of the facultative methylotroph Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate. In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate: succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible beta-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51), lacking 'malate synthase' activity has been isolated and its characteristics indicate that this activity is normally essential for growth, of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.


Defined and semi-defined media for the growth of amoebae of Physarum polycephalum. Amoebae of the true slime mould Physarum polycephalum were cultured in two fully-defined liquid media containing amino acids, glucose, three vitamins and a buffered salts solution. Absolute requirements were demonstrated for methionine, haematin, thiamine and biotin, all of which were known to be specific requirements of the plasmodial stage. Methods are described for large-scale culture in three semi-defined media.


Formation of ethylene by Escherichia coli. Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.


The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium. Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.


Electrodermal, cardiac, and respiratory activity to repeated cold pressor stimulation in drug addicts. Skin conductance responses (SCRs), heart rate (HR), and respiration were recorded in various rest, cold pressor (CP), and CP recovery phases in 20 former drug users (DG) and 20 control Ss (CG), matched on sex and age in a repeated measures design. With regard to the abused drugs (barbiturates and narcotic analgesics), the hypothesis was tested that autonomic responses are decreased after long-term use of barbiturates and narcotic analgesics. The results partially supported that hypothesis: (a) SCRs, HR change, and HR deceleration were significantly lower in DG compared to control Ss; (b) HR baseline, respiration rate, and amplitude, however, tended to be increased in DG in the rest and CP phases. A second hypothesis, addressed to different habituation rates of SCRs and HR in both groups, was confirmed for SCRs and partly for HR, indicating that the habituation rates of SCRs, HR change, and HR deceleration were greater in DG than in CG. A third question concerned the consistency of SCR and HR results in both sessions. A trend for consistent results was found as the direction of the group differences was the same in sessions 1 and 2. The CP results were compared with similar findings in the same Ss during visual and auditory stimulation.


Physiological and pharmacological aids in the differential diagnosis of tremor. Physiological and pharmacological studies of more than 150 patients with movement disorders are reported. Particular attention is paid to the differentiation of various types of tremor on the basis of rate, rhythm, and pattern of EMG activity in antagonistic muscles. The typical 'tremor-at-rest' of Parkinson's disease--3-7 Hz activity which alternates between antagonistic muscles--is suppressed, at least briefly, during voluntary activity, at which time typical 8--12 Hz 'physiological tremor' may be seen. Essential tremor and its familial or senile variants also have a characteristic EMG pattern during voluntary activity--5-8 Hz bursts of activity which are synchronous in antagonistic muscles. This type of tremor may also be present in patients with Parkinson's disease and in certain kinships with a Charcot-Marie-Tooth polyneuropathy. Other tremors in association with polyneuropathy ('neuropathic tremor') have different physiological characteristics. Myoclonus is of essentially two types ('positive' with EMG bursts and 'negative' with brief pauses in ongoing activity, as with asterixis) and may, at times, mimic tremor. Certain specific tremors respond predictably to specific pharmacological therapy.


The reliability of pH-values in fetal blood samples - A study of the second stage. In 119 cases fetal blood analysis (FBA) was done in the last 15 minutes before birth together with blood sampling from the umbilical artery (UA) immediately after delivery before the first breath. Using the measured pH-values we investigated the relationship between these variables, in particular the degree of influence of considerable caput succedaneum on pH-values in peripheral blood. Our investigation was performed during the time just before delivery, when the disturbance of blood circulation in the presenting fetal part whuld have the highest influence on pH-values measured from peripheral blood samples. We kept in mind especially the critiques of FBA by WULF et al. [21] and ASSALI [2]. As other authors have concluded, we found a high degree of correlation between the pH evaluated by FBA and that determined in the umbilical artery blood. The coefficient of correlation was: pHact r=0.82 pHqu40 r=0.78. The relationship between the corresponding pHvalues in peripheral (FBA) and arterial (UA) blood is given in Fig. 1 a and Fig. 1 b. The correlation of quadrants showed for the actual pH as well for the pHqu40 p less than 0.01. As derived form this, a high degree of correlation between the pH-values can be seen. The mean pH-values were: (see article) 42 of the children had a severe or moderate caput succedaneum. Fig. 2a and 2b show the relationship between central and peripheral pH-values in these cases. Again a good correlation between the pH-values was observed as in the whole collective. The coefficient of correlation was: pHact r=0.78 pHqu40 r=0.79. Caput succedaneum seems to have only minimal influence on the pH of blood obtained from the presenting fetal part. These results lead us to conclude that blood sampling from the presenting fetal part in the second stage provides almost indentical pH-values as in the central fetal blood.


