Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.


Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. 1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.


Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Lactic dehydrogenase from pig skeletal muscle (M4) can be reversibly dissociated to the monomer at pH 4-5 depending on the anion applied. Using identical experimental conditions the pH-depending profiles of dissociation, denaturation, and deactivation coincide with each other. Deviations in the pH-dependence of protein fluorescence reflect changes in the microenvironment of specific chromophores rather than significant differences in the structure-function relationship as pH is changed. The dissociation state is characterized by the homogeneous inactive monomer of 35000. The reassociated material consists of up to 85% fully active tetramers, indistinguishable from the initial native enzymes, as shown by hydrodynamic, spectroscopic and enzymic properties. The rest represents a mixture of irreversibly denatured high aggregates. Under optimum conditions of reactivation both recovery of enzymic activity and native fluorescence obey strict second order kinetics with an activation energy of 44 kcal/mol (184 kJ/mol). NAD+ and NADH do not show any significant influence on the yield and kinetics of the refolding process and the recovery of enzymic activity. The kinetic results suggest the reassociation of inactive monomers to be rate-limiting in the refolding and reactivation processes being considered.


Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. 3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.


Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii. The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.


The proton electrochemical gradient in Escherichia coli cells. The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.


[Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]. The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores...


Comparison of the metal-ion-promoted dephosphorylation of the 5'-triphosphates of adenosine, inosine, guanosine and cytidine by Mn2+, Ni2+ and Zn2+ in binary and ternary complexes. The dependence of the rate of dephosphorylation of ATP, ITP, GTP and CTP (= NTP), expressed as first-order rate constants (50 degrees C; I = 0.1 M, NaClO4), on pH (2 to 10), in the absence and presence of Mn2+, Ni2+, and Zn2+, was investigated. The reaction is accelerated by Zn2+ and passes through a pH optimum at about 8 for the system Zn2+-ATP or 9 for Zn2+-ITP and Zn2+-GTP; this is analogous to observations made earlier with the corresponding Cu2+ systems. By computing the pH dependence of the distribution of the several species present in these systems it is shown that the highest rates are observed in the pH regions where the concentration of Zn(ATP)2-, Zn(ITP-H)3-, or Zn(GTP-H)3- dominates. By evaluating the pH dependence evidence is given that the attacking nucleophile is OH- or H2O for Zn (ATP)2- and H2O for Zn (ITP-H)3- or Zn(GTP-H)3-. For all these complexes metal-ion/nucleic-base interactions are known, leading to the formation of macrochelates. These metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion-promoted dephosphorylation; in agreement with this, and the small tendency of the cytosine moiety to coordinate, the CTP systems are rather stable towards dephosphorylation. It should be noted that these experimental results do not necessarily mean that the macrochelates usually described are the reactive complexes, but only that the active complex must be closely related to them (e.g. isomers, etc). Although for the Ni2+ systems with ATP, ITP, and GTP, and for the Mn2+-ATP system a metal-ion/nucleic-base interaction is also known, these systems are not very sensitive to hydrolytic cleavage of the terminal P-O-P bond. The only known significant structural difference between the Ni2+-NTP or the Mn2+-ATP complexes and those of Cu2+ or Zn2+ is that Ni2+ Mn2+ coordinate to all three phsophate groups, whereas Cu2+ and Zn2+ involve only the beta and gamma ones. This structure-reactivity relationship is rationalized by the suggestion that in the active species the metal ion should be coordinated to the alpha,beta-phosphate groups leaving the gamma-group open to nucleophilic attack. Obviously, an initial beta,gamma-coordination is suitable for a shift of the metal ion along the phosphate back-bone into the reactive alpha-beta-position, while for an alpha,beta,gamma-coordination only the less favorable removal of the coordinated gamma-group remains. The metal-ion/nucleic-base interaction is considered as being important for achieving this reactive structure. The connection between trans-phosphorylation in vitro and in vivo is discussed. It is also shown that the formation of mixed-ligand or ternary complexes inhibits the dephosphorylation process. This is on the one hand of interest with regard to the transport of hydrolysis-sensitive phosphates in nature, while on the other it casts doubts on conclusions based on experiments carried out in the presence of buffers, because these contain weak bases and hence potential ligands.


Rat-liver superoxide dismutase. Purification and age-related modifications. Cytoplasmic superoxide dismutase has been purified from livers of young (6 months) and old (27 months) rats. The enzyme purified from old animals shows an age-related reduction in the specific activity, accumulation of antigenically cross-reacting material and increased sensitivity to temperature. No differences were found in the molecular weight, electrophoretic mobility, antigenicity and Ki between enzymes purified from young and old rats. This is the first demonstration of age-related alterations in a purified form of a non-metabolic enzyme, which can be related to reduced activity. The possible role of this reduced activity in age-dependent deterioration of cellular functions is discussed.


Effects of ouabain and hypoxia on the cardiac stimulation threshold in the dog (with special reference to changes in extra- and intracellular potassium concentrations). The influence on the cardiac stimulation threshold of changes in the myocardial intra/extracellular potassium ratio induced with ouabain and hypoxia was investigated in 9 dogs with total atrioventricular block. In spite of significant elevations of extracellular potassium and reductions of intracellular potassium significant changes in stimulation threshold were not seen. It is suggested that different experimental conditions are responsible for the great variations in the results of previous studies of this subject.


The effect of fructose infusions on the oxygen transport system of human blood. In 9 healthy subjects the erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration, which modifies the oxygen affinity of haemoglobin, decreased by more than 25 per cent within 60 minutes of the beginning of a fructose infusion (0.5 g.kg body weight-1.h-1). In contrast erythrocyte adenosine triphosphate (ATP) concentration was unchanged. In spite of the diminished 2,3-DPG concentrations the oxygen affinity of whole blood actually measured remained unaltered. However, at the same time both the arterial and the venous blood pH had fallen by 0.05 or more. In vitro experiments indicated that this fall of erythrocyte 2,3-DPG was not due to a direct effect of fructose on the intra-erythrocytic regulation of 2,3-DPG or to changes indirectly related to the intravenous administration of fructose in vivo, i.e. an increase of the blood lactate/pyruvate ratio or a decrease of plasma inorganic phosphate. It is suggested that two opposing effects on the oxygen transport system of blood are induced by fructose infusions: 1) a displacement of the oxygen dissociation curve to the right due to the Bohr effect 2) a virtually counterbalancing shift of the oxygen dissociation curve to the left due to decreased erythrocyte 2,3-DPG concentrations.


