Formation of sulfone metabolites from chlorpromazine and perazine in man. Didesmethylchlorpromazine sulfone [gamma-(2-chlorophenothiazinyl-10)-propylamine sulfone] has been isolated from the urine of a patient under continuous chlorpromazine therapy and identified by mass spectrometry. The same compound was present in organs of rats after injection of the corresponding sulfide. The Cl-free analogue, gamma-(phenothiazinyl-10)-propylamine sulfone, was excreted by patients receiving perazine and by a volunteer after ingestion of the primary amine sulfoxide.


Comparison of biliary excretory function and bile composition in male, female, and lactating female rats. The rates of excretion of phenol-3,6-dibromphthalein disulfonate, ouabain, indocyanine green, and amaranth into the bile of male and female rats were found to be similar. The rates of bile production in male and female rats were also similar whereas that of lactating rats was 50% greater. The increase in bile production occurred during pregnancy and decreased rapidly after removal of the pups from the dam. The increased bile production was of canalicular origin and was due to an increase in both the bile salt-dependent and independent fractions. The lactating rats tended to excrete some xenobiotics more rapidly than did the other rats, but no difference in the biliary excretion was observed in male and female rats.


Dihydropyrimidinase. Stereochemistry of the metabolism of some 5-alkylhydantoins. The (R)- and (S)-isomers of 5-methylhydantoin (5-MH) and of 5-isopropylhydantoin (5-IPH) were synthesized, and incubations of the individual isomers with a rat liver dihydropyrimidinase preparation (100,000g supernatant fraction) were carried out. Only the (R)-isomer of 5-MH or 5-IPH was ring-opened by the enzyme. Reversibility of the enzymatic ring-opening reaction could be demonstrated with only the (R)-isomer of 2-methylhydantoic acid (2-MHA) or 2-isopropylhydantoic acid (2-IPHA). The results of the present investigation show that the replacement in 5-phenylhydantoin of the phenyl group with an alkyl group does not alter the stereospecificity of the hydantoin substrates in the ring-opening reaction. The results are used to form the concept that (R)-dihydrothymine, the optical isomer previously postulated as the natural substrate to the enzyme, may have a different type of binding at the active site of the enzyme.


Dihydropyrimidinase. Metabolism of some cyclic imides of different ring size. The ability of dihydropyrimidinase (EC 3.5.2.2) to hydrolyze cyclic imides of different ring size was investigated. Succinimide, glutarimide, and adipimide are five-, six-, and seven-membered cyclic imides, respectively. The ring-opened compounds that correspond to these cyclic imides are, respectively, succinamic, glutaramic, and adipamic acid. In incubations of cyclic imides (pH 8, 1 hr) with a rat liver dihydropyrimidinase preparation from which omega-amidase had been removed, adipimide was classed as a good substrate and succinimide and glutarimide were classed as very poor but definite substrates. alpha-Phenylsuccinimide, the N-demethylated metabolite of phensuximide, was a much better substrate than succinimide. alpha-Phenylglutarimide was not a substrate. The in vitro studies of the present investigation were in agreement with observations made in previous in vivo studies.


Metabolism and tissue distribution of mono-2-ethylhexyl phthalate in the rat. The absorption, distribution and metabolic excretion of mono-2-ethylhexyl [7-14C]phthalate (MEHP) were studied in the rat. This compound was readily absorbed from the gastrointestinal tract. Radioactivity following intravenous administration of 14C-MEHP was rapidly distributed in all tissues, with the highest levels occurring in the liver, kidney, and urinary bladder. Excretion of radioactivity was rapid and approximately 80% of the dose was eliminated 24 hr after oral administration, 72% in the urine and 8% in feces. MEHP was extensively metabolized after oral administration, and the major urinary metabolites were identified as an alcohol, a ketone, and an acid resulting from the side-chain oxidation of MEHP. A trace of o-phthalic acid was also identified.


Species, sex, and developmental differences in the O- and N-dealkylation of ethylmorphine by hepatic microsomes. 3-O-[1'-14C]Ethylmorphine and ethylmorphine, respectively, were used to measure O- and N-dealkylase activities of hepatic microsomes. The well-known sex difference in the rate of N-delakylation in mature rats was not observed with O-dealkylation, nor did O-dealkylase activity increase as male rats reached maturity, as is the case with N-dealkylase activity. Accordingly, O-dealkylation represents only about 20% of the total dealkylase activity (O- + N-) in mature male rats, but about 50% in mature female rats. A comparison of the O- and N-dealkylating activities of hepatic microsomes from rats, mice, guinea pigs, and rabbits showed that both the rates of O- and N-dealkylation and the ratio of the two reactions vary greatly among animal species. These studies contribute to the evidence that different cytochrome P-450-dependent mono-oxygenase systems are involved in the O- and N-dealkylation of opium alkaloids.


Levodopa pharmacokinetics. Alterations after benserazide, a decarboxylase inhibitor. Plasma levodopa decay curves appeared to be triexponential up to 6 hr after single intravenous injections of levodopa in rats, rabbits, and dogs. After pretreatment of each of these three species with benserazide (Ro-4-4602), a decarboxylase inhibitor, plasma levodopa concentrations declined biexponentially. Administration of benserazide decreased both total plasma clearance and apparent volume of distribution of levodopa, but did not change the rate constant of the terminal phase of the plasma levodopa decay curve. Thus, decarboxylase inhibition failed to alter the plasma half-life of levodopa.


Biotransformation products of 3,4,4'-trichlorocarbanilide in rat, monkey, and man. 3,4,4'-Trichlorocarbanilide (TCC), uniformly labeled with 14C in the monochloro ring, was administered to rats, rhesus monkeys, and humans. Radioactive materials in the plasma and urine of all three species and in the bile of rats and monkeys were separated by high performance liquid chromatography. The chromatography showed great similarity between the monkey and the human. Principal metabolites common to all species were the sulfate and glucuronide conjugates of 2'-, 3'-, and 6-hydroxy-TCC. The rat also produced the glucuronide and sulfate conjugates of 2',6-dihydroxy-TCC. The major urinary excretion products found in humans and monkeys were the N- and N'-TCC glucuronides.


The absorption, excretion, and biotransformation of 3,4,4'-trichlorocarbanilide in humans. The metabolism and disposition of 14C-TCC (3,4,4'-trichlorocarbanilide) have been evaluated in humans following oral exposure to 2.2 mumol/kg body wt. Fecal elimination (70% of dose) was complete 120 hr after dosing and the urinary excretion (27% of dose) was completed in 80 hr. The maximum plasma level occurred 2.8 hr after dosing and was 3.7 nmol-equivalents of TCC per g of plasma (approximately 1.2 ppm). Biotransformation of TCC was rapid but did not appear to involve splitting of the basic TCC structure. The major plasma metabolites were N- and N'-glucuronides of TCC which were eliminated with t1/2 approximately 2 hr to the urine and 2'-hydroxy-TCC sulfate and 6-hydroxy-TCC sulfate (the o-hydroxy-TCC sulfates) which were removed with t1/2 approximately 20 hr (presumably into the bile). It is concluded that a nonradioactive analytical method based on the urinary excretion of the N-glucuronides would be suitable for the determination of TCC absorption in humans.


Metabolism of cyclobenzaprine in the dog. Ten metabolites of cyclobenzaprine, accounting for approximately 50% of the urinary radioactivity, were identified in the urine of dogs to which the labeled drug had been given orally. These included the 1,2-dihydrodiol, three phenolic derivatives, the N-oxide, the 10,11-epoxide, the 10,11-glycol, desmethylcyclobenzaprine, and the glucuronide conjugates of desmethylcyclobenzaprine and cyclobenzaprine. The metabolites were excreted in both the free and conjugated states. Unchanged cyclobenzaprine was present in only minor amounts.


Pharmacokinetic model of presystemic metabolism. A pharmacokinetic perfusion model is presented that describes the disposition of drugs that are subject to both first-pass hepatic and intestinal epithelial metabolism. Equations are developed to estimate hepatic and intestinal epithelial clearances by determining drug concentrations as a function of time in both portal and peripheral vein blood after oral and iv administration, and by determining or estimating the flow rate of blood perfusing the gastrointestinal tissues. A method that can be used, under the stated conditions, to approximate the systemic availability after oral administration of drugs subject to presystemic metabolism is also developed.