Interfacial properties of polymethyl alpha-cyanoacrylate and polybutyl alpha-cyanoacrylate. Several physical properties of two polyalkyl alpha-cyanoacrylates relevant to their use in pharmaceutical dosage forms have been investigated. Formation of polymer films at several oil-water interfaces reveals films of diverse morphology. The retardation of solute transfer across an oil-water interface caused by the presence of polymer films formed in situ from the monomers has revealed that the methyl derivative forms the more effective barrier to the test solute, gentian violet. The emulsion-stabilizing properties of the methyl polymer have been studied, and the film-forming properties of the monomers spread from benzene onto water surfaces have been examined using a surface balance.


Colorimetric analysis of immunogenic impurities in acetylsalicylic acid. A rapid and convenient colorimetric method is described for the quantitative determination of the immunogenic impurities in acetylsalicylic acid, acetylsalicylsalicylic acid and acetylsalicylic anhydride. The method involves initial aminolysis of the compounds by ammonia to give salicylamide and subsequent coupling of this with 4-amino-phenazone in the presence of an oxidizing agent. In conjunction with a previously described method for analysis of the anhydride the method allows a specific determination of acetylsalicylsalicylic acid in concentrations down to 0-005%. Applying the methods to 15 different commercial acetylsalicylic acid samples and formulations, acetylsalicylic anhydride and acetylsalicylsalicylic acid were found to be present in amounts ranging from 0-001 to 0-024% and from 0-006 to 0-58% (w/w), respectively.


Methylmercury and the skeletal muscle receptor. Methylmercury at bath concentration of 2 X 10(-5) M was capable of inhibiting muscular contractions of the isolated rat phrenic-nerve hemidiaphragm preparation. At the height of inhibition, nerve action potential could still be recorded and the muscles continued to respond to direct stimulation. The inhibition was not reversible with L-cysteine or D-penicillamine but limited protection was possible by prior treatment with (+)-tubocurarine. Treatment of frog rectus muscles with methylmercury (0-2 mM for 15 min) resulted in a shift to the right of 1 log unit in the dose response curve to acetylcholine and a reduction in the maximum response of the tissue. The observed inhibitory action of methylmercury on neuromuscular transmission may be explained by an action on the disulphide bond believed to be present on a cholinergic receptor.


A method for screening diuretic agents in the mouse: an investigation of sexual differences. Acetazolamide, aminophyline, frusemide, ethacrynic acid and triamterene were tested for diuretic action at dosages of 3, 10 and 30 mg kg-1 (s.c.) in male and female mice. Each drug significantly raised sodium excretion and all but acetazolamide elevated urine volume and chloride excretion. Potassium excretion was significantly raised by acetazolamide and frusemide. Acetazolamide and triamterene evoked urinary alkalinization whereas frusemide and ethacrynic acid reduced urinary pH. Female mice were markedly more sensitive than males to the diuretic, natriuretic, chloruretic and urinary acidfying actions of ethacrynic acid.


Anti-inflammatory actions of dapsone and its related biochemistry. Dapsone has been examined by two established animal anti-inflammatory models and found to possess anti-inflammatory activity comparable with established non-steroidal anti-inflammatory drugs. Dapsone also possess some biochemical properties common to other anti-inflammatory drugs.


Brain tyrosine hydroxylase activity and systolic blood pressure in rats treated with either deoxycorticosterone and salt or angiotensin. The hypertension induced in adult male rats by doca/salt was found to be accompanied by a significant rise in whole brain tyrosine hydroxylase (TH) activity. A smaller hypertensive effect, produced by angiotensin (750 ng kg-1 daily) was also accompanied by a proportional rise in whole brain TH activity. The specific antagonists spironolactone and saralasin completely blocked both responses in the doca/salt- and angiotensin-treated animals respectively and spironolactone showed a partial inhibition of the effects of angiotensin. In all the animals treated there was a clear correlation between systolic blood pressure and whole brain TH activity. The significance of these changes is discussed in the light of the central mechanism of hypertension.