A comparison between a melanocyte-stimulating hormone inhibitory factor (MIF-I) and substances known to activate central dopamine receptors. The tripeptide, prolyl-leucyl glycine amide, a melanocyte-stimulating hormone inhibitory factor (MIF-I), which has been reported to be effective in improving symptoms of Parkinson's disease, has been compared with drugs known to activate dopamine receptors in rat and mouse brain. Unlike apomorphine, amphetamine and amantadine it was incapable of producing sterotyped behaviour in the rat and unlike 1-dopa it was also ineffective in rats pretreated with the monoamineoxidase inhibitor mebanazine. Neither did it potentiate apomorphine nor amphetamine in this test. MIF-I did not antagonise chlorpromazine-induced loss of locomotor activity in mice, an effect which was antagonised by apomorphine, amphetamine and amantadine. Chlorpromazine hypothermia in the mouse was antagonised by 1-dopa but not by MIF-I; similar findings were obtained in reserpine-pretreated mice. These results suggest that the reported beneficial effect of MIF-I in Parkinson's disease is unlikely to be due to an interaction with dopamine systems in the brain.


Acute graft-versus-host reaction in mice. 3. Organ distribution of injected 51 chromium labeled lymphocytes. The distribution of labeled lymph node cells, causing an acute GvH reaction in lethally irradiated allogeneic recipients, was studied. Lymph node cells of C57BL mice were labeled with 51Cr and injected into lethally irradiated: a) C57BL mice, b) CBA mice, c) CBA mice sensitized to C57BL antigens prior to irradiation, d) CBA mice splenectomized before irradiation. Two more experimental situations were studied in which C57BL donors of lymph node cells were: e) presensitized to CBA antigens, or f) deprived of T-lymphocytes. The amount of radioactivity was determined in the whole body, blood, liver, spleen, subcutaneous lymph nodes, lungs, femora and kidneys of the irradiated recipient at regular intervals from the time of injection to the 120th hour after it. We found that living cells lodged predominantly in the spleen and the lymph nodes, while dead and dying cells accumulated in the liver. Other organs contained very small amounts of radioactivity. All the results point to the primary role of the spleen in the acute graft-versus-host reaction.


Mitigation of graft-versus-host disease in mice by treatment of donors with bacterial endotoxin. Treatment of DBA/2 (H-2d) mice with bacterial endotoxin prior to transplantation of their spleen and lymph node cells into immunosuppressed AKR (H-2k) mice prevented acute mortality from graft-versus-host (GVH) disease. AKR mice that received immunocompetent cells from untreated DBA/2 mice had a median survival time (MST) of 13 days. In contrast, AKR mice that received immunocompetent cells from endotoxin-treated DBA/2 donors had an MST of 54 days. Endotoxin treatment of AKR recipients was not essential for preventing mortality from acute GVH disease. Chimerism was proved by demonstrating that the lymphoid cells of long-term surviving AKR mice had the characteristics of DBA/2 lymphoid cells as measured by their response in mixed leukocyte culture (MLC) tests. Spleen cells from endotoxin-treated DBA/2 mice were able to stimulate, and to be stimulated by, AKR spleen cells in MLC assays. Furthermore, spleen cells from endotoxin-treated DBA/2 mice did not suppress the responses of DBA/2 or AKR spleen cells in 'three-party' MLC tests.


Influence of diurnal cycles on biochemical parameters of drug sensitivity: the pineal gland as a model. Diurnal cycles in physical parameters in the environment modulate biochemical and physiological circadian rhythms in experimental animals, including cycles in the sensitivity to external influences. Environmental lighting synchronizes cycles of indole metabolism and melatonin synthesis in the rat pineal gland by modulating the activity of postganglionic sympathetic nerves. As a consequence, the sensitivity of pineal N-acetyltransferase to stimulation by isoproterenol or by dibutyryl cyclic AMP varies diurnally. Also, the capacity of actinomycin D to inhibit this induction varies with circadian periodicity. The cycles in sensitivity to isoproterenol reflect cycles in the system that regulates cyclic AMP production, and include variation in the availability of specific B-adrenergic binding sites, and in the sensitivity of receptor-coupled adenylate cyclase to catecholamines. Further, a variation in the response to dibutyryl cyclic AMP indicates in addition the participation of intracellular controls in the regulation of the sensitivity of N-acetyltransferase to catecholamines. The varying sensitivity to actinomycin D suggests a changing requirement for the synthesis of RNA as a function of prior environmental lighting conditions. The basic nature of these sensitivity changes suggests that diurnal cycles of environmental lighting may similarly affect other systems.


Nitrite, nitrosamines, and cancer.

Nitrite, nitrosamines, and cancer. Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution.


Hypothalamic inactivation of thyrotrophin-releasing hormone. Following the demonstration of peptidases in the rat hypothalamus which inactivate thyrotrophin-releasing hormone (TRH), a sensitive and specific radioimmunoassay for the releasing hormone was used to investigate the presence of similar peptidases in the rabbit hypothalamus. TRH was found to be rapidly inactivated by supernatant and particulate hypothalamic fractions, with higher peptidase activity in the supernatant than in the particulate fraction. An optimum pH of 7.3 within physiological limits was obtained for the enzymes in both the fractions examined. The results obtained confirm that the rabbit hypothalamus contains enzymes capable of inactivating TRH, and since it has been found that such peptidases interfere with studies on TRH biosynthesis, it is possible that the peptidases may play a part in controlling the releasing hormone's production. The specificity of the antiserum used in the radioimmunoassay has also suggested that the peptidases may cleave the C-terminal-ProNH2,-NH2 or both from the TRH molecule to cause inactivation.