Studies on metabolism of bromazepam. VI. Reduction of 2-(2-amino-5-bromobenzoyl)pyridine, a metabolite of bromazepam, in the rabbit, rat, and guinea pig. Three urinary metabolites that were formed by cleavage of the benzodiazepine ring of bromazepam, 2-(2-amino-5-bromobenzoyl)pyridine (ABBP), 2-(2-amino-5-bromo-3-hydroxybenzoyl)pyridine (3-OH-ABBP), and 2-amino-5-bromo-2'-azabenzhydrol (ABAB), were measured in urine of rabbits, rats, and guinea pigs. The major metabolite was 3-OH-ABBP in all animals given bromazepam orally. ABAB was also excreted in major amounts in the guinea pig, but was excreted in minor amounts in the rabbit and rat. Moreover, ABAB was excreted in the urine of all animals given ABBP orally. It may be concluded that ABAB was formed by reduction of the carbonyl group of ABBP. ABBP reduction was catalyzed by NADPH-dependent enzymes occurring in rabbit liver cytoplasm, and rat liver microsomes, and guinea pig liver cytoplasm and microsomes. The reductases were inhibited by sulfhydryl group reagents. The optimum pH of the cytoplasmic enzyme ranged from 7.2 to 7.8, and that of the microsomal enzyme was 6.5. The apparent KM value for the reduction of ABBP by guinea pig liver microsomes was the lowest among all of the liver preparations.


Metabolism of levorotary 4,5-dihydrodiazepam in the rat. The metabolism of (-)-4,5-dihydrodiazepam (7-chloro-1,3,4,5-tetrahydro-1-methyl-5-phenyl-2H-1,4-benzo[2-14C]diazepin-2-one) (I) was investigated in rats. Metabolites from urine and bile extracts purified by thin-layer chromatography were identified by mass spectrometry. Diazepam and its main metabolites were found among the biotransformation products of I. Based on these findings, the most important identified metabolic route of the compound studied appears to be the formation of an unsaturated bond between the N4 and C5 atoms, and further transformation of the diazepam formed.


Microsomal N-hydroxylation of trans-4'-alkoxy-4-acetamidostilbenes. Hamster liver microsomes catalyze the N-hydroxylation of the 4'-OCH3, 4'-OCH2CH3, 4'-O(CH2)2CH3, and 4'-O(CH2)3CH3 analogs of trans-4-acetamidostilbene. Other metabolites which were identified were the O-deethylation product of the 4'-OCH2CH3 analog as well as the alpha,beta-epoxides of the 4'-OCH2CH3 and 4'-O(CH2)2CH3 compounds and the (omega-1)-hydroxylation product of trans-4'-n-butoxy-4-acetamidostilbene. The kinetics of metabolism of the 4'-O(CH2)3CH3 analog were determined, but the rates of formation of the metabolites of the other members of the series were not linearly related to substrate concentration.


Microsomal mixed-function amine oxidase. Oxidation products of piperazine-substituted phenothiazine drugs. Oxidation products of fluphenazide, thioproperazine, and trifluoperazine obtained in reactions catalyzed by homogeneous preparations of the microsomal mixed-function amine oxidase have been isolated and identified. Approximately 0.5 g of metabolite of each piperazine-substituted phenothiazine drug was prepared in reactors containing, as catalyst, the purified oxidase covalently attached to glass beads. Nuclear magnetic resonance spectra of the isolated products indicated that with all three substrates the enzyme preferentially catalyzes N-oxidation of the piperazine nitrogen furthest from the phenothiazine nitrogen atom. The enzyme-catalyzed oxidation is quite specific and oxidation of the sulfur or nitrogen atoms in the phenothiazine ring could not be detected. Concentrations of piperazine-substituted phenothiazines required to half-saturate the amine oxidase were in the micromolar range and at pH 8.3 and 37 degrees C, all those tested were oxidized at approximately 2 mumol/min/mg of enzyme. Kinetic constants for the piperazine-substituted phenothiazines were very similar to those obtained with phenothiazines containing a dimethylaminopropyl sidechain.


Influence of pregnancy and folic acid on phenytoin metabolism by rat liver microsomes. Liver microsomal suspensions from pregnant and nonpregnant Sprague-Dawley rats were assayed for NADPH-dependent metabolism of phenytoin to the hydroxylated products, 5-(rho-hydroxyphenyl)-5-phenylhydantoin (rhoHPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)5-phenylhydantoin (H2DIOL). UDP-glucuronic acid-dependent glucuronyltransferase activity for conjugating rhoHPPH was also measured. The specific activity for formation of rhoHPPH decreased by 35% in 21-day pregnant rats compared with nonpregnant rats, whereas specific activity for production of H2DIOL was increased approximately 2-fold on the 7th and 14th but not the 21st day of pregnancy. Because of a 25% increase in liver weight during pregnancy, the total hepatic phenytoin hydroxylase activity in pregnant rats (day 21) was not significantly different from that of nonpregnant controls. Folic acid treatment during pregnancy prevented the decrease in phenytoin hydroxylase specific activity. The KM for production of either rhoHPPH or H2DIOL was similar (90 micrometer) in pregnant (day 21) and nonpregnant animals. Thus, overall hepatic microsomal enzyme activity for metabolizing phenytoin was not significantly reduced during pregnancy in rats, although the ratio of H2DIOL to rhoHPPH was increased during the first two weeks. Folic acid may play a role in pregnancy-associated changes in phenytoin hydroxylase activity.


Quantitation of N-demethylantipyrine in biological samples and isolation and characterization of its glucuronic acid conjugate. An improved method for quantitating N-demethylantipyrine (N-DEM-AP) in urine by gas chromatography or gas chromatography-mass spectrometry has been developed. Recovery of greater than 90% of N-DEM-AP was achieved by extraction of the sample at pH 1 after addition of 3-amino-1-phenyl-2-pyrazolin-5-one. The coefficient of variation of replicate analyses was 8%. N-DEM-AP was excreted in the urine as a glucuronic acid conjugate. This conjugate was isolated from the urine of an individual receiving antipyrine and purified. The NMR and mass-spectral data are consistent with the conjugate being an O-glucuronide of N-DEM-AP in its enol form.


Alterations in the pharmacokinetics of 3H-delta9-tetrahydrocannabinol in mice by bacterial endotoxin. Mice were pretreated with either bacterial endotoxin (3 mg/kg, ip) or saline 3 hr prior to an ip injection of 100 mg of 3H-delta9-tetrahydrocannabinol (3H-delta9-THC) per kg, a combination that results in synergistic lethality. Five hours after the injection of 3H-delta9-THC, the endotoxin-pretreated mice contained a significantly greater amount of radioactivity in kidney, liver, heart, plasma, urine, and brain than those without pretreatment. However, the plasma and brain levels of the animals tested with both endotoxin and 3H-delta9-THC (100 mg/kg) were significantly less than those in mice treated only with 200 mg of 3H-delta9-THC per kg, a sublethal dose. The percentage of radioactivity corresponding to unchanged 3H-delta9-THC in extracts of livers and brains was significantly greater in the endotoxin-pretreatment group than in controls. The brain level of 3H-delta9-THC in the endotoxin-pretreated mice was 50% higher than that in the saline-pretreated mice 5 hr after 3H-delta9-THC (100 mg/kg) administration. However, the brain levels of 3H-delta9-THC in the mice treated with endotoxin and 3H-delta9-THC (100 mg/kg) was one-third of that in brains of mice receiving the LD50 of 3H-delta9-THC (300 mg/kg).


The metabolism of (2-cyclopentyl-6,7-dichloro-2-methyl-1-oxo-5-indanyloxy)acetic acid in chimpanzee and man. The metabolism of the polyvalent saluretic agent (2-cyclopentyl-6,7-dichloro-2-methyl-1-oxo-5-indanyloxy)acetic acid was studied in chimpanzee and man. The drug was well absorbed and extensively metabolized by man. Peak levels of drug (5--8 microgram/ml) occurred within 1.5--4.5 hr of drug administration. The plasma half-life was estimated to be 2 hr; a similar half-life was observed in the chimpanzee. Little unchanged drug (less than 10%) was excreted in the urine of either species. Similar metabolic profiles were obtained for man and chimpanzee. The major urinary metabolites resulted from hydroxylation of the cyclopentyl moiety, giving rise to a number of diastereomers. The alcohol metabolites were subsequently oxidized to the ketone. The excretion of the metabolites coincided with maximal excretion of sodium and chloride ions. The hydroxylated metabolites have intrinsic pharmacological activity.