Correlation of behavioural inhibition or excitation produced by bromocriptine with changes in brain catecholamine turnover. The dopamine agonist, bromocriptine, produced either inhibition or stimulation of motor behaviour in rats depending upon the dose and time after administration. Stimulation of motor activity occurred only with high doses after a 1-2 h delay. Both inhibition and stimulation were associated with decreased turnover of dopamine in the brain. Release of noradrenaline in brain and noradrenaline plus adrenaline in adrenal varied with motor activity. It is suggested that low doses of bromocriptine inhibit behaviour by activating an inhibitory presynaptic receptor, resulting in reduced synthesis and release of dopamine, whilst high doses cause behavioural excitation by activating the post-synaptic dopamine receptor.


Dopamine-sensitive adenylate cyclase in canine renal artery. To characterize further a putative dopamine receptor in the renal artery, the effects of dopamine on canine renal artery adenylate cyclase activity were studied. Since the femoral artery is thought to be devoid of a similar dopamine receptor, the effects of dopamine on the adenylate cyclase activity of the canine femoral artery were also studied. In tissues from dogs with or without phenoxybenzamine pretreatment, renal artery adenylate cyclase was maximally stimulated by 4 muM dopamine, compared to 20 muM required for the femoral artery enzyme. The concentrations of isoprenaline required to maximally stimulate renal and femoral artery adenylate cyclase were 0-04 and 0-2 muM, respectively. In tissue from the phenoxybenzamine-pretreated dog, the sitmulatroy effect of dopamine on the renal artery enzyme was selectively blocked by 0-01 muM haloperidol, but not by 0-2 muM propranolol. In the femoral artery, however, the dopamine stimulation was blocked by both antagonists. Stimulation by isoprenaline of renal and femoral artery adenylate cyclase was blocked by propranolol. These data suggest the concept that dopamine interacts with a specific artery receptor apparently different from alpha-and beta-adrenoceptors.


A four parameter model for oral drug absorption. A method has been developed for assessing absorption parameters from plasma concentration-time results taken over a relatively short time period. In this method, the disposition function is simplified so as to reduce the number of parameters to be evaluated from the five of the two compartment disposition equation of four, and to avoid the requirement for the evaluation of a slow disposition rate constant. The measurments need not therefore be continued over a period long after absorption is complete. A suitable design for kinetic experiments using this method for interpreting the results, is described. A random noise statistical method is proposed for assessing the stability of the calculated parameters.


Carbon-13 magnetic resonance spectroscopy of drugs II: antihistamines. The natural abundance carbon-13 magnetic resonance spectra of a series of antihistamines (pheniramine, chlorpheniramine, methapyrilene, tripelennamine, pyrilamine, and thonzylamine) were detemined using the pulse Fourier transform technique. The chemical shifts were assigned with the aid of long-range carbon-13-hydrogen coupling constants.


Ionization constants of cephalosporin zwitterionic compounds. The microionization constants for two zwitterionic compounds were determined by incorporating two experimental techniques. These compounds have chromophoric changes dependent upon the solution pH. By combining the spectrophotometric measurements with potentiometric mmeasurements, all four microionization constants were calculated. The method used is completely general and is applicable to all diprotic compounds that exhibit this spectrophotometric behavior. The observed pKa's had differences of at most 1.2 units for either compound and were in the 1-4 range. A comparison of the results with each compound and similar compounds indicates that the values are resonable.