Effect of cimetidine on 24-hour intragastric acidity in normal subjects. The effect of H2-receptor blockade on intragastric acidity was studied in nine normal males. The pH of their gastric contents was measured at hourly daytime and two hourly nighttime intervals for 48 hours. The subjects ate identical meals, drank identical volumes of fluid, and smoked the same number of cigarettes during the two study days. Their physical activity was unrestricted in a ward environment. Blood cimetidine and plasma gastrin were measured in serial blood samples. The nine subjects were treated in random sequence with cimetidine 0-8-1-0 g on one day and placebo capsules on the other. The drug was given in four divided doses: four subjects received it before, and five after, the three main meals. All took the fourth dose at bedtime. Replicate studies in an additional subject given placebo on both study days showed good reproducibility (r=0-80, P less than 0-01). Cimetidine therapy decreased intragastric acidity in all nine subjects. The decrease was similar in the two groups taking the drug before or after meals, mean 24 h intragastric hydrogen ion activity being lowered by 70 and 72% respectively. Nocturnal anacidity was recorded in only two of 45 samples. Administration of cimetidine before meals produced earlier and higher drug blood levels than post-prandial medication, but when it was taken after food the blood levels were highest at the time when the buffer capacity of the food was waning. Blood concentrations of cimetidine exceeded the secretory IC50 level for most of the time between doses. The results show that cimetidine 0-8-1-0 g/day in four divided doses produces a striking and consistent decrease of intragastric acidity. Although variation in the timing of the dose in relation to meals did not affect the decrease of acidity, the absorption data suggest that patients should take the drug after meals.


The effects of some factors on the growth and morphology of Naegleria sp. and three strains of the genus Acanthamoeba. The effects of various biophysical and chemical factors on the cytology of vegetative stages of Naegleria sp., Vitek strain, Acanthamoeba culbertsoni, Acanthamoeba castellanii, Neff strain and Acanthamoeba polyphaga, No. 1289, were studied. The amoebae were cultured in a liquid medium under axenic conditions. The optimum temperature was 37 degrees C for pathogenic strains of Naegleria sp. and Acanthamoeba culbertsoni and 20 degrees C for A. castellanii. No changes were observed in the growth of A. polyphaga at the temperatures 20 degrees and 37 degrees C. The strains investigated grew at pH values of 5.6 to 7.7 using Soerensen's buffer. At the limit values the growth was inhibited and the morphology of cells was markedly changed. All of the four strains grew still at pH 8.4 kept by NaHCO3. A. polyphaga grew at partial anaerobiosis. The three tested strains of the genus Acanthamoeba grew in liquid axenic medium with 0.89% NaCl. The growth of Naegleria sp., Vitek was inhibited already at 0.2% concentration of this salt. The addition of 3 X 10(-2) m KCl to the culture medium had a harmful effect on the growth and morphology of three tested strains, except A. polyphaga. In the culture medium containing 2 X 10(-3) m CaCl2 the encystment of both pathogenic strains was stimulated. The cytological changes under experimental conditions were manifested by atypical movement of trophozoits and their intracellular structure.


[Potassium substitutes during tocolysis]. The level of serum potassium during tokolytic therapy with Partusisten and Isoptin decreases in the first 24 hours of therapy. This decrease is not due to an increase in renal potassium elimination. During tokolytic treatment the serum level of potassium returns to its normal value after 48 hours without potassium substitution. While there is a decrease in serum potassium noticeable changes in electrocardiogram which are typial for hypopotassemia are observed. These changes disappear afer 48 hours of tokolytic treatment but never the less the initial serum potassium drop should be balanced by potassium substitution within these 48 hours.


[Ambulatory therapy of trichomoniasis vaginalis. Clinical trial of a new Trichomonas-active azide substance]. Report on the results of the clinical trial of a new trichomonacide in ambulatory patients in a gynaecological practice. A report is given on a new drug for the treatment of vaginal trichomoniasis, which has been tried in 87 patients of a gynaecological practice under outpatient conditions. The rate of failures was 5.7% (5 of 87 cases). The advantages over alternative preparations is to be seen in the short duration of treatment (24 hours at the most), and in the favourable tolerance in spite of high doses.


Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10- or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a Kav of between 0.4 and 0.6 and were further increased in potency by two- or fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mug/ml and that of the filtrate was less than 0.1 mug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of 5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin.


Activity of two Streptococcus mutans bacteriocins in the presence of saliva, levan, and dextran. The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology of the oral cavity.


Cephacetrile, a new cephalosporin: in vitro, pharmacological and clinical evaluation. Cephacetrile, a parenteral cephalosporin, was evaluated for in vitro antibacterial activity, clinical pharmacology and effectiveness in the treatment of severe infections. The antibacterial activity against 187 isolates was determined by an agar-dilution technique. The MICs were 0.06 to 0.5 mug/ml for Group A Streptococcus, D. pneumoniae, and Staph. aureus, 4-6 mug/ml for E. coli and Klebsiella-Enterobacter 8-32 mug/ml for Pr. mirabilis and more than 500 mug/ml for Ps. aeruginosa. A few strains of Klebsiella and E. coli had MICs of more than 125 mcg/ml. Serum levels after 0.5 and 1 g of i.m. cephacetrile were respectively 14.6 and 18.6 mug/ml after 1 hr, and 1.5 and 2.5 mug/ml after 6 hr. Serum levels after i.v. infusion of 0.5 and 1 g were respectively 16 and 25 mug/ml after 1 hr., and 1 and 2 mug/ml after 6 hr. Urine levels after 0.5 and 1 g i.m. cephacetrile were respectively 500 and 650 mug/ml in the 0-3 hr period, and 250 and 300 mug/ml in the 3-6 hr period. Renal clearance was 166 +/- 5 ml/min/1.73 m2; renal excretion was about 20% of the dose 6 hr after i.m. injection. Cephacetrile was well tolerated when administered i.m. with lidocaine. Mild phlebitis occurred sometimes after i.v. infusions. The clinical response, evaluated in 36 patients with severe systemic, respiratory and urinary infections, was good in all but two cases.


Pituitary gonadotropin response to luteinizing hormone-releasing hormone (LH-RH) in males with azo- and oligospermia. Twenty-one males with azo- or oligospermia presenting with infertility and with no evidence of organic disease were studied with luteinizing hormone - releasing hormone (LH-RH). A pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) response was present in all cases and was normal in the majority of the subjects. Serum testosterone and 17 beta estradiol levels were normal in all cases studied. No significant correlations were found between the gonadotropin estimations and sperm count, basal serum testosterone or testosterone response to human chorionic gonadotropin. It is concluded that LH-RH is of limited diagnostic use in the investigation of this group of patients with male infertility and provides no further insight into the pathogenesis of this condition.