Urinary metabolites of 3,5-di-(1-[13C]methyl-1-methylethyl)4-hydroxytoluene (BHT-13C) in man. Butylated hydroxytoluene (BHT) containing the stable isotope 13C was synthesized from 2-[13C]methylpropan-2-ol. A minor constituent of urine following ingestion of BHT-13C by a human volunteer was identified as 3,5-di-(1-[13C]methyl-1-methylethyl)-4-hydroxybenzoic acid, The major metabolite detected was 13C-labeled 5-carboxy-7-(1-carboxy-1-methylethyl)-3,3-dimethyl-2-hydroxy-2,3-dihydrobenzofuran. Detailed spectral analysis provided the basis for structural assignment. No evidence was found for intermediate metabolites leading from BHT-13C to the above compounds. Analysis of one fraction indicated the presence of traces of S-(3,5-di-(1-[13C]methyl-1-methylethyl-4-hydroxy-benzyl)-N-acetylcysteine. Analysis of serum samples by gas chromatography detected only trace quantities of unchanged BHT. No evidence was found for the presence of other nonpolar or unconjugated metabolites.


Elimination of radioactivity following administration of [15,16-3H]naltrexone to rats and guinea pigs. The elimination of radioactivity after [15,16-3H]naltrexone administration was studied in rats and guinea pigs. An average of 42% of the dose was eliminated in urine and 55% in feces following administration of 1 mg/kg iv to each of three rats. Analysis of radioactivity in the excreta of one rat that received the same dose im yielded similar results. On the other hand, four guinea pigs that received 1 mg/kg iv excreted only 14% of the dose in feces and 84% in urine. Similar results were obtained following im administration to guinea pigs at 1 and 20 mg/kg doses. In guinea pig excreta, an average of 64% of the dose corresponded to naltrexone and conjugates, 19% to beta-naltrexol and conjugates, and 2% to alpha-naltrexol and conjugates. In urine, the radioactivity corresponding to alpha-naltrexol and naltrexone was present mainly in conjugated form, whereas apparent beta-naltrexol was mainly unconjugated. The radioactivity in feces corresponded principally to unconjugated naltrexone and beta-naltrexol.


Lipid-soluble inhibitors of dihydrofolate reductase. I. Kinetics, tissue distribution, and extent of metabolism of pyrimethamine, metoprine, and etoprine in the rat, dog, and man. With the aim of developing anticancer compounds which overcome some of the clinical limitations of the polar dihydrofolate reductase inhibitor, methotrexate, the physicochemical properties, kinetics, and metabolism of a series of lipid-soluble 2,4-diamino-5-phenylpyrimidine folate antagonists have been studied. Metoprine and etoprine, potent inhibitors of mammalian dihydrofolate reductase, were compared with pyrimethamine, a widely used antimalarial drug. The development of assay procedures in our laboratory and the synthesis of radiolabeled compounds have enabled a comparison of the kinetic characteristics and tissue distribution of these compounds in several species. The relative lipophilicities as indicated by the octanol/water partition coefficient are: etoprine (log P = 3.19) greater than metoprine (log P = 2.82) greater than pyrimethamine (log P = 2.69). Etoprine has the greatest affinity for plasma proteins, but all three compounds are bound to human plasma protein by 87% or more at therapeutic concentrations. Pharmacokinetic studies in the mouse, rat, dog, and man indicate that metoprine has the longest plasma half-life in all four species. The mean plasma half-lives in man are: pyrimethamine, 85 hr; metoprine, 216 hr; etoprine, 176 hr.


[Assessment of vigilance].

[Assessment of vigilance]. Two aspects of vigilance are to be distinguished: the quantitative one refers to sleep-wakefulness dimension and the qualitative one refers to focusing of attention. Their combination leads to a curvilinear relationship between activation and performances. After describing the psychometric characteristics of vigilance tests, some examples are given of simple paper-pencil or more sophisticated methods. Physiological indices are also approached. The sources of variation influencing vigilance measurement are presented and also some useful experimental designs. At last some remarks are made on statistical exploitation and interpretation of results.


Pharmacokinetics and steady-state myocardial uptake of disopyramide in the dog. The pharmacokinetics and steady-state myocardial uptake of the antiarrhythmic drug disopyramide (DP) were determined in dogs after oral or intravenous administration of [14C]disopyramide phosphate. DP was absorbed rapidly and its absolute oral bioavailability was about 70%. Significant dose-dependent kinetics were not apparent after 7.5- to 30-mg/kg po doses. Plasma half-lives of DP were about 2.9 and 1.2 hr after the po and iv doses, respectively. DP and its N-dealkylated metabolites were largely excreted in the urine and their composition was qualitatively similar after the po and iv doses. Marked differences in the protein binding of DP in human and dog plasma were found. In the papillary muscle, ventricular septum, and ventricles of one dog, the steady-state concentrations of DP and its less active mono-N-dealkylated metabolite were about twice those in plasma, whereas in the atria and tricuspid and mitral valves they were similar to those in plasma.


[Influence of pipotiazine on schizophrenia]. This study is an account of thirty cases of schizophrenia treated experimentally in open with Pipotiazine (19366 R.P.), these cases, of the Kraepelin's Dementia Praecox type, in serious or complicated forms necessitating important measures of hospital care. The quantitative and qualitative therapeutic results, very favourable and original, consist in the transformation of these serious forms into simple or latent forms of schizophrenia. The undesirable effects, rare and always regressive, were all the smaller in the measure as the favourable effects were greater, which indicates a precise impact on certain determinants (central control of affectivity and the representation of the autonomous body). Pipotiazine develops a curative process in the personality characterized mainly by the emergence of lucidity, narcissistic euphoria (anti-persecutory and anti-splintering) and by the apprehension of intrapsychic splitting, setting in motion the necessary psychological task of reconstruction.


Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Effect of phenylalanine, NaCl and pH on the protein conformation. The effects of phenylalanine, NaCl and pH on the conformation of chorismate mutase/prephenate dehydratase have been investigated, using measurements of far and near-ultraviolet circular dichroic spectra and ultraviolet difference spectra. At pH 8.2 in 20 mM Tris-Cl buffer the enzyme was found to contain 10-20% helix and 40-50% beta-structure. There was little or no change in these values on the addition of 1 mM phenylalanine (the allosteric effector) or 0.4 M NaCl or by decreasing the pH to 7.4. Both phenylalanine and NaCl caused significant changes in the conformation of the enzyme. The most prominent of these was the movement of a tryptophan residue into a more hydrophobic environment. There was also a slight perturbation of this tryptophan when the pH was decreased to 7.4. The conformational changes can explain sigmoidal kinetic behaviour observed previously [Gething et al. (1976) Eur. J. Biochem. 71, 317-325].


Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Binding studies with the allosteric effector phenylalanine. The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.


Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split. Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I  in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.


Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats. Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.


Formation of complexes between microsomal cytochrome P-450-Fe(II) and nitrosoarenes obtained by oxidation of arylhydroxylamines or reduction of nitroarenes in situ. A cytochrome P-450 complex exhibiting a Soret peak at 454 nm is formed by direct interaction of nitrosobenzene with NADPH-reduced rat liver microsomes in anaerobic conditions, by reaction of phenylhydroxylamine with aerobic microsomes or during nitrobenzene reduction by NADPH-reduced or dithionite-reduced microsomes. In the latter conditions, the complex formation is only transient as it is unstable to dithionite. Analogous reactions with myoglobin lead to the previously described myoglobin-Fe(II)-nitrosobenzene complex which has similar properties to those of the 454-nm-absorbing cytochrome P-450 complex. This analogy, together with the various conditions of its formation, strongly indicates that it is a cytochrome-P-450-Fe(II)-nitrosobenzene complex. The corresponding complex with the 4-chloro-nitrosobenzene ligand is formed in similar conditions. Cytochrome P-450-Fe(II) complexes with nitrosoarenes seem less stable than the previously described complexes with nitrosoalkanes.