Correlation of in vitro and in vivo methodology for evaluation of antacids. The rate and extent of acid consumption of an antacid suspension and tablet were evaluated by in vitro and in vivo techniques. Four different test procedures were used to estimate in vitro antacid reactivity. In vivo effects were determined in the fasted and postcibal states in normal human subjects by a radiotelemetry procedure. The duration of elevation of intragastric pH greater than 3 was in agreement with in vitro estimates of total acid consumption of the antacid. There was also good correlation between onset, extent, and duration of in vivo antacid activity and a modified in vitro Beekman antacid test procedure. There was no significant difference in antacid activity of the tablet or suspension in either in vitro or in vivo test procedures. A wide variation in antacid activity was observed between subjects and also in the fasted versus postcibal states. These studies emphasize the requirements for standardization of antacid products by comparactive in vitro and in vivo evaluations to facilitate individualized dose titration  of the antacid in each patient and correlation of the acid secretion rate in various types of GI disease with the antacid dose.


Effect of solvent on tetrazolium reaction. The rate of color development of a tetrazolium formazan is shown to be inversely proportional to the dielectric constant of the solvent medium and directly proportional to the hydrogen-bonding capability of solvent mixtures having the same dielectric constant. The geometric isomers of the formazans have different absorbance maxima, and the wavelength of maximum absorbance of a mixture of formazans in different solvents depends upon which isomer predominates in that solvent. The trans-syn-isomer (blue form) of blue tetrazolium has a maximum absorbance at 625 nm in dimethylformamide while the trans-anti-isomer (red form) absorbs at 517 nm in methanol. The absorbance maxima of the corresponding isomers of the formazans of triphenyltetrazolium occur at 535 and 485 nm, respectively. Water and/or methanol (to a lesser extent) are important in the stabilization of the trans-anti-isomer, since the small size of these two substances allows them to form strong intermolecular hydrogen bonds with one or both nitrogen atoms of the azo linkage, thereby preventing the formation of the intramolecular hydrogen bonding exhibited by the trans-syn-isomer. The formazan produced by the reaction of corticosteroids with tetrazolium in strongly basic media can lose a reduction unit and be reoxidized to the tetrazolium. This reaction is solvent dependent and occurs at a much faster rate in chloroform than in alcohol USP.


Aggregation of antihistamines in aqueous solution: effect of counterions on self-association of pyridine derivatives. The effects of electrolytes on the self-association of the antihistaminic drugs, tripelennamine hydrochloride, thenyldiamine hydrochloride, pyrilamine maleate, pheniramine maleate, chlorpheniramine maleate, and brompheniramine maleate, in aqueous solution were examined by light-scattering from tripelennamine bydrochloride and thenyldiamine hydrochloride in 0.154 mole of sodium chloride/kg and 0.150 mole of sodium maleate/kg indicated a micellar pattern of aggregation. Higher aggregation numbers and lower CMC's were determined in the presence of the maleate ion. No significant discontinuity in the concentration dependence of the light scattering of the remaining compounds in either of the two electrolytes was evident, and the aggregation of these compounds was treated using a stepwise association model. Values of the association constants and the limiting number of associating species were, in general, increased by the addition of electrolyte in the order water less than sodium chloride less than sodium maleate. An apparently nonmicellar pattern of aggregation could be induced by chemically changing the counterion from chloride to maleate.


Enthalpies of hydrogen bonding in psychotropic drugs. The enthalpy of hydrogen bonding of some antipsychotic, antidepressant, anticonvulsant, and antianxiety agents with phenol, as determined from IR and NMR spectroscopic measurements, was shown not to be responsible for differences in activity within the drug classes. These results support a theoretical prediction advanced for anticonvulsant activity.


Synthesis, physicochemical parameters, and in vitro evaluation of N1-p-chlorophenyl-N5-alkylbiguanides. A series of N1-p-chlorophenyl-N5-alkylbiguanides were synthesized as potential inhibitors of dental plaque. Partition coefficients and pKa values were determined by standard methods. Biological activity was evaluated against Streptococcus mutans, a pure strain of plaque-forming bacteria. All compounds were compared to chlorhexidine acetate.