Age at the menopause and onset of the climacteric in women of Martin District, Czechoslovkia. Statistical survey and some biological and social correlations. In this study, 6877 women were analysed whose ages ranged between 38 and 58 (born between 1909 and 1929) and who had had no artificial menopause. This is 88.04% of the total female population in this actual period of life, living in Martin District in 1967. The mean age at the menopause was found, by status quo method, to be 51.21 years (standard deviation 4.4), and by the method of weighted arithmetical means, 48.81 years, (standard deviation 3.9). The mean age at the onset of the climacteric, calculated by the same methods, was 47.55 years, or 46.74 years, respectively. The mean age at menarche was 14.6 years. The average birth-rate was 2.8. The mean period of fertility for the series as a whole was 36.6 years. Women with menstrual disturbances had their menopause about 1 year earlier. We have noted a similar tendency in nulliparas and primiparas. We could find no great difference in the age at menopause between those who had had an early or a late menarche. Menstrual disturbances also influenced the onset of the climacteric. It was at least one year earlier than with regular menstruation. Age at menarche and parity had no effect on the age at the onset of climacteric. Women working in agriculture and housewives had their menopause a little later than mean age of the series, whereas manual workers and those in other occupational categories had their menopause and onset of the climacteric about 1 year earlier. Furthermore, single women had their menopause about one year earlier than the married ones. Widows had their menopause twice so often as the married women and they got it very soon after the husbands's death.


Fibrinolytic activity of the rat ovum, appearance during tubal passage and disappearance at implantation. Fertilized rat ova were histochemically examined for their fibrinolytic activity. Activity was found during tubal passage, which disappeared at implantation. Simultaneously the fibrinolytic activity of the endometrium disappeared at deciduation. These changes might be a prerequisite for implantation of the zygote.


Juvenile diabetes and human sperm quality. Spermiological findings in 25 juvenile diabetics in the age range 16 to 22 years (average 18.5) have been compared with normal spermiological findings in 24 individuals of the same age (16 to 22 years, average 18.7). A drift to lower sperm values in the group of the juvenile diabetics became evident in all parameters observed. As concerns motility and morphology, the differences between the groups reached statistically significant values.


On round-headed human spermatozoa.

On round-headed human spermatozoa. In an infertile man and his only, likewise infertile, brother the sperm contained only round-headed spermatozoa. Electron microscopic examination of embedded ejaculate revealed a malformation, viz. absence of acrosomes in all the spermatozoa with consequent lack of normal development of the heads of the spermatozoa. Acrosomeless spermatozoa were also found in the testicular tubuli. No disturbance of the endocrine functions could be demonstrated. The blood cells were of normal male karyotype. Investigation of meiosis, on the other hand, showed rudimentary second division. Two other unrelated infertile men showed the same isolated disturbance in their spermiogram. Treatment with chlomiphene had no effect on the structure of the spermatozoa or on the infertility. The rare disorder, which seems to be a special syndrome, the first hitherto distinguishable among the various types of teratospermia, and its probable background are discussed.


Assessment of the therapeutic effect of epimestrol and epimestrol associated with clomiphene in female sterility. In 24 women with disturbances of ovulation treated for sterility with Epimestrol, ovulation was achieved in 3 patients but none of these became pregnant after therapy. Since it has been suggested that the association of Clomiphene with a weak estrogen might improve the pregnancy rate, we decided to administer Clomiphene associated with Epimestrol. Using this combined therapy in 58 patients, 32 out of the 58 women ovulated and 17 conceived. The overall rate of pregnancy using the combined therapy was no better than that obtained when Clomiphene alone is administered. From this study it is concluded that: (1) Epimestrol is not an effective method for the induction of ovulation, and (2) addition of Epimestrol to Clomiphene is of no clinical benefit.


Improvement of fertility and semen quality in men treated with a combination of anticongestive and antibiotic drugs. The effects of a combined therapy of antibiotics and antiinflammatory drugs was evaluated in 344 men referred to our clinic for treatment of infertility. Physical signs of congestion, usually not severe, were detected in 244 men. The treatment caused significant improvement in semen quality, especially in sperm concentration, morphology and motility. Forty percent of the wives became pregnant. There was a striking relationship between increase in morphologically normal spermatozoa and incidence of pregnancy. It is speculated that the therapy had its major effect at the level of the epididymis and/or testis.


Genital organs. Auto and homotransplantation in forty dogs. Auto and homo transplantation of the uterus and ovaries was studied in 40 bitches divided into four groups. Group A served as a control. In group B the omentopexy technique of auto-transplantation was used. This method did not prove very rewarding and all 12 grafts were found to have degenerated and were non-functioning when relaparotomy was performed, 3 months after transplantation. In group C, auto transplantation with vascular anastomosis was performed in 12 dogs, yielding satisfactory results including one successful pregnancy and the delivery of 3 healthy puppies. In group D homotransplantation of the internal genital organs was performed in two sets of twin dogs. No immunosuppressive treatment was used. These grafts survived for a limited period as proved by the functional tests and X-ray examination but after 3 months were found to be necrotic and degenerated. Graft function and viability was established by: X-ray examination, cytology of vaginal smears, the effect of gonadotropins on the grafted ovaries and finally relaparotomy undertaken after 3 weeks and 3 months.


Crossed immunoelectrophoresis of sperm antibodies in human serum and cervical mucus. Sperm agglutinating antibodies are purified from sera and cervical mucus of women with unexplained causes of infertility which were positive in the FD-test. Fractionation was performed by affinity-chromatography in a batch device and the sperm agglutinating activity controlled by the Franklin and Dukes test. This sperm antibody fraction was determined via crossed immunoelectrophoresis by migration into an anti-human serum containing gel. In all cases only one big peak resulted. The negative control serum and mucus samples demonstrated no precipitation peaks. By absorption studies it was shown that the sperm agglutinating antibodies in sera were IgM and in cervical mucus IgA. The concentration of IgA and IgM was determined by comparison with standard human IgA and IgM. Thus only one serum- and one cervical mucus antibody seems to be responsible for agglutination. The number of experiments, however, is still too small for general conclusions. This method is easily and quickly performed and can therefore be used as a routine method for the determination of sperm agglutinating antibodies. Its application for sperm-immobilizing or cytotoxic activity remains to be tested.