Neurotransmittor changes in the rat brain after portacaval anastomosis. Portacaval anastomosis in rats elevated the brain serotonin content by about 30%, the greatest increase being noted in the brain stem. A minor increase in norepinephrine content but no change in dopamine concentration was also noted. Low dose L-dopa treatment reversed the serotonin changes but did not affect catecholamine concentrations. Protein load tended to lower most transmittor concentrations without clinically affecting the animals. The formation of 5-HT from tryptophane in vitro in rat cortical slices was reduced by about 35% whereas the formation of 5-HIAA in the same slices was increased with about 60%. This in vitro study indicates a change in the activity of serotonin neurons caused by the protacaval shunt. The findings suggest that changes in brain serotonin metabolism and brain serotonin neurons may be an explanation of hepatic encephalopathy.


Effect of capsule colour and order of administration of hypnotic treatments. Ninety-six hospitalized insomniac patients were asked to compare by a daily questionnaire the effect on sleep of the colour of the capsules and the order of administration of two treatments--heptabarbital and placebo. A balanced incomplete-block design was used; sleep onset time and sleep duration time were the main criteria of assessment. There was a significant interaction of colour and order of administration with sex. The placebo response was positive in 36% of instances.


Subpopulations of mouse T lymphocytes. II. Suppression of graft-vs.-host reactions by naturally proliferating splenic T cells. The immunological role of a naturally proliferating subpopulation of splenic T cells was investigated using the graft-vs.-host (GvH) reaction on the mouse. Normal parental spleen cells, purified splenic T cells or lymph node cells were pulse-treated for one hour in vitro with tritiated thymidine of high specific activity ([3H]dThd, "thymidine suicide"). The treatment specifically and selectively kills proliferating cells which are actively synthesizing DNA, i.e. cells in S phase. Following treatment, the cells were transferred to F1 recipients and the GvH reaction measured by the splenomegaly assay. The results showed that the GvH effector cells in the donor spleen and lymph node are nonproliferating T cells. Furthermore, donor spleen cells treated with [3H]dThd consistently had enhanced GvH reactivity when compared to the controls, while the phytohemagglutinin response of these same treated cell suspensions was significantly inhibited. When purified splenic T cells were used, treatment with [3H]dThd also caused an increase in the GvH reaction, showing that a T cell population was being affected by the cycleactive agent. These results indicated that some naturally proliferating T cells have suppressor functions, and their specific inactivation allows nonproliferating effector T cells to mount a more vigourous GvH reaction.


Investigation of cardiac beta-adrenoceptors using 125I-labelled 1-(4-iodophenoxy)-3-isopropylaminopropan-2-ol. 1-(4-iodophenoxy)-3-isopropylaminopropan-2-ol (IIP) is a potent beta-adrenergic antagonist which has been labelled to high specific activity with 125I and used to bind to rat myocardial membranes. The characteristics of binding were consistent with the known properties of beta-receptors. Thus, binding was highly stereospecific for the L-stereoisomer since L-propranolol was two orders of magnitude more potent than the D-isomer in competing for these sites. The beta-adrenergic agonists isoproterenol, epinephrine and norepinephrine competed for binding with potencies paralleling their pharmacological potencies as beta-adrenergic effectors. The dissociation constant for binding of IIP was 4--5 nM as measured either by direct binding studies or by its inhibition of isoproterenol stimulated adenylate cyclase. Binding was saturable with 0.06 pmoles of IIP per mg of membrane protein binding at saturation. 125IIP is a high affinity, high specific activity ligand suitable for use as a selective probe for the detection and quantitation of cardiac beta-receptors. Its introduction should help solve the problems involved in the investigation of myocardial beta-adrenergic receptors.


Benzodiazepines: potentiation of a GABA inhibitory response in the dorsal raphe nucleus. Based on evidence that the dorsal raphe nucleus (DR) has specific and independent receptors for 5HT, GABA and glycine (Gallager and Aghajanian, 1976; Wang and Aghajanian, 1977), alterations in the firing rate of DR neurons following the administration of benzodiazepines (BZ) were evaluated to determine whether they were the result of a direct interaction with 5HT receptors or due to interactions of these drugs with GABA and/or glycine. The effects of BZs after both direct and systemic application were tested in rats using microiotophoretic and single-cell recording techniques. Although the BZs did not alter the spontaneous firing rate of the DR, both the systemic and iontophoretic administration of these drugs were found to potentiate the inhibitory response produced by GABA. The data suggest that this potentiation is mediated postsynaptically. Since the effects of BZs on the spontaneous activity of the DR are only apparent following pretreatments with AOAA, it is speculated that these drugs may only have pronounced effects when GABAergic input is prominent.


2,5-Dimethoxy-4-methylamphetamine (DOM)- a central component of its cardiovascular effects in rats; involvement of serotonin. The hallucinogen DOM produces a rise in blood pressure and heart rate when injected into the cerebral ventricles of anesthetized rats. These effects are abolished in rats with transected spinal cords but are unaffected by prior treatment with hexamethonium. Central but not peripheral administration of the serotonin antagonist BOL reduces the response. Tachyphylaxis to the response develops rapidly and is accompanied by a decrease in the ability of 5-HT to induce a centrally mediated cardiovascular change. It is concluded that the response is mediated by direct stimulation of central 5HT receptors. Tachyphylaxis may be the result of irreversible binding of DOM to 5-HT receptors or to 5-HT receptor densensitization.


Biphasic effect of chlorpromazine on rat paradoxical sleep: a study of dose-related mechanisms. The experiments reported here investigated the effect of chlorpromazine (CPZ) alone or after inhibition of catecholamine (CA) synthesis on paradoxical sleep (PS) in the rat. The dose--response curve for CPZ was biphasic with enhancement of PS after low doses, and depression of PS after higher doses. In contrast, low doses of CPZ after inhibition of CA synthesis markedly decreased PS. This decrease was greater after tyrosine hydroxylase inhibition than after dopamine-beta-hydroxylase inhibition. These results support the view that low doses of CPZ produce increased activity in brain CA synapses, and that both dopamine and noradrenaline participate in the control of PS in the rat.


Studies on the mechanism of the vasodilator effects of prazosin in dogs and rabbits. In pentobarbital (35.0 mg/kg) anaesthetised dogs, bolus injections of prazosin into the femoral artery (3.0--300.0 microgram) provoked a dose-related fall in the vascular resistance of the innervated hind limb. In contrast to papaverine, prazosin failed to produce the same effect in dogs under spinal anaesthesia even when the intrinsic femoral vascular tone was increased with vasopressin. However, vasodilator effects of prazosin were again observed when the tone of the limb was elevated by either stimulating the sympathetic lumbar chain or by infusing alpha-adrenoceptor agonists. A significant reduction of both aortic blood pressure and pressor response to bilateral carotid artery occlusion was noted in a group of normotensive dogs anaesthetised 12 h after the last dose of prazosin given twice daily at 0.5 mg/kg, p.o., for 3 day period. This short-term treatment modified neither the resting heart rate nor the positive chronotropic effect induced by either intravenous noradrenaline or electrical stimulation of pre- and post-ganglionic nerve fibres of the right stellate ganglion. However, it prevented the larger increase in heart rate in response to bilateral carotid occlusion in placebo-treated dogs after section of the vagi. A decrease in baseline sympathetic tone of the perfused hind limb as well as vasoconstrictor effects produced by i.a. injections of several alpha-adrenoceptor agonists and electrical stimulation of the lumbar sympathetic chain was observed in prazosin-treated animals. The dose--pressor response profiles to these alpha-adrenoceptor stimulants after prazosin were not parallel to those obtained in the control group. The vasoconstrictor response to angiotensin II was not changed by prazosin. In rabbit aortic strips, prazosin (0.1--3.0 micrometer) produced competitive antagonism of the contractile responses induced by cirazoline, noradrenaline and phenylephrine. In contrast to papaverine, prazosin in concentrations up to 100.0 micrometer neither relaxed the aortic strips contracted by potassium ions nor modified the concentration-response curve to calcium ions. These studies indicate that blood pressure lowering effects of prazosin given acutely or for three days can be accounted for by a clear-cut functional impairment of vascular postsynaptic alpha-adrenoceptors. No evidence for a direct myorelaxant property of prazosin could be obtained in these studies.


Positive chronotropic and inotropic responses to guanosine in the isolated dog atrium. The effects of guanosine on chronotropism and inotropism in isolated dog atria were studied in spontaneously beating preparations which were suspended in a bath perfused with arterial blood from a carotid artery of a heparinized support dog. Guanosine administered into the cannulated sinus node artery in a dose range of 30 microgram to 3 mg produced a dose-related positive inotropic and chronotropic effect. The positive responses to guanosine were not inhibited by treatment with propranolol or a non-depressant beta-blocker, carteolol, in doses which blocked responses to norepinephrine. From these results, it is concluded that guanosine has a direct effect on atrial rate and contractility.