A quantitative study of metiamide, a histamine H2-antagonist, on the isolated whole rat stomach. 1. An isolated whole stomach preparation from immature rats is described. The lumen of the stomach was perfused and the hydrogen ion activity of the perfusate recorded continuously. 2. The mean basal acid secretion before stimulation was 4-19 +/- 0-31 X 10 (-8) mol min-1. Metiamide did not significantly reduce this spontaneous secretion. 3. The preparation gave dose-dependent responses to histomine (10(-5)-10(-4)M) which were readily reversed on washing. 4. The effect of metiamide on histamine-stimulated acid secretion was investigated. Metamide, at doses of 3 X 10(-6)M, and 3 X 10(-5)M, caused a parallel displacement of the histamine dose-response curve, indicating competitive antagonism. These dose-response curves were used to calculate dose ratios (DR) for metiamide. 5. A plot of log10 (DR - 1) against log 10 [antagonist] gave a pA2 value for metiamide of 5-91.


Evidence that brain prostaglandin synthesis is not essential in fever. 1. We have tested the hypothesis that a fever caused by pyrogen depends upon the synthesis of prostaglandin E in the brain and that the prostaglandin in turn acts on the hypothalamus to produce fever. 2. In rabbits, fever was produced by the injection of leucocyte pyrogen in a lateral cerebral ventricle. The latency, rate of rise and magnitude of the fever was unaffected by the simultaneous intraventricular injection of two prostaglandin antagonists, SC 19220 and HR 546. 3. Both antagonists effectively attenuated the fever caused by the intraventricular injection of prostaglandin E2. 4. This evidence is not consistent with the hypothesis that prostaglandin E is the principal mediator of fever.


Non-uniform probabilities of quantal release at the crayfish neuromuscular junction. 1. Transmitter release at the neuromuscular junction of the crayfish walking leg has been found to deviate from binomial predictions immediately after the onset of repetitive stimulation of the presynaptic nerve at frequencies of at least 15 Hz. 2. After several minutes of continuous stimulation, and at lower rates of stimulation, however, the number of quanta released could be described quite well by binomial or Poisson statistics. 3. Deviations from the theoretical expectations were characterized by (a) fewer release failures than predicted, (b) occasions in which the number of quanta released was more than the estimated number of quanta available for release, and (c) a tendency for the data to be underdispersed. 4. Each of these three characteristics are consistent with the hypothesis that different releasable quanta may have different probabilities of responding to a nerve impulse. 5. Using two different methods, different values of the non-uniform probabilities were estimated from the data. At each synaptic site at least one of the estimated probabilities was very high. 6. The need for caution in interpreting statistical description of quantal release is emphasized.


Caudate stimulation and substantia nigra activity in the rat. 1. The responses of spontaneously active single neurones in the substantia nigra and overlying mesencephalic reticular formation have been analysed during the electrical stimulation of the ipsilateral caudate nucleus. Experiments were performed in rats anaesthetized with urethane or pentobarbitone. All recordings were made extracellularly with multi-barrelled glass micropipettes which were also used to test neuronal responsiveness to electrophoretically administered substances. The micropipette tip position was marked and the distribution of neurones studied has been analysed. 2. Single shock stimulation of the caudate nucleus inhibited neuronal activity in the substantia nigra (270/320 cells: mean latency 5-4 msec) and in the mesencephalic reticular formation (62/72 cells: mean latency 16-6 msec). However, these effects were often accompanied by periods of excitation. In pentobarbitone anaesthetized animals the latency and duration of these substantia nigra inhibitions was increased. 3. Compared with the zona reticulata, fewer neurones in the zona compacta of the substantia nigra responded to caudate stimulation in both urethane or pentobarbitone anaesthetized animals. 4. The activity of most cells was depressed by electrophoretically administered GABA or glycine and increased by acetylcholine or glutamate. Neurones of the mesencephalic reticular formation were less sensitive to GABA and glycine than substantia nigra neurones. Within the substantia nigra, both zona compacta and zona reticulata neurones were more sensitive to GABA than to glycine. Over-all, glutamate was a more potent excitant than acetylcholine (ACh). 5. Electrophoretic bicuculline methochloride (BMC) consistently reduced GABA but not glycine depression of substantia nigra neurones. Approximately twice as much BMC was required to reduce the endogenous inhibition of the same substantia nigra neurones and the amplitude of concomitantly evoked positive field potential as was required to abolish exogenous GABA responses. Some evoked substantia nigra inhibitions were resistant to BMC. 6. Electrophoretic strychnine consistently reduced glycine but not GABA depression of substantia nigra neurones, and did not modify caudate evoked inhibition of these neurones or the accompanying field potential. 7. The results support the concept of a slowly conducting caudato-nigral pathway which has both facilitatory and inhibitory components. The inhibitory pathway uses GABA as the neurotransmitter. The identity of the possible excitatory transmitter is unknown. The monosynaptic nature of this pathway is uncertain and the possible contribution of other bicuculline insensitive nigral inhibitory processes is discussed.