Microbiopsy of the fallopian tube as a method for clinical investigation of tubal function in infertility. Microbiopsy of the fimbrial end of the fallopian tube may prove to be a valuable method for investigating the tubal pick-up and transport mechanism in infertility patients. Counting of the percentage of ciliated cells on semithin sections shows that in normal fertile women a percentage of such cells is present that is higher than in abnormal conditions such as peritubal adhesions, ectopic pregnancy and amenorrhoea following prolonged progestogen treatment. It is also suggested that ultrastructural examination of the biopsy may provide additional valuable information.


Improvement of sperm motility in patients with asthenozoospermia by kallikrein treatment. Parenteral and oral application of kallikrein (EC 3.4.21.8)--a kinin-releasing proteinase from porcine pancreatic tissue--significantly stimulates quantitative and qualitative sperm motility in subfertile males with semen criteria of asthenozoospermia. Improvement of sperm motility was found to exist for at least 3 months following termination of the 7 week duration kallikrein treatment. Additionally, a significant increase in the number of spermatozoa was observed 3 and 5 months after starting parenteral application of kallikrein.


Oligozoospermia: a seven-year survey of the incidence, chromosomal aberrations, treatment and pregnancy rate. No sperm count should be regarded too low to consider extensive treatment in order to improve semen or to correct any possible abnormalities in femal partners. The infertile couple should be given devoted care and advice to improve their sexual relations and their psychological attitude towards the problem of infertility. A pregnancy rate of 51.9% where the husband's count was less than 10 million/ml, offers adequate support for this statement. In cases of mental trauma inflicted on patients by a verdict of inability to achieve parenthood--on people who have already suffered severe psychological shock and tension resulting from a period of infertility--discouragement lessens the already doubtful chances of achieving pregnancy. In no circumstance should oligozoospermic patients receive treatment for infertility unless a chromosomal analysis has been completed and found normal, since our rate of chromosomal anomalies (11.4%) in a group of oligozoospermic patients is considered to be too high.


Further studies on the fertility promoting factor from human seminal plasma. A small molecular weight substance from human seminal plasma has been further purified by chromatography. The fertility promoting action of this factor on epididymal sperm has been confirmed in mouse in vitro and in vivo. Experimental evidence indicates that the factor acts on the sperm and not on the eggs. Its possible mode of action is by improving the motility and survival of the epididymal sperm.


The presence of paternal H-2 antigens on hybrid mouse blastocysts during experimental delay of implantation and the disappearance of these antigens after onset of implantation. The presence of paternal H-2 antigens on hybrid mouse blastocysts before and during implantation was investigated by means of the isotope anti-globulin technique. It was found that experimentally delayed blastocysts possess paternal H-2 antigens whereas these antigens can no longer be detected 14 hours after estradiol activation of delayed blastocysts.


Effect of copper and plastic intra-uterine devices on the fibrinolytic activity of the endometrium in the rat. The effect of copper and plastic intrauterine devices (IUD) on the fibrinolytic activity of the endometrium was studied in the rat. A copper or a plastic device was placed in one of the uterine horns, while the other horn served as a control. Biopsy specimens were obtained from both horns and examined histochemically. The copper concentration was determined by atomic absorption spectroscopy. The fibrinolytic activity of the control horn was found to be localized to small vessels in the outer layer of the uterine wall, while that of the endometrium was low. Plastic as well as copper IUDs increased the fibrinolytic activity which, in contrast with what was seen in the controls, was localized to the endometrium. Compared with the effect of the plastic device, the increase in the fibrinolytic activity induced by the copper device was more widespread in the endometrial area and was accompanied by an increase in the concentration of copper. These findings might help to explain why the contraceptive effect of IUDs is more reliable when they are partly coated with copper.


Participation of vitamin A in the maturation of rabbit spermatozoa. Vitamin A concentration was fluorometrically measured in epididymal and ejaculated rabbit spermatozoa and in some of the sperm cells subcellular components. The concentration of vitamin A in the epididymal cells was about one-half that observed in the ejaculated spermatozoa (2.68 as against 1.05 mug/10(8) cells) and seemed to be the same in the sperms obtained from both the head and the tail of the epididymus. The concentration of vitamin A was also found to be significantly higher in the seminal plasma than in the epididymal secretion (0.06 as against 0.039 mug/mg protein respectively). Practically all the vitamin A was found in the fractions obtained by treatment with hypotonic MgCl2 (acrosomal region) and/or with hyamine and dithiothreitol (plasma membrane). It was concluded that the sudden increase in the sperm concentration of vitamin A that occurs upon ejaculation may be required for the stabilization of the acrosomal and plasma membranes.


Evaluation of d-norgestrel 1.0 mg as a post-coital contraceptive. Two hundred and ninety-eight women were followed for 2578 months (2739 'bleeding intervals') of treatment with d-Norgestrel 1.0 mg given as a post-coital oral contraceptive. Fourteen pregnancies were recorded (general failure rate, 6.5 per 100 woman/years); at least 6 of these patients did not miss any tablet (corrected failure rate, 2.8). The acceptability rates (life table method) were 0.58 and 0.40 after 6 and 12 months of follow-up. The most important medical reason for drop-out was cycle irregularities. The cycle pattern is deeply disturbed by this method of oral contraception.


"Genital dyscrinism" as a cause of subfertility in mice of the CBA strain. Following four generations of inbred mating (brother-sister) in three direct lineages of CBA strain mice, sterility appeared which from that generation forward became more frequent. The genital organs in animals of both sexes were altered. There was a noticeable occurrence of cysts in the ovaries of female animals already following the third month; in mice approximately one year of age this condition was followed by cystic glandular hyperplasia of the endometrium, sometimes complicated by disturbances in blood circulation, inflammation or even malignancy. In some female animals, manifesting, due to cysts, completely degenerated ovaries, the rest of the genital system was severely atrophic. Male animals frequently showed severe atrophy of the seminiferous epithelium along with preserved interstitial cells and hypertrophic seminal vesicles. These pathological changes represent an independent nosologic unit, for which the label "genital dyscrinism" has been proposed. The authors have considered an endocrine mechanism as the possible cause of these pathological changes which are presumed to be genetically conditioned.