Graft versus leukemia. VIII. Selective reduction in antihost reactivity without loss of antileukemic reactivity by treatment of donor mice with lipopolysaccharide. Spleen and lymph node cells from DBA/2 (H-2d) donor mice treated with multiple injections of bacterial lipopolysaccharide (LPS) were tested in vivo for reactivity against normal tissues of host AKR (H-2k) mice against an AKR long-passage, acute lymphoblastic leukemia (BW5147). LPS treatment of donor mice resulted in a reduction in graft-versus-host (GVH) reactivity without loss of graft-versus-leukemia (GVL) reactivity. Immunocompetent cells from LPS treated DBA/2 donors were effective when used for adoptive immunotherapy (in combination with chemoradiotherapy) of BW5147 leukemia. GVH associated mortality decreased as the dose of spleen cells from LPS treated histoincompatible donors was increased as much as four times the number necessary to eliminate leukemia. The mechanism by which LPS reduced GVH reactivity without eliminating GVL reactivity is unclear; however, it does not appear to be the result of a dilution in the number of GVH reactive cells by nonlymphoid elements in the donor spleen nor of the adjuvant effects of LPS on resistance to bacterial infections.


Acid release at meiotic maturation of oocytes in the polychaete annelid Sabellaria alveolata. ABout 1 pmole of acid per egg is released when prophasic oocytes undergo maturation under the action of sperm, proteases or ionophore A 23187. No similar acid release occurs at fertilization of matured oocytes. These findings are compared with data on Urechis and sea urchin.


[Effect of tropaphene and fentolamin on the spasmogenic effects of adrenaline, noradrenaline, serotonin and other biologically active substances]. Tests conducted in vitro and in vivo demonstrated that tropaphen, tropine ether of alpha-phenyl-beta-(p-acetoxyphenyl)-propionic acid, an original alpha-adrenoblocking agent, display a marked antiserotonin activity exceeding that of phentolamine. Moreover, tropaphen is more active that phenolamine by its inhibiting influence on the spasmogenous effects of histamine, angiotensin, bradykinine and prostaglandin E2. In doses of 0.1 and 0.25 mg/kg both drugs had no effect on changes of the arterial pressure in anesthetized rats, caused by the above mentioned biologically active substances.


[Comparative assessment of the effect of sympatholytics and beta-adrenoblockaders on carbohydrate-phosphorus metabolic indices in the myocardium of growing rats]. The sympatholyitic ornide and beta-adrenoblocking agent alpheprol were introduced intraperitoneally in single doses of 5 and 1 mg/kg, respectively to 7--30 day and 3--5-month old adult rats. The content of ATP, ADP, AMP, inorganic phosphorus glycogen and also active P32 incorporation were determined. The 7-day old rattlings are shown to be more sensitive to ornide than to alpheprol by comparison with older animals.


[Metabolic effects of ethyl alcohol the ways to correct them]. Tests conducted on albino male-rats demonstrated that with its intragastric administration in a dose of 3 g/kg ethanol produces a hypoxy-like situation in the organism, i.e. the oxygen tension in the liver and skeletal muscles decreases and the redox potential is activation during the 1st hour with succinate-cytochrome-c-oxidase of the liver mitochondria with concomitantly activated oxidation of succinic acid. The activity of NAD-dependent dehydrogenases goes down and in the blood occur acidotic shifts in the acid-base equilibrium (ABE). Twenty four hours after a single administration of ethanol the activity of all the study liver enzymes declines with the acidotic ABE shifts remaining unchanged. The used mixture of glutamate and sodium succinate appreciably decreases the blood ethanol level, corrects metabolic effects of ethanol and increases the survival of rats receiving lethal doses of ethanol.


[Ethological analysis of the action of medazepam and diazepam on the zoosocial behavior of isolated mice]. Tests set up with isolated mice of two groups (aggressive and "fearful") evidenced that diazepam and medazepam weaken the behavioral manifestations of the partner's avoidance, increase sociability in "fearful" mice and help to regain the ability for elementary intraspecies contacts. These drugs have a biphasic action on the behavior of aggressive mice, small doses (medazepam 0.1 mg/kg) facilitating aggression and large one (medazepam, diazepam--5 mg/kg) suppressing it.


[Effect of a new central adrenolytic on cardiac metabolic indices in neurogenic damage]. It is shown that in rabbits subjected to an electric stimulation of the aortic arch there developed concurrently with depletion of tissular norepinephrine reserves in the myocardium also gross metabolic disorders. A preliminary introduction of the drug izm-611 by blocking central alpha-adrenoreseptors at the same time prevented depletion of the tissular norepinephrine reserves and development of disturbances of the energy exchange in the myocardium caused by its neurogenic damage.


[Action of analeptics in acute alcoholic intoxication]. Tests conducted on rabbits in a state of acute ethanol poisoning (2.5 g/kg per os) of a medium degree demonstrated that caffein (10 mg/kg) and bemegride (5 mg/kg) introduced one time intravenously at the height of alcoholic intoxication raise the activity of aerobic oxidative processes, but fail to eliminate metabolic acidosis and do not accelerate the excretion of ethanol. Unlike caffein, bemegride shows a tendency toward respiratory compensation of metabolic acidosis and lowers the activity of the alcohol-dehydrogenase.


[Pharmacokinetics of the new Soviet, tritium-labelled, antihistamine preparation, phencarol]. As evidenced no less than 45 per cent of tritium-labelled antihistaminic drug phencarol (quinuclidine-3-diphenylcarbinol hydrochloride) introduced intragastrically to rats is absorbed. The maximal radioactivity in various organs is reached in 1--3 hours, with high specific radioactivity being recorded in the lungs and liver and low--in the brain. The drug or radioactive products of its tranformation are eliminated largely via the gastrointestinal tract and kidneys.


[Effect of amisyl and spasmolytin on the blood supply and oxygen metabolism of the brain]. The effect of benactyzine and spasmolytin adiphenine on the blood circulation in the oxygen consumption by the brain were investigated in experiments on dogs. The resulting data point to a biphasic action of benacyzine and spasmolytin on the blood supply of the brain (after a short-lived increase of the blood flow a clearcut decline of it is noted). The oxygen intake from the blood varies irregularly. In chlorophos-poisoned dogs the oxygen absorption by the brain changes under the effect of benactyzine and rises after administration of spasmolytin. The cerebral circulation then changes non-uniformly under the effect of benactyzine, while following spasmolytin injections it dicreases in the majority of experiments.


[Possible neuropharmacological regulation of chemical carcinogenesis caused by diethylnitrosamine]. The effect of a number of neurotropic drugs on diethylnitrosoamine (DENA)-induced chemical carcinogenesis was studied by applying pharmacological, morphological and biochemical techniques. Atropine stimulated significantly the rat liver and esophagus carcinogenesis, whereas the alpha-adrenoblocking agent, a pyrrhoxane analogue, and, particularly, proserine inhibited these processes. All animals treated with DENA demonstrated a significant increase of liver mitochondrial monoaminoxidase activity. These findings as well as the evidence of a stimulating effect of exogenous norepinephrine on carcionogenesis, together with the literature sources on carcinogenic and modifying carcinogenesis action of neurotropics allow the authors to formulate a concet of the trigger role played by catecholamines in the DENA-induced cell transformation and carcinogenesis.


The pathophysiology of hyperprolactinemic states and the role of newer ergot compounds in their treatment. Studies of prolactin secretion in humans have confirmed the concept, derived originally from animal investigations, that prolactin is predominantly controlled by tonic inhibition from the hypothalamus. The locus of action of dopamine and dopaminergic agents such as the ergot alkaloids inhibiting prolactin secretion appears to be primarily at the pituitary level, though a hypothalamic action to increase secretion of prolactin inhibitory factor may also contribute. Prolactin hypersecretion, through any of several possible mechanisms, is frequently but not always found in patients with galactorrhea. Recent studies have shown that hyperprolactinemia is considerably more common than was previously appreciated among patients without galactorrhea. It is present in at least two-thirds of all patients with pituitary tumors and in a significant minority of patients with secondary amenorrhea. Its clinical measurement in these conditions is therefore of considerable diagnostic importance. Whatever the pathophysiology of its production, hyperprolactinemia of all forms is responsive to treatment with the newer ergot alkaloids. The potential use of these agents for therapeutic purposes, particularly in the treatment of infertility, appears to be wider than was originally anticipated.