Eye protection from light radiation.

Eye protection from light radiation. It appears that green absorptive lenses are highly desirable for the purposes of shielding the eyes against light radiation from molten metal during the casting and soldering procedures. The dental laboratory technicians have experienced no after-images, less eye fatigue, and no adverse symptoms when using these lenses, as were reported previously. The lenses not only effectively protect the eyes from the injurious light wavelengths, but they also provide for enough normal (as well as individually corrected) vision to accomplish any procedure necessary in the dental laboratory. We propose that the green absorptive lenses, with individual corrections if needed, be considered for dental laboratory technicians during dental laboratory procedures when needed, such as during casting and soldering, to protect their eyes from light radiation.


Giant cell arteritis in general practice. The clinical syndrome of polymyalgia rheumatica is reviewed. The relationship of this disease with temporal arteritis is discussed, and I consider both syndromes have a pathological basis of generalised giant cell arteritis.Seven cases of polymyalgia and four cases of temporal arteritis were recorded during the six-year period (1969-1975) in one general practice.The outlines of management are discussed, with a plea for earlier recognition of the syndromes of polymyalgia rheumatica and temporal arteritis in general practice.


[Arteriography in polyarteritis nodosa. 15 cases (author's transl)]. Despite its imperfections, arteriography, when it demonstrates aneurysms, is an important element in diagnosis in all those cases in which histological findings are not definitive. It should be carried out before renal biopsy, given the risk of traumatic rupture of a possible aneurysm. The discovery of isolated distal arterial stenoses is not characteristic, though in the presence of a suggestive clinical picture, amy be considered as an argument in favour of the diagnosis. They occur frequently in the arteries of the digestive tract even in the absence of any abdominal symptoms or signs. Arteriography also has a prognostic value in establishing the extent of arterial lesions and, finally, is useful in the diagnosis of certain complications such as visceral haematomas and digestive haemorrhages. We thus feel that polyarteritis nodosa should be the object of a complete vascular exploration, including aortography with selective renal studies but also coelio-mesenteric opacification. In the light of the clinical context, the distal limb arteries may also be explored. Thus the diagnosis of polyarteritis nodosa is not purely histological but also arteriographic.


Bifidobacteria in the intestinal tract of infants: an in-vivo study. Weekly faecal specimens from 18 babies were examined during the first 8 weeks of life. Eight infants were breast fed, ten were bottle-fed. All suckling infants received supplementary feeds for the first 8 days. A buffer consisting of acetic acid and acetate was demonstrated in the faeces of all the breast-fed infants at some time during the period of examination. This buffer was rarely detected during the 1st week of life when supplementary feeds were given, and buffer already present gradually disappeared with the introduction of mixed feeding. In contrast, at no time was an acetate buffer demonstrated in the faeces of bottle-fed infants. Babies receiving breast milk produced faeces with low pH, high counts of saccharolytic organisms including bifidobacteria and Streptococcus faecium, and low counts of Escherichia coli, bacteroides and clostridia. Bottle-fed infants on the other hand produced faeces with a high pH and high counts of E. coli and putrefactive bacteria, but with low counts of bifidobacteria.


Bifidobacteria in the intestinal tract of infants: an in-vitro study. In-vitro studies showed that a number of factors are likely to influence the production and maintenance of a bifidobacillary flora and low pH in the faeces of newborn infants. Considerable importance is attached to the nature of the end products of bacterial metabolism in the large intestine. Thus, there is evidence to suggest that acetic acid and other metabolites of intraluminal bacterial growth suppress the growth of gram-negative organisms, but are without effect upon that of bifidobacteria. This mechanism in turn is controlled by the nature of the feed; important factors in breast milk include high lactose, low protein and low phosphate content.