Motility of the rat oviductal tract isolated in different stages of the sex cycle. Effects of catecholamines. Physiological and pharmacological characteristics of the spontaneous motility of rat oviductal tracts (the coiled oviduct plus its mesosalpinx), isolated in proestrus, estrus or metestrus, are described. The initial contractile tension (recorded following isolation) was comparable in the three stages of the cycle, but its decrement with time was greater in metestrus than in proestrus; the opposite being observed regarding the rate of contractions. Norepinephrine and phenylephrine depress motility in proestrus and metestrus, but not in estrus. The inhibition of motility during proestrus, produced by added norepinephrine, phenylephrine or isoproterenol was not modified by phenotolamine but was abolished by propranolol. During estrus, norepinephrine and phenylephrine inhibited tubal contractions of preparations incubated with phentolamine, whereas it produced a distinct stimulation in the presence of propranolol. It is concluded that: (a) the rat mesosalpinx might play some role in the motility of the whole isolated oviductal tract; (b) there are variations in the decrement with time of contractile tension and frequency of contractions in different stages of the sex cycle; (c) the effects of catecholamines upon the rat oviductal tract also varies within the cycle, probably due to influences imposed by sex hormones.


The effect of prostaglandins and prostaglandin inhibitors on spermatogenesis. The effect of the prostaglandin inhibitors, aspirin and indomethacin and of prostaglandins PGE1 and PGE2 on spermatogenesis in the mature male mouse has been studied. Aspirin at 100 mg/kg and at 200 mg/kg, and indomethacin at 1.0 mg/kg given orally twice a day for fifteen days produced a marked increase in spermatogenesis. The number of step 7 spermatids increased significantly over controls at about the same rate in all three groups. No significant changes in seminal vesicle weight or testicular weight was noted, although testicular weight did show an increase. Administration of prostaglandins E1 and E2 subcutaneously in doses of either 2 mg/kg or 3 mg/kg once a day for fifteen days produced a marked decrease in spermatogenesis. Step 7 spermatids decreased significantly at both dosage levels of PGE2 and at the higher dosage level of PGE1. Spermatocyte showed a significant decrease at the higher dose of PGE2. Testicular weight showed a significant decrease at the higher dose of PGE2. Seminal vesicle weight showed a significant decrease at the lower dose of PGE1 and at the higher dose of PGE2. Epididymal weight decreased at the higher dose of PGE2. Increased numbers of exfoliated immature germ cells and mature spermatozoa were observed in the epididymus of both the PGE1 and PGE2 treated animals.


Low fertility rate in vasovasostomized males and its possible immunologic mechanism. Contradictory views have been expressed about the role of the various antisperm antibodies which develop after vasoligation. The present study was conducted in 50 normal fertile males, 50 vasectomized subjects and 25 subjects after recanalization of their vas deferens in order to investigate the development of various anti-sperm antibodies after vasectomy, along with their incidence, their persistence after successful relief of vaso-obstruction by vasovasostomy and their role in the causation of infertility in vasoanatomized normospermia males. Sperm agglutinating, immobilizing and haemagglutinating antibodies showed rises in titres with increase during the post-vasectomy period, indicating continuous antigenic stimulus. Age, post-operative complications and blood group did not seem to alter the results. 86% of subjects developed antisperm agglutinins, mostly tail-to-tail type (54.5%), 1-12 years after vasoligation, while only 2% of fertile men had circulating spermagglutinins. A lower incidence of positive sperm in the immobilization test than in the agglutination test suggests either that different antibodies are detected by these two tests or these tests have differing sensitivities. Of the 25 vasovasostomized subject, 13 (52%) cases became normospermic and 4 (16%) oligospermic while 8 (32%) remained azoospermics. Except for 3 oligospermic subjects, all had circulating spermagglutinins. Among the 13 normospermic vasovasostomized persons, a significant correlation was found between the titres of circulating antisperm agglutinins and autoagglutination of spermatozoa in their ejaculates; and also between the sperm immobilization values of their sera and the degree of their sperm motility. Three normospermic recanalized men, having low levels of sperm agglutinins and haemagglutinins with normal seminogram and no sperm immobilizing antibody, successfully impregnated their wives. Another 10 vasovasotomized infertile subjects had sperm agglutinins in significant titre; 5 showed positive sperm immobilization values, a similar number showed autoagglutination of sperm, while a decreased degree of motility of sperms was noted in 6 cases. Thus there was a significant correlation between the titres of anti-sperm antibodies and autoagglutination of spermatozoa, which might be an important cause of male infertility after successful anatomic relief of vasoobstruction. Histological studies of testicular biopsy showed normal spermatogenesis in azoospermic recanalized subjects, although they had high levels of antisperm antibodies. This suggests that these antibodies do not affect normal spermatogenesis, and sperm counts.


Studies on urinary arylsulphatase activity in vitamin A deficient rats. Urinary arylsulphatases (E.C.3.1.6.1) A and B were increased in male rats fasted for 24 hours. Excretion of non dialysable protein nitrogen decreased whereas creatinine excretion increased. On refeeding diet arylsulphatase A activity was restored to normal whereas arylsulphatase B was not normalised. A single oral supplementation of vitamin A acetate (20 000 IU) to rats fasted for 24 hours resulted in a significant reduction of both arylsulphatase A and B eventhough no further reduction of protein nitrogen excretion was evident. In vitamin A deficient male rats significant reduction in urinary excretion of both arylsulphatases A and B occured. In a smaller number of female rats depression of only arylsulphatase A was observed. This effect of vitamin A deficiency leading to reduced urinary arylsulphatase activity was evident even at the "weight plateau" stage when no reduction in food intake or growth had  occurred. These results suggest a possible direct or indirect role for vitamin A on urinary excretion pattern of arylsulphatases presumably released from lysosomes of tissues.


Effect of long-term starvation on the rat liver lysosomes. The effect of 120- and 240-h starvation on rats hepatocytes ultrastructure and particularly the changes of the lysosomes were studied. Eelectronmicroscopically and cytochemically there have been observed diminution of the number of mitochondria and degranulation and vacuolzation of the ER. At the same time Golgi complex was hypertrophied and the number of lysosomes was much increased, mainly those of the autophagic type. Biochemically was shown, that the activity of some acid hydrolases (beta-glucosidase, alpha- and beta-galactosidases, beta-N-acetylglucosaminidase, beta-glucuronidase and arylsulphatases A and B) in the liver of starved rats was markedly expressed. The sedimentation properties of the lysosomes and the lysosomal membrane stability was damaged as well. The data received have been discussed in the light of the reconstructive role of lysosomes.