Evaluation of testicular function in prepubertal boys by means of the luteinizing hormone-releasing hormone test. Luteinizing hormone (LH)-releasing hormone (LH-RH) tests (50 microgram/sq m intravenously) were performed in 112 prepubertal boys ages 13/12 to 11 years (mean +/- standard deviation, 75/12 +/- 16/12 years) suspected of having a testicular disorder because of improperly located testes (77 boys) or hypogonadism (35 boys). Four of the patients were retested within a period ranging from 6 to 16 months. Of the 112 boys tested, 17% were found to have high basal levels of follicle-stimulating hormone (FSH) and 23% were found to have an abnormally high release of FSH after LH-RH administration. Only three patients had abnormally high basal levels of LH and/or elevated LH responses to LH-RH. The basal plasma testosterone levels were found to be normal in all 112 bosy. The fact that plasma FSH levels were elevated more often than LH levels suggests that the tubular elements are damaged more frequently than are the Leydig cells. The surprisingly high incidence of an abnormal response of plasma FSH to LH-RH in boys with mobile testes calls for an increased awareness of the importance of regular examination of these patients until full puberty has been achieved. It is concluded that determination of basal plasma FSH levels and the response to LH-RH stimulation is a useful diagnostic tool for evaluating testicular function in prepubertal boys with suspected pathology of the testes.


[Modulation of autonomic correlates of emotional stress and adaptive responses]. The character and dynamics of the cardio-vascular response to psychogenic stress (confrontation of cat and dog) depends on active or passive type of behavioral response. Tranquilizers inhibit long-lasting hypertension otherwise occurring after the stress stimulus. Adaptive cardio-vascular responses and baroreceptor reflexes did not alter after tranquilizers administration. Effects of psychotropic drugs correspond to the type of individual behavioral response.


Local graft-versus-host reaction in mice evoked by Peyer's patch and other lymphoid tissue cells tested in a lymphocyte-induced angiogenesis assay. The competence of murine Peyer's patch cells to evoke a local GVH reaction was compared with that displayed by lymphoid cells of spleen, lymph nodes, thymus, bone marrow, peritoneal cavity and peripheral blood. The local GVH reaction was assessed in a lymphocyte-induced angiogenesis assay in which an intradermal injection of lymphoid cells results in a new blood vessel formation at the injection site, and the number of vessels corresponds to the number of the immunocompetent cells injected. Peyer's patch cells were capable of mounting a local GVH reaction of intensity comparable to that evoked by the corresponding number of thymus cells or a four times lower number of spleen cells. The highest activity was exhibited by lymph node and peripheral blood cells while bone marrow cells were the least active. A striking increase in angiogenic response was observed after X-irradiation of the recipients with 700R.


Serum polyphenol oxidase activity (ceruloplasmin) in conventional laboratory animals and man. The optimum pH for ceruloplasmin as polyphenol oxidase (EC 1.10.3.2) activity was determined in human serum (pH 5.4) and the serum of conventional laboratory animals--the rat (pH 5.2), mouse (pH 5.2), hamster (pH 5.3), guinea pig (pH 5.4), multimammate mouse (pH 5.2) and rabbit (pH 5.4). Determined at the optimum pH in 0.1M acetate buffer polyphenol oxidase activity fell in the sequence: rat--man--rabbit--mouse--multimammate mouse--hamster--guinea pig. Ceruloplasmin polyphenol oxidase activity was inhibited by 0.1M phosphate buffer in the mouse, rat and multimammate mouse, but not in the other species. It was inhibited by 0.05M citrate and 0.1M phthalate buffer in all the species tested.


Evaluation of in vitro and in vivo activities of isoantibodies directed against idiotypes and recognition structures for transplantation antigens. Anti-idiotype sera were prepared by injection of CBA anti-A/JAX serum with or without adjuvant into CBA mice. These sera were tested in allocluster inhibition and passive enhancement-facilitation. While the anti-idiotype sera inhibited allocluster formation, particularly when prepared with complete Freund's adjuvant, they had virtually no effect on enhancement of Sa I grafted in CBA mice except for a marginal prolongation of graft survival in one out of three experiments. Anti-RS serum was prepared by immunizing (CBA X A)F1 mice with CBA thymus or spleen cells. The sera were tested in local graft-versus-host inhibition and passive enhancement. F1 anti-CBA thymus serum inhibited the local graft-versus-host reaction but had no enhancing effect. F1 anti-CBA spleen serum was virtually ineffective in both tests although a slight tendency to graft-versus-host inhibition was noted. The relative contribution of anti-idiotype and/or anti-RS antibodies to transplantation reactions is discussed.


Interaction of glucocorticoid receptors from lymphoid cell lines with their nuclear acceptor sites. Procedures have been developed which provide simple means of determining binding constants of steroid receptors for glucocorticoids in mouse lymphoid cell lines and of characterizing the interaction of the steroid--receptor complex with the nucleus. An average of 70% of the steroid--receptor complexes is found associated with the nuclear fraction in three investigated cell lines, whereas 30% of the steroid--receptor complexes is found in the cytosol fraction. This distribution of the steroid-receptor complex within the cell is independent of whether steroid uptake of the cells is performed at low or at high steroid concentration. Part of the binding of the steroid receptor to the nuclear fraction is sensitive to high ionic strength and to high pH. A larger fraction of the steroid--receptor complex binding to the nuclear fraction is insensitive to high ionic strength and pH when the steroid uptake is performed at low steroid concentrations than when performed at high steroid concentrations. Steroid--receptor complex is released from the nuclear fraction by DNAase treatment but not by RNAase treatment. The possible correlation between the sensitivity to ionic strength and pH and the specificity of the binding is discussed.


Effect of cimetidine on ion fluxes and potential difference across the human stomach. The H(2)-receptor antagonist, cimetidine, reduces acid output regardless of the means of stimulation. It is not known in man whether this is due entirely to a reduction in acid secretion or whether increased back diffusion of hydrogen ions is also occurring. We studied fluxes of H(+), Na(+), K(+), and Cl(-) ions after acid instillation into the human stomach in six healthy subjects with and without prior administration of 300 mg cimetidine orally. Potential difference across gastric mucosa was measured continuously throughout each study. Cimetidine caused a significant reduction in hydrogen ion secretion (5.05 mEq per 15 minutes controls versus 2.70 cimetidine, p < 0.05), and consequently a significant reduction in net hydrogen flux into the gastric lumen (2.01 mEq per 15 minutes versus 0.02, p < 0.05). There were no significant differences between sodium ion fluxes in control and cimetidine studies, suggesting that the gastric mucosal barrier remained intact. Cimetidine alone caused a highly significant rise in intragastric pH (to 7) and of potential difference (p < 0.001). Addition of intragastric acid (pH < 1.0) did not reverse the rise in potential difference caused by cimetidine, suggesting that factors other than change in intragastric pH were involved. In conclusion, our studies support the concept that reduction in acid output by cimetidine is due to inhibition of acid secretion, and not to increased permeability to hydrogen ion.


p-Nitrobenzyl p-toluenesulfonyl-L-arginine: a chromogenic substrate for thrombin, plasmin and trypsin. p-Nitrobenzyl p-toluenesulfonyl-L-arginine is hydrolyzed by thrombin, plasmin, and trypsin to p-nitrobenzyl alcohol and tosyl-L-arginine. The absorption of p-nitrobenzyl alcohol formed is measured at 271 nm (AmM 8.89). With 0.10 mM of the ester in 0.1 M Tris-HCl at pH 8.4 and 30 degrees C, the hydrolysis catalyzed by thrombin, plasmin, and trypsin is linearly proportional to time up to consumption of 60% of the substrate. Km is 14 micron and Vmax is 0.037 mumol/min/NIH unit for bovine thrombin, Km is 78 micron and Vmax is 0.31 mumol/min/CTA unit/ml for human plasmin, and Km is 12 micron and Vmax is 138 mumol/min/mg protein/ml for bovine trypsin. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2,133 NIH units/mg protein showed esterase activities ranging from 0.15 to 0.4 mumol p-nitrobenzyl alcohol formed/10 min/NIH unit. Useful ranges for assay of enzymes were (per milliliter): 0.05-0.2 NIH units (thrombin), 0.005-0.02 CTA units (plasmin), and 0.01-0.04 microgram (trypsin).


Chromogenic substrate assay of plasma prekallikrein. With a note on its site of biosynthesis. A method for assaying plasma prekallikrein has been developed applying the chromogenic substrate Chromozym PK. Different variables have been investigated, and the final assay system was found to give a reproducible and reliable assay procedure. A normal value of 99.0 +/- 18% was found, the coefficient of variation being 7.9. Studies on material from patients with various liver diseases indicated that the main site of prekallikrein biosynthesis is the liver.


Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. New chromogenic tripeptide substrates have been used for the determination of kallikreins and urokinase. The conditions have been optimized. It is possible to determine prekallikrein in plasma after activation with Cephotest. No significant loss in activity caused by plasma kallikrein inhibitors is observed at the dilutions used.


[Regional myocardial blood supply in coronary disease]. 1. Value and limitations of the precordial Xenon-clearance technique are analyzed. The results of this technique are compared to the scintigraphic display of the initial Xenon distribution and to microsphere scintigraphy (Engel et al. 1976a). 2. The effects of coronary obstructions and of segmental LV dysfunction on regional myocardial blood flow are analyzed (Engel et al. 1977). Independent of coronary obstructions, a clear reduction of regional myocardial blood flow in areas of hypokinesis and akinesis was observed. Thus in coronary disease a decrease of regional myocardial blood flow may represent either a cause or a result of abnormal segmental wall motion. 3. The effects of nitroglycerin, beta-blockade (Atenolol), calcium-antagonists (Nifedipine) and coronary dilatation (Dipyridamole) on regional myocardial blood flow were investigated (Engel et al. 1976b). Regional flow at rest decreased after nitroglycerin and beta-blockade, increased mildly after Nifedipine and markedly after Dipyridamole.


[On the acute toxicity of labetalol (AH-5158), a combined alpha-and-beta-adrenoceptor-blocking agent (author's transl)]. Acute toxicity studies in labetalol were performed using mice, rats and rabbits, An oral administration of labetalol produced an increase in salivary secretion due to local irritation. Death followed convulsions by large doses of labetalol. Pathological examinations suggested that the cause of death by labetalol was circulatory disturbances due to the damage of heart muscles and a subsequent respiratory paralysis. Parenteral LD50 showed almost the same values among mice (50 mg/kg iv, 117 mg/kg ip), rats (66 mg/kg iv, 115 mg/kg ip) and rabbits (43 mg/kg iv). However, species difference was seen in oral LD50 values which were enormously large as compared with iv LD50 values.


Gastric lesions in familial adenomatosis coli: their incidence and histologic analysis. In order to detect accompanying gastric lesions, we examined 22 patients with familial adenomatosis of the colon belonging to 14 families. Various gastric lesions were confirmed in 15 patients (68.2 per cent) belonging to 13 families. The lesions were found histologically to be adenoma in nine cases, fundic gland polyposis in six, carcinoma in three, and microcarcinoid in two. Fundic gland polyposis consisting of simple hyperplasia of the fundic glands seems to be the gastric lesion specific to familial adenomatosis of the colon and rectum. Familial adenomatosis coli not only affects the colon and rectum, but is also capable of inducing tumorigenicity in other organs. The stomach is one of the organs in which extracolonic lesions occur.


Morphologic aspects of bone marrow transplantation in patients with aplastic anemia. Pre- and post-transplant bone marrow samples from 20 patients with aplastic anemia were studied. Morphologic evidence of marrow reconstitution was noted in 18 patients one to three weeks following transplantation. In most instances the engrafted marrow elements in early weeks appeared as small clusters of erythroid or myeloid precursors. Bone marrow biopsy or clot sections obtained four to eight weeks after transplantation were more cellular with larger clusters of hematopoietic cells, which were most often composed of mixed cellular elements, including megakaryocytes. Two patients with morphologic evidence of engraftment died shortly after transplantation and were excluded from further analysis. In four of the remaining 16 patients grafts were "rejected" three to eight weeks after transplant. A fifth patient who received a marrow graft from his identical twin showed a transient increase in marrow cellularity without clinical improvement. However, the second marrow transplantation in this patient using the same donor after conditioning with cyclophosphamide resulted in moderate clinical improvement. In four of the five patients developing graft "rejection" or failure there was an increase in marrow mast cells in pre- and post-transplant marrow samples. In contrast, only two of 11 patients with successful engraftment had an increase in mast cells. Although the pathophysiologic role of mast cells in marrow transplantation is unclear, the present study suggests a possible inverse correlation between the numbers of marrow mast cells and the likelihood of successful engraftment. Bone marrow samples in patients with graft versus host disease displayed a slight increase in the number of eosinophils, lymphocytes, and plasma cells.


Mice bearing the ob/ob mutation have impaired immunity. The obese mutant mouse C57BL/6J ob/ob showed impaired ability to reject skin grafts or react to a contact-sensitising agent in comparison with littermate controls (either +/ob or +/+). Ability of spleen cells from mice bearing the ob/ob mutation to produce a graft-versus-host reaction in C57BL/6J X DBA/2J F1 hybrid mice was not impaired.


Interaction of cholera toxin and toxin derivatives with lymphocytes. III. Modulating effects in vivo by cholera toxin on the graft-versus-host reactivity of lymphoid cells: suggested inhibition of suppressor cells. The influence of cholera toxin (CT), and thus probably of cyclic AMP, on the capacity of parental lymphoid cells to elicit a graft-versus-host reaction (GVHR) was studied. Toxin-treated DBA/1 mice were used as cell donors and untreated DBA/1xC57B1/6 F1 hybrid mice as recipients, and the GVHR reactivity of the transferred cells was estimated by their ability to induce spleen enlargement or stimulation of antibody formation ('allogenic effect') in the recipients. Spleen cells from donors intravenously injected with 1 microgram CT 1-3 days earlier, gave a significantly stronger GVHR than did spleen cells of untreated mice. Choleragenoid, a toxin analog devoid of the toxin's ability to activate plasma membrane adenylate cyclase even though it binds efficiently to cells, had no effect on the GVHR-inducing capacity of the spleen cells. The enhanced GVHR by spleen cells from toxin-treated DBA/1 animals was reduced to the normal level when the donor cells were transferred along with lymphoid cells from untreated animals of the same strain. Spleen was the most powerful source of the suppressive influence. No evidence for a redistribution of suppressor cells following administration of CT was found. Spleen cells from mice syngeneic with the recipients had no suppressive effect. The results suggest that parenterally administered CT, directly or indirectly, can inhibit a cell population in spleen which normally exerts an antigen-specific suppressive regulatory influence on the development of GVHR.


[Acute mucocutaneous lymph node syndrome (Kawasaki-syndrome) with fatal periarteritis nodosa of the coronary arteries. Another case from Europe]. Case report of a 1 8/12-year-old boy suffering from an acute febrile disease with phlegmonous cervical lymphadenopathy, skin rash and other septic symptoms. Bacterial cultures were sterile, and antibiotics had no effect, whereas prednisone made an immediate improvement. On the 23rd day of illness the patient died unexpectedly. Autopsy revealed a ruptured coronary aneurysm due to periarteritis nodosa. This curious disease of yet unknown cause is quite frequent in Japan, but very rare in Europe.


Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature. The histochemical activities of succinic dehydrogenase (SDH) and Ca++-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. 'Red' and 'white' muscle fibres, as distinguished by SDH, showed high and low Ca++-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K2-EDTA solution. The ultrastructural investigation of Ca++-ATPase reaction at pH 7.4 by the Ca++-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and 'Z' bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb++ precipitation technique demonstrated Mg++-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail 'red' muscle fibres are possible 'slow,' and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, 'red' muscle fibres of the anuran tai- musculature are not equivalent to 'Type I' fibres of higher chordates.


Microbial metabolism of anthracycline antibiotics daunomycin and adriamycin. It has been shown that the antitumor antibiotics daunomycin (1) and adriamycin (4) are metabolized by microorganisms in a fashion similar to their metabolism by mammalian cells. Both the fungus Mucor spinosus and its cell-free extracts reduce the 13-keto group of daunomycin to give daunomycinol (2) by a TPNH-dependent process. Cell-free extracts of Streptomyces steffisburgensis convert adriamycin and daunomycinol to their 7-deoxyaglycones (5) and (3) by DPNH-linked reductive glycosidic cleavage. Cell-free extracts of the latter organism convert 7-deoxyadriamycinone (5) to 7-deoxyadriamycinol aglycone (6) by TPNH-linked 13-keto reduction.