Hypogastric carotid bypass for Takayasu's disease. Two cases of Takayasu's disease are presented which were successfully resolved by a Dacron graft from the right hypogastric artery to the right internal carotid artery. We recommend this bypass procedure of connecting the hypogastric artery to one of the aortic branches. Medical treatment with antibiotics, steroids and anti-coagulants has not been satisfactory.


Psychopharmacology research ward. Ten years' experience. The differences between the results of controlled clinical research and the experiences of the practitioners can be attributed to a certain extent to the artificiality of the setting in which controlled studies are performed. The system developed in the wards of the Department of Psychopharmacology of the Institute of Psychiatry in Prague is an attempt to overcome some of these difficulties. Certain similarities exist between this system called continuous controlled trial and 'silent' trial proposed by Ross et al. The main characteristics of the system are: (1) all patients admitted to the ward are assigned to the trial without exception; (2) the trial goes on continuously without interruption for more than 10 years; (3) except that the patients do not know the quality of drugs administered they do not realize any difference between this research setting and the usual routine treatment. The advantages of this system are: reduction of artificiality; the possibility to perform early clinical trials in controlled conditions and to shorten the usual three-phase testing of a new drug; the possibility to perform long-term, longitudinal studies with individual patients admitted several times in double-blind conditions.


[Comparative study of three psychotropics for treatment of depressions]. The effect of three anti-depressive psychotropes (Clorimipramine, Doxepine and Dibenzepine) was studied in 107 depressed patients. In each patient the mean value of twelve symptoms was evaluated and compared weekly (for 4 weeks), by statistical methods. In addition, the effect of each drug was analysed in personality stratus. A thymeretic and thymoanaleptic rapid action on 'corporality' and 'endotimic-vital' layer was found with Clorimipramine. Doxepine acts rapidly with sedative and anxiolitic actions on reactive symptoms related with personality super-structures having long term anti-depressive effects. Dibenzepine has a thymeretic rapid and intensive action and a slow thymoanaleptic effect on the same personality stratus of Clorimipramine.


Properties of rat lens phosphofructokinase. Two interconvertible forms of phosphofructokinase (PFK) have been eluted from a DEAE-cellulose column from the supernatant fraction of rat lens homogenates centrifuged at 96,000 x g for 1 hour at 0 to 4 degrees C. The interconversion can be manipulated by a change in the pH of the extracting and eluting buffers. PFK-I is the dominant form at pH between 7.4 to 7.05, while PFK-II dominates at pH 7.4 to 8.2. PFK-II is believed to be the functional form; it is inhibited by high concentrations of ATP and the inhibitory effect is enhanced by more acidic pH. Fructose-6-phosphate counteracts ATP inhibition, but the most potent de-inhibitors are ADP and AMP. Among the inorganic ions tested, sulfate, phosphate, ammonium, and potassium also de-inhibit, whereas calcium further inhibits the enzyme. The behavior of PFK under physiologic conditions and the significance of the presence of two forms of PFK in the lens are discussed.


Effects of pregnancy on the development of acute uremic syndrome in the rat. We evaluated the contributory role which gestational metabolic adaptions may play in the development of acute uremic syndrome in rats by studying the effects of nephrectomy in pregnant and nonpregnant animals. Whereas no significant differences in creatinine and uric acid concentrations were found between control and pregnant rats, urea concentrations were significantly lower in the pregnant animals before, as well as well as 6, 12 and 24 h after, bilateral nephrectomy. It is suggested that creatinine production, which is related to muscle mass, is not altered during late gestation, whereas urea synthesis, which is affected by catabolic and anabolic factors, is significantly reduced.


Blood-gas analysis and the assessment of acid-base status. In this article the more common kinds of acid-base disorders have been discussed. To accurately assess the kind of acid-base disturbance found in a patient, the clinician must know the arterial pH, PaCO2, and the [HCO-3] in arterial blood. Acid-base disturbances are associated with fluid and electrolyte disturbances. Potassium balance is often upset in acid-base disturbances. Accurate determination of K+ balance requires serial or repeated determinations of plasma K+ concentration as well as careful clinical monitoring.


Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study. The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.


Arterial blood-gas interpretations in the respiratory intensive-care unit. The role of the nurse in the respiratory intensive-care unit requires increased sophistication as our knowledge of the patient becomes more complex. This expanded role should include a thorough understanding of disturbances in acid-base balance, the relationship of PaCO2 to ventilation, the difference in acute and chronic respiratory problems, and the causes and treatment of hypoxemia. The ability to analyze and evaluate blood-gas determinations is simply one more important tool the nurse may utilize in the care and treatment of the critically ill patient.


Antimycin A fermentation. II. Fermentation in aerated-agitated fermenters. Fermentation characteristics, previously studied in shake flasks, were reproduced in aerated-agitated fermenters, using three strains of Streptomyces sp. which had been selected for their high antimycin A productivity in shake flasks. Fermentation in fermenters was run in three stages. The medium consisted of soy flour, glucose, ammonium sulfate and calcium carbonate; initial pH was 7.2 approximately 7.5, and temperature 25 degrees C. The course of fermentation was then modified to encourage maximal growth and eliminate the intermediate lag period observed in shake flasks. Useful corrections included continuous addition of soybean oil at 1.25 %/day and maintenance of pH at 6 by addition of ammonium hydroxide on demand. The ammonium hydroxide added also served as a rapidly utilized nitrogen source and could not be replace by NaOH or KOH. Under optimal conditions antimycin A was produced at constant rate from the second to the sixth day, when maximum yields of more than 9 g/liter were attained. A procedure for antimycin A extraction is described.