Semiautomated turbidimetric microbiological assay for cefazaflur. A quantitative semi-automated turbidimetric bioassay for cefazaflur, using Streptococcus faecium as the indicator, is described. Assays were run at pH 6.5 approximately 7 for 3.75 hours at 37 degrees C using 2 approximately 12 microgram cefazaflur per ml assay broth for standards. The dose response line was plotted point to point using the natural log of the absorbance vs natural log of the concentration. This assay is both accurate and precise and is more rapid than traditional plate assays for antibiotics.


Salmonella typhimurium peptidase active on carnosine. Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.


Aspartokinase of Streptococcus mutans: purification, properties, and regulation. Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine.


Carboxypeptidase displaying differential velocity in hydrolysis of methotrexate, 5-methyltetrahydrofolic acid, and leucovorin. An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extraced from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate.


Glutamate-induced uptake of proline by Streptomyces antibioticus. Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized.


Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase. The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.


Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase. A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.


Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism.


Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella. When synchronous cells of the eucaryotic microorganism Chlorella sorokiniana growing in nitrate medium were challenged to synthesize an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) at frequent intervals during the cell cycle the initial rate of induction (i.e., enzyme potential) of this enzyme increased in an approximately linear manner until the period of DNA replication (i.e., S phase). During the S phase, NADP-GDH potential exhibited a positive rate change proportional to the step increase in DNA level. The timing of this rate change was insensitive to large changes in cellular growth rate. This rate change could be blocked within the first cell cycle by specific inhibition of DNA replication with 2'-deoxyadenosine. The approximately linear increase in NADP-GDH potential and also of total cellular protein observed before and after the S phase is proposed to be a result of the increasing photosynthetic capacity of the cell during the cell cycle.


Mutations that alter the covalent modification of glutamine synthetase in Salmonella typhimurium. glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction.


Genetics and physiology of Neurospora crassa glutamine auxotrophs. This work reports on the isolation and characterization of two glutamine auxotrophs in Neurospora crassa. The mutations responsible for the glutamine-requiring phenotype were very closely linked, and one of them proved to be recessive to wild type. The mutations impaired the conversion of glutamic acid to glutamine and resulted in changes of both the activity and oligomeric structure of the enzyme glutamine synthetase.


Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate. Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum oxidation rates at pH 3.0, but produced substrate inhibition at other pH values. Tetrathionate-grown cell suspensions also oxidized thiosulfate at pH 2.0 to 4.0. Apparent Km values (1.2 to 25 mM) were of the same order as for tetrathionate, but kinetics were complex. Continuous culture on growth-limiting tetrathionate at pH 2.5, followed by continuous culture on growth-limiting thiosulfate at pH 2.5, indicated true growth yield values (grams [dry weight] per gram-molecule of substrate) of 12.2 and 7.5, and maintenance coefficient values (millimoles of substrate per gram [dry weight) of organisms per hour) of 1.01 and 0.97 for tetrathionate and thiosulfate, respectively. Yield was increased on both media at low dilution rates by increase in CO2 supply. The apparent maintenance coefficient was lowered without affecting YG, suggesting better energy coupling in CO2-rich environments. Prolonged continuous cultivation on tetrathionate or thiosulfate did not affect the ability of the organism to grow subsequently in ferrous iron medium.


Sodium ion-proton antiport in a marine bacterium. Alteromonas haloplanktis ejected protons in response to a brief respiratory pulse; the rate of decay of the resulting pH change was accelerated when Na+ was present in the suspension medium. The addition of an anaerobic NaCl solution to an essentially Na+-free anaerobic bacterial suspension induced the acidification of the suspension medium. These results and others discussed provide substantial evidence for the existence of an Na+-H+ antiporter in this organism.


Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma). The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure.


Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida. The primary amine dehydrogenase of Pseudomonas putida NP was purified to homogeneity as judged by polyacrylamide gel electrophoresis. Cytochrome c or an artificial electron acceptor was required for amine dehydrogenase activity. The enzyme was nonspecific, readily oxidizing primary monoamines, benzylamine, and tyramine; little or no measurable activity was detected with isoamines, L-ornithine, L-lysine, and certain diamines or polyamines. The pH optima for n-butylamine, benzylamine, and n-propylamine were 7.0, 6.5, and 7.0, respectively. The molecular weight of the enzyme was 112,000 as determined by gel filtration and 95,300 as analyzed by sedimentation equilibrium. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested that the enzyme was composed of two nonidentical subunits with molecular weights of 58,000 and 42,000. The absorption spectrum of the purified enzyme was indicative of a hemoprotein, exhibiting absorption maxima at 277, 355, and 408 nm. Reduction with sodium dithionite or amine substrates resulted in absorption maxima at 523 and 552 nm and a shift in the Soret peak to 416 nm. These results suggested that the enzyme is a hemoprotein of the type c cytochrome. There was no evidence that flavins were present.


Organic fluorides: implications for psychiatry. The widespread use of synthetic organic fluorides has recently received attention as a potential health hazard. There are a number of organic fluorides which have become important considerations in psychiatry. Therapeutically the organic fluorides include the neuroleptics trifluoperazine, fluphenazine, triflupromazine, and haloperidol, the benzodiazepine flurazepam and the polyfluorinated inhalant convulsant indoklon. On the negative side, deliberate inhalation of fluorocarbon aerosol propellants has become a modern form of drug abuse among the young. A review is presented on the biochemistry and toxicology of organic fluorides with special emphasis on implications to the field of psychiatry.


Enzymatic oxidation of disulfides and thiolsulfinates by both rabbit liver microsomes and a reconstituted system with purified cytochrome P-450. Both rabbit liver microsomes and reconstituted system with purified cytochrome P-450 and cofactors enzymatically oxidized o-dithiane (1, 2-dithiane), 3-methyl-o-dithiane, thiane and 2-methylthiane to the corresponding mono-oxygenated products; sulfides or disulfides were oxidized to the corresponding sulfoxides or thiosulfinates, while thiosulfinate was oxidized to thiolsulfonate. The reconstituted systems required oxygen and NADPH and were not affected by the catalase which decomposes H2O2, or by 1,4-diazabicyclo-[2,2,2]octane (DABCO), which is a good quencher of singlet oxygen. The differences in the binding of substrates such as sulfides and disulfides with the enzyme system are discussed in connection with differences in the spectra of the substrates in the reconstituted system with pure cytochrome P-450. A correlation was found between the rates of oxidation of the substrates and the rates of oxidation of NADPH.


New chromogenic and fluorogenic substrates  for pyrrolidonyl peptidase. L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate.


Acceptor specificity of ATP:nucleoside-5'-phosphate pyrophosphotransferase from Streptomyces adephospholyticus. Synthesis of the 3'-pyrophosphates of pyrimidine nucleotides, some oligoribonucleotides, 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am. ATP: nucleoside-5'-phosphate pyrophosphotransferase [EC 2.7.6.4] of Streptomyces adephospholyticus synthesizes not only 3'-pyrophosphates of 5'-purine ribomononucleotides but also those of pyrimidine mononucleotides, some short oligonucleotides, a variety of 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am.


Purification and characterization of alkaline phosphatase in cultured rat liver cells. Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.


Purification of lysophospholipase of Vibrio parahaemolyticus and its properties. Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.


Studies on the formation of gamma-aminobutyric acid from putrescine in rat organs and purification of its synthetic enzyme from rat intestine. The formation of gamma-aminobutyric acid (GABA) from putrescine was examined in organs of rats using radioactive putrescine. Radioactive GABA was detected in all the organs of a rat injected intraperitoneally with radioactive putrescine, and the highest radioactivity of GABA was observed in the small intestine. The enzyme involved in this formation was purified from small intestine and identified as a diamine oxidase, histaminase, from the properties of the enzyme. Activity of the enzyme was found in all the organs of rat, and the highest activity was observed in the small intestine.


Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma. Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.


Monomer-dimer equilibria of a Bence Jones protein and its variable fragment. The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains.


A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin. Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.


3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.