Nutritionally defined conditions for germination of Streptomyces viridochromogenes spores. Spores of Streptomyces viridochromogenes were removed from the surface of solid media with glass beads and suspended in a buffer-detergent solution. Addition of yeast extract and glucose resulted in rapid loss of refractility of the spores. Appearance of germ tubes followed. Germination was accompanied by a decrease in the optical density (OD) of the suspension. The OD decrease was used as an assay for germination. A defined germination medium (DGM) comprised of L-alanine, L-glutamic acid, adenosine, para-aminobenzoic acid, and calcium and magnesium ions provided a germination rate nearly equal to that of complex media. The germination rate was essentially the same if D-alanine and D-glutamate replaced the L-isomers. The optimum pH and temperature for germination were 7.0 and 35 C. Germination was absolutely dependent on the presence of CO2. Spores harvested after growth for longer periods than the usual time (10 days) became less germinable in DGM. The same was observed for spores grown at 37 C as compared with 30 C. Spores incubated in DGM for various time periods before being transferred to a buffer solution did not continue to germinate. Spores harvested after growth of eight species of Streptomyces did not show a decrease in OD when incubated in yeast extract medium. Another strain of S. viridochromogenes did exhibit an OD decrease in the medium. Comparative properties of spores of streptomycetes, fungi, and bacilli are discussed.


Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky). Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF.


Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells. Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino) strain of Saccharomyces cerevisiae.  Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-1-phosphate (IP synthethase, EC5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC3.1.3.25).  The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubaion time and enzyme concentration.  The pH optimum was 7.0.  The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD) and was stimulated byNH4C1 and MgC12.  NADP substituted poorly for NAD, and NADH inhibitedthe reaction.  Phosphorylated inositol accumulated in the absence of MgC12, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg ions stimulate the dephosphorylation of inositol-phosphate.  IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM.  The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 muM.  IP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol.  The specific activity of the enzyme increased with time in logarithmically growing cultures lacking inositol andapproached the fully depressed level as the cells entered stationary phase.


Heat activation of Streptomyces viridochromogenes spores. The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.


Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum. Active transport of glucose in prosthecae isolated from cells of Asticcacaulis biprosthecum was stimulated by the non-physiological electron donor N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride. Glucose uptake was mediated by two transport systems; the apparent Km of the high-affinity system was 1.8 muM and that of the low-affinity system was 34 muM. Free glucose accumulated within prosthecae at a concentration 60 to 200 times above that present externally, depending on the Km of the system being observed. The glucose transport system in prosthecae was stereospecific for D-glucose, and neither methyl alpha-D-glucopyranoside nor 2-deoxyglucose was transported. Uptake of glucose was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB), and the inhibition by PCMB but not by NEM was reversed by dithiothreitol. Glucose uptake was also inhibited by the uncoupling agents 5-chloro-3-t-butyl-2'-nitrosalicylanilide (S-13), 5-chloro-3-(p-chlorophenyl)-4'-chlorosalicylanilide (S-6), and carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and by the respiratory inhibitor KCN. Efflux of glucose from preloaded prosthecae was induced by PCMB and KCN, but not by S-13 or CCCP. Glucose uptake was not affected by arsenate or an inhibitor of membrane-bound adenosine triphosphatases, N, N'-dicyclohexylcarbodiimide. The lack of inhibition by these two compounds, combined with the extremely low levels of adenosine 5'-triphosphate present in prosthecae, indicates that adenosine 5'-triphosphate is not involved in the transport of glucose by prosthecae.


Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes. Membrane preparations obtained from a stabilized L-form of Streptococcus pyogenes are incapable of synthesizing peptidoglycan from uridine-5'-diphospho-N-acetyl-D-muramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala and uridine-5'-diphospho-N-acetyl-D-glucosamine, in contrast with similar preparations from the parental streptococcus. Furthermore, 50-fold higher levels of lipid intermediates which serve as membrane-bound substrates for peptidoglycan synthesis are synthesized in reaction mixtures containing streptococcal membranes than with similar preparations from the L-form. These observations suggest that the inability of this stabilized L-form to form a cell wall in vivo lies, at least in part, in its failure to synthesize significant quantities of the lipid substrates for peptidoglycan synthesis.


Protonmotive force as the source of energy for adenosine 5'-triphosphate synthesis in Escherichia coli. Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).


Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium. Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane.


Effect of colicin K on a membrane-associated, energy-linked function. The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E. coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi. Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source. The ATP-linked reaction was partially inhibited in French press extracts of E. coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant. Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells. The extent of inhibition increased with increasing concentration of colicin. Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect. A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin. The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical. Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase. It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction.


Ribonucleic acid synthesized in meiotic cells of Saccharomyces cerevisiae: effect of culture medium pH. Pulse-labeled ribonucleic acid (RNA) was extracted from polysomes of sporulating cells of Saccharomyces cerevisiae and characterized in sucrose gradients and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transfer RNA, ribosomal RNA, and heterodisperse RNA, presumed to be messenger RNA, were synthesized during a 20-min pulse at T4 and T6 when labeling was performed in sporulation medium adjusted to pH 6.0. Furthermore, ribosomal RNA was processed into functional ribosomes during the pulse. The specific activity of pulse-labeled RNA of cells labeled in sporulation medium where the pH was unadjusted at T4 (pH 7.8) and T9 (pH 8.6) was 20- to 50-fold lower than RNA from cells labeled at pH 6.0. The low specific activity resulted from a 50-fold reduction in uptake of labeled precursors when the medium pH was greater than 7.2. However, heterodisperse RNA ranging from 4-17S in size and transfer RNA were synthesized during the pulse at T4 (pH 7.8),but the low specific activity of ribosomal RNA prevented a thorough analysis of its synthesis. Cellular impermeability at T9 (pH 8.6) resulted in minimal uptake of label, and an analysis of pulse-labeled transcripts was impossible. A comparison of the percantage of polysomal material indicate, however, that these cells were at least as active in translation as cells pulse-labeled at pH 6.0.


An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis. Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.


Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus. Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.


Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.


Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins. Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable to gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of sialidase I, II, and III activities were 310,000 +/- 10,000, 105,000 +/- 4,000 and 64,000 +/- 2,000, respectively, the first figure being an approximate value only.


Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities. Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed.


Development of Microbodies in the yeast Kloeckera growing on methanol. A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.


Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants. The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.


Autolysis of Neisseria gonorrhoeae.

Autolysis of Neisseria gonorrhoeae. Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.


Glucose-6-phosphate dehydrogenase. Purification and partial characterization. Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm.


A kinetic study of glucose-6-phosphate dehydrogenase. The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively.


alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis. The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes.


Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties. gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.


Kinetics of the hemerythrin-oxygen interaction. The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units.


Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture. To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.


Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked. The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.


Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase. Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM.