Bile salt 3 alpha- and 12 alpha-hydroxysteroid dehydrogenases from Eubacterium lentum and related organisms. Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3alpha- and 12alpha-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12alpha-HSDHase only (10 strains), (B) those containing 3alpha- and 12alpha-HSDHase (13 strains), (C) those containing 3alpha-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H(2)S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H(2)S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H(2)S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated "phenotypically similar organisms." Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12alpha-HSDHase of category B organisms was unstable unless 10(-3) M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12alpha-HSDHase and H(2)S production. Growth curves and the effect of arginine on growth and the production of 3alpha- and 12alpha-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3alpha- and 12alpha-HSDHase, respectively. Purified 12alpha-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.


Production of L-serine by Sarcina albida. Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.


Alcohol dehydrogenase from Rhizopus javanicus. Alcohol dehydrogenase of Rhizopus javanicus was purified, and its physical and chemical characteristics were determined. The intact enzyme was shown to have a molecular weight of approximately 60,000. Since the smallest apparent subunit was 14,000, the enzyme was presumed to be composed of four subunits. The crude mycelial extract contained multiple forms of the enzyme, which were separated by ion-exchange chromatography.


Pasteurization of salted whole egg inoculated with Arizona or Salmonella. Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.


Effects of pH of the medium on flagellation of Vibrio parahaemolyticus. Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.


Method for Salmonella concentration from water at pH 3.5, using micro-fiber glass filters. A method is described for the concentration of Salmonella from water. As is done with enterovirus, Salmonella bacteria were concentrated from water in two steps: by pH 3.5 adsorption on and pH 9.5 elution from 8-micron porosity micro-fiber glass filter tubes. This method worked in less than 30 min, and Salmonella typhimurium was inactivated only slightly in spite of rapid pH variations (pH 3.5 to 9.5). It was demonstrated that the retention by the filters stems from two phenomena: a low retention in the micro-fiber glass labyrinth for small filtered volumes, and a high retention by adsorption at pH 3.5 for any filtered volume (experiments done with 15- and 80-liter samples). Addition in tap water of trivalent ions like Al3+ did not increase Salmonella adsorption. In most of the trials, Salmonella recovery varied from 42 to 93%. Preliminary field investigations indicate that enterovirus and Salmonella may both be concentrated from the same water sample by this procedure.


Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria. Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.


Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methane- and methanol-grown microbes. Cultures of methane- or methanol-utilizing microbes, including obligate (both types I and II) and facultative methylotrophic bacteria, obligate methanol utilizers, and methanol-grown yeasts were isolated from lake water of Warinanco Park, Linden, N.J., and lake and soil samples of Bayway Refinery, Linden, N.J. Resting-cell suspensions of these, and of other known C1-utilizing microbes, oxidized secondary alcohols to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Succinate-grown cells of facultative methylotrophs did not oxidize secondary alcohols. Among the secondary alcohols, 2-butanol was oxidized at the highest rate. The optimal conditions for in vivo methyl ketone formation were compared among five different types of C1-utilizing microbes. Some enzymatic degradation of 2-butanone was observed. The product, 2-butanone, did not inhibit the oxidation of 2-butanol. The rate of the 2-butanone production was linear for the first 4 h of incubation for all five cultures tested. A yeast culture had the highest production rate. The optimum temperature for the production of 2-butanone was 35 degrees C for all the bacteria tested. The yeast culture had a higher temperature optimum (40 degrees C), and there was a reasonably high 2-butanone production rate even at 45 degrees C. Metal-chelating agents inhibit the production of 2-butanone, suggesting the involvement of metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble extract of sonically disrupted cells. The cell-free system requires a cofactor, specifically nicotinamide adenine dinucleotide, for its activity. This is the first report of a nicotinamide adenine dinucleotide-dependent, secondary alcohol-specific enzyme.


Reduction of endogenous nucleic acid in a single-cell protein. The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.


Biochemical properties of penicillin amidohydrolase from Micrococcus luteus. Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.


Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C. From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.


Effect of SO2 and bisulfite on heterotrophic activity in an acid soil. Glucose oxidation was inhibited in a forest soil (pH 4.01) previously exposed by 1.0 microliter of SO2 per liter, the extent of inhibition and the decline in pH being directly related to the length of exposure. The phase of rapid CO2 evolution in protein hydrolysate-amended soil previously treated with 5.0 microliter of SO2 per liter for 24 h or 1.0 microliter/liter for 48 h was delayed, but the degradation of the amino acid mixture then proceeded rapidly. Bacterial numbers in soil incubated for 48 h with 1.0 microliter of SO2 per liter were reduced, but the bacteria grew rapidly if glucose or an amino acid mixture was added after the exposure period. Low levels of bisulfite inhibited amino acid decomposition in soil at pH 3.89, but the effect was less pronounced in soil at pH 4.01. Comparable levels of sulfate were not toxic to carbon mineralization. Approximately 1.0 microgram of bisulfite S and about 20 microgram of sulfate S per g of soil appeared when the soil was treated with 1.0 microliter of SO2 per liter for 48 h. Bisulfite added to the soil disappeared readily. The possible ecological significance of the findings is discussed.


Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment. This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.


An 11 year evolution of coronary arterial surgery (1968-1978). All patients who underwent isolated myocardial revascularization procedures from 1967-70 (n = 741) were compared with the first 1,000 patients who received similar elective operations each year from 1971 through 1978. Data from these eight years were processed through a computerized cardiovascular information registry. Median age increased from 50 to 56 years, multiple-vessel disease increased from 44 to 89%, and left ventricular asynergy from 41 to 54%. The number of grafts per patient increased from 1.5 to 2.5 and yet morbidity declined in every category except neurologic deficit. Operative mortality was 1.1% from 1967 through 1978 and 0.9% from 1971 through 1978. Graft patency was determined for 475 patients from 1967-70, 553 patients from 1971, 519 from 1972, and 540 from 1973. Patency rates after a mean catheterization interval of 21 months were 77, 77, 84, and 87% respectively. Higher graft patency coincides with introduction of the internal mammary artery graft. Five year follow-up was completed for the 1967-1970 series and 1971, 1972, and 1973 cohorts. Actuarial five year survival was 89.6, 91.6, 93.2, and 91.7%. Five year survival comparisons between 1967-1970 patients and 1971-1973 patients in single-, double-, and triplevessel disease categories show significant extended longevity in the later experience. Abnormal ventricular function and incomplete revascularization adversely influenced longevity (p < 0.05) in all years surveyed. In those series the percentage of asymptomatic patients at five years was 66, 65, 69, and 67%. Lower risk and higher five year survival are attributed to greater technical experience, changing technology, and improved management rather than to selection of lower risk cases.


Testicular tumors in undescended testes. Twelve cases of malignant tumors in undescended testes were studied. Early diagnosis of these tumors was usually difficult as the testes were intrabdominal and the symptoms vague. Sixty percent of the seminoma cases were of the anaplastic aggressive variant. Early treatment of undescended testes included either orchiopexy or orchidectomy in early childhood. Exploratory laparotomy and frozen section examination of the abdominal nodes, followed by silver clip application, aided in better staging and the planning of further treatment.


Tissue levels of several radiolabelled beta-adrenoceptor antagonists after intravenous administration in rats. The uptake of radioactively labelled propranolol, oxprenolol, metoprolol, acebutolol, practolol and atenolol into brain, liver and lung tissue was studied five min after intravenous administration (1.0 mg/kg) in normotensive Wistar rats anaesthetised with nitrous oxide and halothane. All of the beta-adrenoceptor antagonists including the least lipophilic compounds atenolol and practolol were detected in brain tissue five min after systemic administration. However, the level of propranolol (as measured by total radioactivity) in the bran was 40 and 67 times greater than the levels found for atenolol and practolol, respectively. Additionally, significantly more radioactivity was detected in lung tissue compared to that in liver tissue for the lipophilic, non-selective beta-adrenoceptor antagonists propranolol and oxprenolol. Levels of radioactivity in blood, brain, liver and lung were measured 5, 15, 30 and 60 min after administration of either propranolol or atenolol (1.0 mg/kg, i.v.) to both conscious and anaesthetised rats and a tendency towards high tissue levels of radioactivity was found in the animals which received the beta-adrenoceptor antagonists under anaesthesia. Pretreatment of groups of rats for 7, 14 and 21 days with unlabelled atenolol (1.0 mg/kg/day, i.p.) caused an increase in the subsequent central uptake of labelled atenolol whilst both the blood levels of radioactivity and the uptake into peripheral tissues were significantly lower in the 2 and 3 week pretreated rats compared to the control animals. These results are consistent with the hypothesis that a central action may contribute to the antihypertensive effect of beta-adrenoceptor antagonists.


[Noisy circulation syndrome. Study of 28 cases]. Twenty eight subjects with noisy circulation sindrome (NCS) defined as cases at least, with 4 murmurs in different arteries were studied. They were divided in groups A, B, C formed by 9 normal children, 9 peripheral vascular atherosclerosis adults and 10 Takayasu's arteritis cases, respectively. In the whole population a complete chronometric arterial auscultation and in groups B and C an arteriography of at least 3 arteries with murmurs, were performed. Groups A, B and C had 40, 50 and 37 arterial murmurs, respectively, which were predominantly localized in supraortic trunks, pelvic and phemoral arteries, and supraortic and abdominal regions, respectively. 100, 19.1 and 21.1% of


Effects of halopemide, a new psychotropic agent, on the uptake of serotonin by blood platelets. The influence of halopemide, a new psychotropic agent, and some putative metabolites on the uptake of 14C-5-HT into blood platelets of rat and man were studied and compared to the effects of imipramine, sulpiride and clozapine. Halopemide, its putative metabolites R 38570 and R 29676 and sulpiride inhibited the uptake in a dose-dependent manner, but the compounds were less active than imipramine. Clozapine and p-fluorohippuric acid, another putative metabolite of halopemide, were totally devoid of activity.


Antihypertensive effects of intra-cisternal beta-blocking agents in dogs with acute nerogenic hypertension. Dl-propranolol, acebutolol, atenolol, bupranolol, oxprenolol, pindolol, practolol, sotalol and d-propranolol were injected into the cisterna magna of anesthetized dogs with neurogenic hypertension induced by deafferentation and vagotomy. Dl-propranolol and bupranolol correct the hypertension and the tachycardia of the debuffered dog, whereas oxprenolol has only an action on the heart rate. The other beta-blocking drugs and d-propranolol were inactive at the doses used. Our results show that a mere central effect cannot explain the antihypertensive properties of all the tested beta-blocking agents.


Relationship between the hypotensive and alpha-adrenoceptor blocking actions of prazosin. The relationship between the hypotensive and alpha-adrenoceptor blocking actions of prazosin was investigated in anaesthetized rats. Pressor responses to norepinephrine and angiotensin II were determined before and after intravenous administration of prazosin, 0.0005 to 0.7 mg/kg. The prazosin-induced reduction in mean arterial pressure was recorded immediately before measurement of 87 such pairs of pressor reponses. The percentage antagonism of norepinephrine-induced pressor responsivity, corrected where necessary for non-specific changes in pressor sensitivity, was used as an index of alpha-adrenoceptor blockade. Linear regression analysis of the data revealed that there was a highly significant correlation between the degree of alpha-adrenoceptor blockade afforded by prazosin and the hypotensive response to the drug. The findings extend those of previous studies in which alpha-blocking properties of prazosin were demonstrated using doses of the drug 10(2) to 10(4)-fold greater than those producing significant hypotensive effects. The present results show that prazosin exhibits alpha-adrenoceptor blocking properties at much lower doses, such that a close relationship exists between its alpha-blocking activity and its hypotensive effects.


Cardiovascular profile of 2-(3,4-diethyoxy-beta-methoxyphenethyl) imino-1-methylpyrrolidine fumarate (McN-2840-46), a preferential atrial anti-arrhythmic agent. McN-2840-46, 2.5 mg/kg, i.v., protected against atrial tachyarrhythmias induced by three different methods in dogs and monkeys. The compound was inactive against ventricular arrhythmias produced by ouabain and by chloroform-epinephrine interaction at a four-fold higher dose. Significant reversal of ventricular arrhythmias produced by occlusion of the left anterior descending coronary artery in dogs was achieved by infusion of 8.8 +/- 2.4 mg/kg, i.v. of McN-2840-46. Myocardial electrogram studies confirm that the atrium is preferentially affected. McN-2840-46 does not possess beta 1- or beta 2-adrenergic blocking activity when evaluated on isolated rabbit atrial and guinea-pit tracheal chain preparations. McN-2840-46 is vagolytic but not anticholinergic. The vagolytic activity is attributed to its local anesthetic effect. Depression of myocardial function was observed in anesthetized dogs and in the heart-lung preparation. However, the isolated cat papillary muscle was stimulated by McN-2840-46 and doses considerably above the effective anti-arrhythmic dose did not significantly decrease cardiac output in the non-anesthetized dog. The results of these experiments suggest that McN-2840-46 is a potent "preferential" atrial anti-arrhythmic agent.


beta-Adrenergic blocking action of halonitrophenethanolamines. A series of phenethanolamines with N-isopropyl and N-tertbutyl substituents and ring-substituted with nitro- and halogen groups has been prepared. Using guinea-pig isolated atrial and tracheal preparations, the influence of the nitro-group on the beta 1- and beta 2-antagonist actions of the mono-halogen compounds was determined, and the antagonist and partial agonist effects of the halo-nitro-compounds on beta-adrenoceptors in these tissues measured to help elucidate structure-activity relations in this series. The halonitro compounds did not show enhanced activity compared with the mono-halogen substituted analogues. Several of the new compounds showed slight but significant beta 2-antagonist selectivity of action, and one compound was significantly beta 1-selective.


Modification of behavioral and neurochemical effects of cocaine by haloperidol. Cocaine (20 mg/kg, i.p.) stimulated spontaneous motor activity (SMA) and induced stereotypy (ST) in rats. Haloperidol at 0.015 mg/kg, i.p. dose reduced or blocked cocaine-induced ST, but did not affect, drug-induced hyperactivity. At 0.03 mg/kg, i.p. dose of haloperidol, both behavioral effects were blocked. Cocaine decreased the norepinephrine (NE) and serotonin (5-HT) contents of diencephalon-midbrain (DM) and pons-medulla (PM) and increased dopamine (DA) contents in the DM and caudate nucleus (CN) at 20 min after its administration. Haloperidol (0.03 or 0.015 mg/kg) at 30 min postdrug produced opposite effects on the levels of NE, DA and 5-HT in the respective brain areas compared to cocaine. Given in combination, haloperidol reversed the effects of cocaine on the levels of NE, DA and 5-HT. Thus the cocaine-induced behavioral changes and their modification by haloperidol can be correlated to the neurochemical changes produced by these drugs alone or their combination.


Effect of strophantin on the isolated guinea-pig ileum. The effect of strophantin on isolated guinea-pig ileum was investigated. It was found that strophantin produces a dose-dependent contraction that is not blocked by atropine, ganglionic blockade, but is potentiated by prostigmine. Depolarization of the guinea-pig ileum by a high potassium solution does not change the effect of strophantin. Strophantin response is competitively antagonized by verapamil. The effect is significantly reduced, 5 to 10 min, after the preparation is immersed in zero calcium Tyrode solution. These results support the view that the effect of strophantin is dependent on the influx of calcium through the cell membrane.


A comparison between lick or lever-pressing contingent reward and the effects of neuroleptics thereon. Rats implanted with an electrode in the lateral hypothalamus were trained to obtain brain-stimulation by either pressing a lever or by licking a steel drinking tube in subsequent 15-min sessions. Half of the rats began the sessions with lever-pressing, the other half began with licking. After stabilization of the response rates, rats were run in saline- or drug-sessions. During the drug sessions rats were treated with 4 doses of the following neuroleptics: haloperidol, pimozide, pipamperone, azaperone. These neuroleptics dose-relatedly inhibited licking for brain-stimulation but suppressed lever-pressing only at the highest dose tested. The lick response was thus inhibited at lower doses than lever-pressing. This differential sensitivity to neuroleptics appears not to be due to a difference in baseline response rates, schedule differences or to the motor activity involved in both responses, but rather to the different thresholds of reinforcement produced by licking and lever-pressing for brain-stimulation.


Potentiation of D- and L-amphetamine effects on copulatory behavior in female rats by treatment with alpha-adrenoreceptor blocking drugs. The influence of noradrenergic mechanisms on the effects of D- and L-amphetamine on lordosis behavior and stereotype activity was studied in ovariectomized estrogen + progesterone treated rats. Phenoxybenzamine, phentolamine and prazosin, which all block alpha-adrenergic receptors potentiated the inhibitory effect of D-amphetamine on lordosis behavior. Phenoxybenzamine and prazosin also potentiated the effect of L-amphetamine, while phentolamine did not increase the effect. No augmented effect of D- or L-amphetamine was obtained on stereotype activity after pretreatment with phenoxybenzamine or phentolamine. L-propranolol, which blocks beta-adrenergic receptors and pimozide, a dopamine receptor blocker did not influence the effect of D- or L-amphetamine on lordosis behavior. Clonidine an alpha-receptor stimulant drug inhibited lordosis response in estrogen treated rats. This is probably due to a presynaptic effect, decreasing the release of NA from nerve terminals. The possibility that D- and L-amphetamine activates a NA system which influences lordosis behavior either by a direct facilitation or by decreasing the activity of an inhibitory serotonergic system is discussed.


Effects of chemoreceptor stimulating agents on reflex bradycardia. Effects of carotid body chemoreceptor stimulation on reflex bradycardia were studied by injecting various pharmacological and physiological agents into the external carotid artery of rabbits. Reflex bradycardia was induced by ACh and nicotine in doses from 3 micrograms to 10 micrograms. The response to ACh was markedly potentiated by physostigmine and small doses of atropine, while it was completely blocked bymecamylamine and a large dose of atropine. The nicotine-induced bradycardia was also inhibited by mecamylamine and a large dose of atropine. After reserpinization, the responses to ACh and nicotine were depressed, but were elicited by increasing the doses. NaCN, low pH solution and high CO2 saline induced weaker bradycardiac responses which were not affected by atropine and mecamylamine, but were abolished in the reserpinized animals. The results indicate possible participation of dopamine in the chemoreceptor transmission.


Studies on avian infectious bronchitis virus (IBV). I. Resistance of IBV to chemical and physical treatments. The resistance of avian infectious bronchitis virus (IBV) to several chemical and physical treatments was studied. Ten strains, including four Japanese strains, were used. 1. All strains were sensitive to heating at 56 degrees C for 15 minutes; although two of them, KH and Massachusetts-41, were resistant to heating at 45 degrees C for 90 minutes. 2. All strains were resistant to pH 3.0 and most of the strains were sensitive to pH 11.0. 3. All strains were completely inactivated by chloroform and sodium deoxycholate and all except Beaudette-42 and Connaught were relatively stable to ether. 4. All strains rapidly lost their infectivities upon ultraviolet irradiation. 5. Trypsin did not affect the infectivity of any strain. 6. From these results, the ten strains were classified into three groups based on their stabilities to exposure to heating at 45 degrees C for 90 minutes and to ether.


The effect of ribonuclease on the replicative forms of Sindbis virus RNA. Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.


[Pulmonary lesions in systemic connective tissue diseases with immune disorders (collagen diseases)]. The data from the literature and the authors' own studies on changes in the lungs in systemic diseases of the connective tissue with immune disorders: systemic lupus erythematosus, progressive systemic sclerosis, rheumatoid arthritis, and periarteriitis nodosum are presented. Changes in the lungs in the above diseases have some common features: damage of the microcirculatory bed, increased vascular permeability, impregnation with plasma of alveolar septae and vessel walls, cellular reactions, and septo-alveolar sclerosis. Specific features of each of the diseases under study were demonstrated. The time course of morphological changes in the lungs was followed in relation to the severity, duration, and form of the disease.


Dipivefrin and epinephrine treatment of elevated intraocular pressure: a comparative study. Every 12 hours 0.1% dipivefrin was administered to one eye and 2% epinephrine hydrochloride was administered to the fellow eye of 42 patients with primary open-angle glaucoma or ocular hypertension in a randomized, double-masked study lasting three months. Dipivefrin produced similar percent reductions in intraocular pressure (18.6%) to epinephrine (21.0%), as well as similar increases in outflow facility and pupil diameter. A significantly lower incidence of burning and stinging after drug instillation was noted with dipivefrin therapy. This study supported the contention that dipivefrin is an effective and safe alternative to epinephrine therapy for the reduction of elevated intraocular pressure.


Solubilization of human erythrocyte membrane glycoproteins by triton X-100. 1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.


The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate. 1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572--4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.


NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH-ubiquinone reductase preparation. 1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O(2) (-)) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O(2) (-) by NADH-ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O(2) (-) production by the mitochondrial inner membranes.


Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase. Liver of rat foetuses from 14 to 19 days of gestation and cultured hepatocytes derived from foetuses of 14 or 15 days gestation show a limited capacity to transaminate tyrosine. This low tyrosine transamination activity can be ascribed to aspartate aminotransferase. Definitive tyrosine aminotransferase can be demonstrated in 1-day-old cultures of hepatocytes taken from 19-day foetuses, but not from 15-day foetuses. However, after 3 days of culture hepatocytes from 15-day foetuses are able to synthesize tyrosine aminotransferase. Induction studies reveal that dexamethasone is capable of increasing tyrosine aminotransferase activity once it is detectable in culture.


The porphyrin pigmentation of subspecies of Bacteroides melaninogenicus. Various subspecies of Bacteroides melaninogenicus differ in their pigmentation. Subsp. asaccharolyticus produces protohaem almost exclusively, subsp. intermedicus both protohaem and a smaller proportion of protoporphyrin, and subsp. melaninogenicus mainly protoporphyrin with a trace of protohaem. As a consequence young colonies can be differentiated by their red fluorescence in u.v. light (365nm): subsp. asaccharolyticus does not fluoresce, subsp. intermedicus shows a limited fluorescence, and subsp. melaninogenicus shows a bright fluorescence. The pigments were isolated as the dimethyl esters of protohaemin and of protoporphyrin and identified by electronic spectroscopy, mass spectrometry and comparisons by t.l.c. Incorporation of delta-aminolaevulinate into these pigments was not detected, nor was porphobilinogen formation observed. Subsp. melaninogenicus grown in the presence of [14C]protohaemin formed [14C]protoporphyrin. This appears to represent a novel biological demetallation.


Re-examination of the subcellular localization of thyroxine 5'-deiodination in rat liver. We describe the existence of at least two thyroxine 5'-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5'-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5'-deiodination. However, this process is not mediated by only a single enzyme.


Subcellular localization and ionic properties of acetyl-coenzyme A synthetase in the electric organ of Torpedo marmorata. Acetyl-CoA synthetase activity was shown to be present in pure cholinergic synaptosomes from electric organ of Torpedo marmorata. After osmotic disruption of synaptosomes a substantial part of the activity was recovered in the soluble fraction. The effects of varying pH and increasing K+ concentrations on the synaptosomal enzyme activity were shown to differ from those observed with the mitochondrial enzyme. Whereas this latter enzyme showed optimal activity above pH 8.5, and a maximal activation in the presence of 120 mM-K+, the synaptosomal enzyme exhibited an optimal activity at pH 7.9 and a moderate K+ stimulatory effect with an optimal concentration of 30 mM.


Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A. 1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1--0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 x 10(4) litre-mol-1; for oestrone sulphate, 7.8 x 10(2) litre-mol-1; for haem, 4.5 x 10(5) litre-mol-1; and for bilirubin, approximately 1.2 x 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.


The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. 1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4--5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.


Tyrosine aminotransferase induction in hepatocytes cultured from rat foetuses treated with dexamethasone in utero. 1. The administration of dexamethasone to foetal rats in utero does not result in the appearance of specific tyrosine aminotransferase activity even after 24 h. 2. When foetal hepatocytes are cultured in vitro from animals treated in utero with dexamethasone, significantly higher activities of specific tyrosine aminotransferase are found than in untreated controls. 3. Dexamethasone in vitro induces specific tyrosine aminotransferase in cells cultured from control animals and the effect is maximal at 10 nM in the culture medium. 4. Actinomycin D at 0.2 microgram/ml in the culture medium completely prevents the induction of activity in vitro. 5. In cultures established from animals treated with dexamethasone in utero, the increase in specific tyrosine aminotransferase activity over the control cultures is only marginally decreased in the presence of actinomycin D. 6. The results can be interpreted to mean that dexamethasone in utero stimulates the transcription of enzyme-specific mRNA, which is not rranslated until a translational block in the foetal liver is removed by the conditions of culture in vitro.


The role of insulin in the regulation of stearic acid desaturase activity in liver and adipose tissue from obese--hyperglycaemic (ob/ob) and lean mice. The relationship between the hyperinsulinaemia of obese--hyperglycaemic (ob/ob) mice and their high activity of stearic acid delta 9-desaturase compared with lean mice has been investigated. The concentrations of plasma insulin in obese mice were decreased by 71, 88 and 96% after treatment either with alloxan or food restriction to maintain the same weight as lean mice, or treatment of the weight restricted mice with alloxan followed by feeding ad libitum. The concentration of plasma insulin produced by the latter treatment was the same as in normal lean mice. After treatment the hepatic desaturase activities were 24, 68 and 19% less respectively on a cell basis than in livers from untreated obese mice, and the total epididymal fat-pad activities were lower by 16, 62 and 57%. These results suggest that hyperinsulinaemia is not essential for the increased hepatic desaturase, controlling the hepatic desaturase activity, but even this may be subject to overriding regulation by the concentration of esterified linoleic acid in the liver lipids, which was negatively correlated (r = 0.91, P less than 0.001) with desaturase activity.


The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine. Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.


Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver. Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.


Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta. 1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.


Partial purification and properties of L-asparagine synthetase from mouse pancreas. l-Asparagine synthetase was partially purified from mouse pancreas to a final mean specific activity of 0.10 unit/mg of protein. The enzyme exhibited an l-glutaminase activity which was not affected by l-asparate, NH(4)Cl, ATP-MgCl(2), l-glutamate, AMP (sodium salt) or sodium pyrophosphate. The l-glutamine-dependent l-asparagine synthetase activity of the partially purified enzyme from mouse pancreas was markedly decreased by freezing for 7 days at -87 degrees C in the presence of 1mm-dithiothreitol, but effectively protected from inactivation by high concentrations (10mm) of the thiol reagent. The l-glutaminase activity of the enzyme was inhibited by antagonists of l-glutamine (e.g. 6-diazo-5-oxo-l-norleucine, 5-chloro-4-oxo-l-norvaline, 5-diazo-4-oxo-l-norvaline and NSC-163501) and thiol-reactive compounds (e.g. 2-amino-4-arsenophenol hydrochloride, maleimide, mucochloric acid and ZnCl(2)), but not by aminomalonic acid, the next lower homologue of l-aspartate, nor by l-homoserine beta-adenylate, an analogue of the presumed transitory covalent intermediate. The complete forward reaction catalysed by l-asparagine synthetase from mouse pancreas appears to be irreversible and essentially stoicheiometric under the conditions examined. Mouse pancreas contains a proteolytic inhibitor of l-asparagine synthetase separable from the enzyme by ion-exchange column chromatography. The inhibitor is activated by incubation at 4 degrees C for 110h and inactivated by soya-bean trypsin inhibitor, di-isopropyl phosphorofluoridate and boiling.


Lymphocytotoxic antibodies in systemic lupus erythematosus and clinically related diseases. Sera from patients with systemic lupus erythematosus (SLE) and clinically related diseases were examined for cold-reactive lymphocytotoxic antibodies (LCA). The incidence of LCA was significantly increased in SLE (93%), discoid lupus (50%), and "lupus-like" syndromes associated with congenital complement deficiencies (63%) as compared to normal controls (3%) and patients with drug-induced lupus (11%), mixed connective tissue disease (MCTD) (17%), and necrotizing vasculitis (19%). The diagnostic and pathogenetic implications of these differences are discussed.


Synthesis and pharmacological properties of eterylate, a new derivative of acetylsalicylic acid. A description is given of the synthesis and main physical and chemical properties of a new derivative of acetylsalicylic acid which we have called eterylate and which is 2-(p-acetamidophenyloxy)-ethyl-o-acetoxy-benzoate. The anti-inflammatory and analgesic action of the preparation was studied and found to be comparable to that of acetylsalicylic acid and benorylate. Nevertheless, eterylate is tolerated better than the latter product, as is shown by toxicity tests and studies of the digestive system.


Fluorimetric determination and pharmacokinetic studies of fazadinium bromide in dogs. A rapid fluorimetric method for the estimation in biological fluids of the neuromuscular-relaxant drug 1,1'-azobis-3-methyl-2-phenyl-1H-imidazo-[1,2-a]-pyridinium dibromide (fazadinium bromide, AH 8165) is described. The drug is hydrolized by alkali to give 3-methyl-2-phenyl-1H-(1,2-a)-imidazo-pyridinium bromide, which is extracted by cyclohexane and measured for fluorescence. Intravenous administration (1 mg/kg) in dogs gives initial blood levels of unchanged drug of 4.34 +/- 0.25 microgram/ml. The decay of serum concentrations is rapid. The extent of urinary excretion of unchanged drug is 15% within 6 h.


Pharmacological properties of 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e] [1,4]diazepine (Y-7131), a new anti-anxiety drug. 6-(o-Chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thienol[2,3-e][1,4]diazepine (Y-7131), a new derivative of the thienodiazepines, had marked activities in antipentylenetetrazole effect in mice, attenuation of conflict behavior in rats, inhibition of aggressive behavior induced by hypothalamic stimulation in cats and muscle relaxant effects in normal and decerebrate cats. Y-7131 had weak activities in anti-MES effect in mice and loss of righting reflex in mice. The acute toxicity of Y-7131 was considerably lower than that of diazepam. No significant autonomic or neuroleptic effects were noted. Y-7131 appears to be a characteristic and potent anti-anxiety agent different from the benzodiazepines.


Effects of 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo [3,4-c]thieno[2,3-e][1,4]diazepine (Y-7131) on the metabolism of biogenic amines in brain. A new derivative of thienodiazepine, 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e][1,4]diazepine (Y-7131) decreased the turnover of 5-hydroxytryptamine (5-HT) but not that of dopamine (DA) in the rat brain. The potency of Y-7131 in inhibiting the 5-HT turnover was stronger than that of diazepam. Imipramine decreased the turnover of 5-HT, whereas chlorpromazine increased the turnover of DA. Y-7131 decreased the turnover of norepinephrine (NE) in the mouse brain. With diazepam, such an action was not recognized. Imipramine did not affect the turnover of NE, whereas chlorpromazine increased it. Y-7131 inhibited the uptake of NE in the mouse brain, although it did not inhibit the uptake of 5-HT. Diazepam was devoid of such an action. Imipramine inhibited the uptake of both amines. The increase of the turnover of 5-HT, NE and DA due to foot-shock stress in the rat brain was suppressed by the administration of Y-7131. In this antagonistic effect, Y-7131 was more potent than diazepam. Neither imipramine nor chlorpromazine showed these antagonistic effects. The results obtained suggest that Y-7131 has biochemical profiles similar to diazepam but differs from it in exhibiting inhibitory effect on the NE turnover and its uptake.


Absorption, distribution and excretion of a new thienodiazepine derivative (Y-7131) in rats and mice. The synthesis of radioactive 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo[3,4-c]thieno[2,3-e][1,4]diazepine (Y-7131), a new psychotropic agent, is descirbed. The labelled compound was rapidly and completely absorbed following oral administration to rats and mice. The blood levels of radioactivity reached maximum at 0.5 h in rats, and 1 h in mice, respectively, and then declined rapidly with biological half-lives of about 1.5 h in both animals, although the level was higher in mice than in rats. Approximately 45% of the radioactivity in the serum was bound to the serum protein at 1 h after oral administration. The dosed radioactivity was almost completely excreted within 3 days. In rats, more radioactivity was excreted in feces than in urine, while the reverse was noted in mice. An extensive biliary excretion of radioactivity was evidenced in rats after oral dosing. The highest concentrations of radioactivity were found in the liver, kidney, and adrenals, while relatively low levels in the brain of rats. The distribution patterns of radioactivity in mice were similar to those in rats except for the serum and liver. No remarkable accumulation of radioactivity in rat tissues was observed by repeated oral doses of the labelled compound for periods up to 21 days. The metabolic pathways of Y-7131 were qualitatively similar in rats and mice, and one of them was demonstrated to be the hydroxylation at alpha-position in the ethyl side chain.


[Pharmacokinetic studies on interactions between dipotassium clorazepate and alcohol after oral application (author's transl)]. Pharmacokinetic investigations concerning possible interactions of an orally administered single dosage of 20 mg dipotassium clorazepate and ethanol were conducted with 14 male subjects. Blood alcohol concentrations did not appear to be influenced by dipotassium clorazepate or its metabolites nordiazepam and oxazepam. The increase in oxazepam glucuronide excreted in the urine, however, was statistically significant. Nordiazepam concentration in the serum differed considerably between the group with ethanol administration and the control group. This metabolization pattern is apparently the result of the inhibitory effect of ethanol on hydroxylization processes during biotransformation of the metabolite nordiazepam.


Studies on the effects of MJ-9184-1 in man. A new beta 2-adrenergic receptor stimulating drug, 2-hydroxy-5-[1-hydroxy-2-(2-methyl-1-phenyl-2-propyl-amino)-ethyl]-methanesulfonanilide hydrochloride (MJ-9184-1), was found to be a potent bronchodilator in man. In double-blind cross-over study including placebo, 0.25 mg and 0.50 mg MJ-9184-1 and involving ten patients with stable chronic obstructive lung disease with reversible airways obstruction, MJ-9184-1 caused a fast-appearing and long-lasting bronchodilator effect. The oral intake of 0.50 mg MJ-9184-1, compared to 0.25 mg, caused a more rapid effect and a more pronounced bronchodilation; 0.50 mg MJ-9184-1 also caused some elevation of the pulse rate. The effect was not seen after 0.25 mg MJ-9184-1.


Microbiological activity of cefazedone as compared to cefazolin and cephalothin. In a comparison of the antibacterial activity of (6R,7R)-7-(2-[3,5-dichloro-4-oxo-1(4H)-pyridyl]-acetamido)-3-([5-methyl-1,3,4-thiadiazol-2-yl)-thio]methyl)-8-oxo-5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid (cefazedone, Refosporen) as compared to other cephalosporins it can be seen that cefazedone possesses good activity against gram-positive bacteria; as regards activity against Enterobacteriaceae, in particular strains of E. coli, Klebsiella species and Proteus mirabilis, they fall within the spectrum of activity of the substance.


[Influence of Silybin-dihemisuccinate on fatty acid synthesis in rat liver (author's transl)]. 1. The influence of silybin-dihemisuccinate, a derivative of the flavonolignane silybin from silybum marianum L. Gaertn., on fatty acid biosynthesis of rat liver was studied measuring the radioactivity incorporation of [1-14C]-acetate and 3H2O in fatty acids of the postmitochondrial supernatant of liver homogenates and in fatty acids of liver slices as well as the activities of enzymes involved in do novo synthesis of fatty acids. 2. In the postmitochondrial supernatant of liver homogenates or in liver slices, prepared 30 or 60 min after i.v. injection of 150.6 mg/kg silybin-dihemisuccinate, radioactivity incorporation of 14C-acetate or 3H2O in fatty acids was lowered by about 25%. Adding silybin-dihemisuccinate to incubation mixture in vitro in the concentration of 0.45--0.6 mmol/l silybin the radioactivity incorporation was linearly diminished with increased concentration of silybin. 3. After in vitro addition of varying concentrations of silybin to incubation mixtures in the presence of 0.1 mmol/l silybin activities of acetyl-CoA-carboxylase, fatty-acid-synthetase and ATP-citrate-lyase were diminished by about 50%, while activity of NADP-malate-dehydrogenase was lowered by 20% in the presence of 1 mmol/l silybin. 4. Our results suggest that silybin caused an unspecific and, under in vivo conditions, transitory inhibition of fatty acid synthesis in rat liver.


[Synthesis of several phenoxymethylphenyl derivatives with local-anesthetic activity (author's transl)]. 1. Several 4-phenoxymethyl-procaine derivatives (IV) were synthetised in order to investigate the influence of the intermediate chain on the activity. 2. By replacing the CH2-CH2-group in fomocain by a COO-group we succeeded in doubling the efficacy of this local anesthetic. 3. The type and position of the hetero atoms, N,S and O in the intermediate chain of the procaine (I), xylocaine (II), cinchocaine (III) and phenoxymethyl-procaine (IV) series have in each of these series quite a different effect due to the structural specificity of the remaining part of the molecule. 4. Other examples confirm the significance of the ester group in the intermediate chain for the liposolubility and reactivity. In ketones these properties are altered unfavourably.


[Synthesis and H2-antihistaminic activity of ring methylated metiamide analogues. 4th communication: H2-antihistaminic agents (author's transl)]. In studies on structure-activity relationships of histamine H2-receptor antagonists, the influence of different imidazole ring methylation on the H2-antagonist activity was investigated. 2-Methyl-, 5-methyl-, 2,5-dimethyl- and unsubstituted analogues of metiamide were prepared and tested. While 5-methyl- and unmethylated analogues with a thiourea group proved to be quite active, the 2-methyl- and 2,5-dimethyl-derivatives, as well as those with a urea group, had only low H2-antihistaminic activity.


[On the mechanism of the positive inotropic effect of triamterene on the myocardium (author's transl)]. The influence of triamterene on the contractile force and the cellular electrolyte content and exchange was studied in isolated, electrically stimulated guinea-pig atria. 1. Triamterene (40 to 200 mumol/l) produces a positive inotropic effect which is most pronounced when the extracellular calcium concentration is reduced (1.2 to 0.6 mmol/l) or the contractile force is diminished by pentobarbital (400 mumol/l). The inotropic action of triamterene was shown to be independent of the rate of stimulation (1--2 Hz). 2. By pretreatment with the adrenergic beta-receptor blocking drugs bupranolol and pindolol or by reserpinization the positive inotropic effect of triamterene was not significantly altered. 3. After incubating the atrial preparations with 45calcium for 30 min the intracellularly exchangeable calcium fraction was significantly enhanced by triamterene (100 mumol/l). If triamterene was added to the bathing medium after the 45calcium exchange process had already reached a steady-state, no further increase in the extent of the intracellular calcium exchange was observed during an additional 30-min period of incubation. Since the cellular total calcium content shows remained unchanged it is postulated that the increase in calcium influx is balanced by a simultaneous elevation of calcium efflux. In addition, triamterene did not show any influence on the intracellular potassium, sodium and magnesium content as well as on the extracellular space volume.


A comparative study of the effects of prostaglandins and H2-receptor antagonists on gastric acid secretion, mucosal blood flow and ulcer formation. The prostaglandins E2 and 16,16-dimethyl-PGE2 methyl ester were compared with the H2-receptor antagonists burimamide and metiamide for their effects on gastric acid secretion (GAS) and gastric mucosal blood flow (MBF) in rats and dogs, and on ulcer formation in rats. Orally, both 16,16-dimethyl-PGE2 methyl ester (20 microgram/kg) and metiamide (6 mg/kg) were markedly effective inhibitors of GAS stimulated by histamine acid phosphate or pentagastrin in Heidenhain pouch dogs, producing a reduction both in volume of gastric juice and in the concentration of titratable acid. In anaesthetised rats, however, the H2-receptor antagonists, when perfused into the gastric lumen, did not consistently inhibit the increased GAS caused by various secretagogues. In contrast, the prostaglandins, under the same conditions, caused marked inhibition of GAS provoked by all secretagogues. Intravenously, both burimamide and metiamide were effective in inhibiting GAS in rats but were less potent than the prostaglandins. The order of potency was: 16,16-dimethyl-PGE2 methyl ester greater than PGE2 greater than metiamide greater than burimamide. By the oral route, the H2-receptor antagonists were found to be very weak inhibitors of indometacin-induced gastric ulcer in rats, as compared to the prostaglandins. MBF studies in rats and in Heidenhain dogs showed that i.v. or p.o. administration inhibited both GAS and MBF in most cases. The ratio r = [MBF (ml/min)/GAS (mumol H+/min)] was generally increased by both types of compounds, suggesting a preferential effect on GAS.


Studies of cholesterol esterase in rat arterial wall. Cholesterol esterase activity was estimated in homogenates of rat arterial wall using radioactive cholesteryl oleate incorporated into phospholipid vesicles as a substrate. The labeled oleic acid was separated from the ester by addition of benzene-chloroform-methanol mixture. Under these conditions, two pH optima were found at about 4.5 and 7.5. Most of the activities at pH 4.5 and 7.5 were found in the lysosomal and microsomal fraction, respectively. No enzyme activity was detected when the substrate vesicles were prepared with phosphatidylethanolamine or sphingomyelin, but the activity was higher when the substrate vesicles were prepared with phosphatidylserine and highest when they were prepared with phosphatidylcholine. The relationship between enzyme regulation and lipid deposition in the arterial wall is discussed.


Attachment of Neisseria gonorrhoeae to human sperm. Microscopical study of trypsin and iron. Pilated Neisseria gonorrhoeae of colony type 1 (T1) and non-pilated bacteria of colony type 4 (T4) were observed by transmission (TEM) and scanning electron microscopy (SEM). No pili were observed on T4 gonogocci, but two types of pili--straight, type a, and bent, type b--were seen on T1 by TEM. When incubated with human sperum and examined by either TEM or SEM, T1 gonococci were seen to attach by individual pili, by several pili wound together as a rope, or by direct contact. Gonococci from T4 colonies attached only by direct contact. Treatment with typsin (1 mg/ml) damaged or removed pili from gonococci. After incubation with trypsin, attachment of pilated gonococci to sperm was decreased significantly, but such treatment did not affect attachment of non-pilated gonococci. Incubation of gonococci from either colony type in 0.1 mmol/l ferric nitrate, followed by incubation with sperm, significantly increased attachment of only T4 bacteria. No pili were seen on T4 gonococci treated with ferric nitrate; thus, it appears that factors other than pili alone are concerned in attachment of these gonococci to sperm.


Haemodynamic effects of a new beta 1-receptor agonist in acute myocardial infarction. A useful antidote to unwanted cardiac effects of beta-blocking agents. The haemodynamic effects of a new beta 1-receptor agonist, 1-(4 hydroxyphenoxy) 3-isopropylamino-2-propanol, were studied in 25 patients after acute myocardial infarction using non-invasive methods. The drug caused an increase in systolic blood pressure and pulse pressure, without change in diastolic blood pressure, and a slight increase in heart rate and reduction in the pre-ejection period. These changes were greater in patients without a history of left heart failure. It is suggested that this cardioselective drug possesses positive inotropic activity but only slight positive chronotropic activity. The substance has been further investigated as a possible antidote to unwanted cardiac side effects of the cardioselective beta-blocker, metoprolol. The changes in the cardiovascular dynamics caused by metoprolol in patients with acute myocardial infarction were promptly reversed by this new beta 1-agonist. With its positive inotropic properties and its efficacy in reversing the effects of a cardioselective beta-blocker, the drug is a potentially useful pharmacological agent to support an acutely depressed myocardium in patients on beta-blocking agents.


The effect of beta-adrenoceptor blockade on factors affecting exercise tolerance in normal man. 1 We have studied the effects of single oral doses of 80 mg propranolol and 100 mg metoprolol on the cardiovascular and respiratory responses to progressive exercise in nine healthy men in double-blind, placebo-controlled experiment. As judged by their effects on exercise heart rate and cardiac output the doses of the two drugs used were equivalent. 2 Beta-adrenoceptor blockade reduced oxygen consumption by 3.5% over the whole work range with an increase in the respiratory exchange ratio of 0.056 units. Carbon dioxide production and exercise ventilation were unchanged. The two drugs had similar effects. Possible mechanisms for these observations are discussed. 3 Perceived exertion during exercise was increased by both the beta-adrenoceptor blocking drugs and this may be of relevance to the symptom of fatigue reported by patients on these drugs. Endurance, assessed as either total work done or maximal work achieved, was reduced by 15%.


Dermatitis as a side-effect of long-term topical treatment with certain beta-blocking agents. Three groups of glaucoma patients, treated topically with various beta-blocking agents, were studied for mucocutaneous side-effects of long-term therapy. In five of eleven patients with ocular and/or periocular dermatitis as an adverse reaction to long-term treatment with metoprolol eye drops a dermatitis, reproducible by patch tests with pure metoprolol 3%, was demonstrable. Histopathological examination of positive patch tests examined in three cases showed a picture compatible with a delayed type of hypersensitivity. Four atenolol treated patients showed adverse reactions, but negative patch tests to atenolol were found. In addition new data are reported in favour of cross-reactivity between certain beta-blocking agents.


Kinetics of carboxymethylation of histidine hydantoin. The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.


Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin. Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.


Functional interactions of lipids and proteins in rat intestinal microvillus membranes. Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.


Formation of unilamellar lipid vesicles of controllable dimensions by detergent dialysis. Vesicles are formed by solubilizing mixtures of phosphatidylcholine and cholesterol with sodium cholate and removing the detergent by rapid (hollow fiber) dialysis [e.g., Goldin, S. M. (1977) J. Biol. Chem. 252, 5630--5642]. Characterization of the vesicle size distribution by agarose gel filtration, and determination of the intravesicular aqueous compartment, demonstrates that the vesicles are relatively homogeneous in size and are primarily unilamellar. The mean diameter of the vesicles can be varied from 340 to 1280 A by varying the conditions under which they are formed; increasing the mole fraction of cholesterol and lowering the pH of the dialysate tend to produce larger vesicles. The gentle detergent treatment required for vesicle formation and the ability to control vesicle size distribution reproducibly may make this method particularly useful in studies of reconstitution of membrane proteins and in use of vesicles as vehicles for delivery of materials to living cells.


Protein mobility and self-association by deuterium nuclear magnetic resonance. Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.


A study of the surface-active properties of the Mg2+-activated ATPase from cytoplasmic membranes of Streptococcus faecalis. The surface activity and enzymic properties of the factor F1, the catalytic moiety of Streptococcus faecalis H+-ATPase, has been studied at the air-water and phospholipid-water interfaces. F1 does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F1 molecule at the air-water interface have been estimated. In the presence of Mg2+, base area is S = 1.8 . 10(4) A2, height h = 27 A. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F1 subunits should all lie in the same plane. The ATPase activity of F1 at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution. Adsorption of F1 at the interface shifts the isoelectric point of tiscussed relative to the proton-active transport mechanism.


Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo. The equilibrium oxidation-reduction mipoint potential (Em) of isolated Rhodopseudomonas sphaeroides cytochrome c2 exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197-208). However, as with mammalian cytochrome c (Brandt, K.G. Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180-4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the Em of cytochrome c2 before and after the conformational change. The Em of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium Em is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c2 to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent Em from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c2 in vivo involve protons.


On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria. Different approaches have been used to evaluate the stoichiometry of proton translocation linked to cytochrome c oxidase in rat liver mitochondria. A mathematical model was designed that successfully describes the kinetics of redox-linked proton translocation provided that the rate of electron transfer is not too high. With ascorbate as reductant, an essentially pH-independent (in the pH range 6--8.5) proton ejection stoichiometry (H+/e-) is obtained from either initial rates of H+ ejection (0.86 +/- 0.12), or the model (0.87 +/- 0.14). Similar results are obtained with either ferrocyanide, N.N.N',N'-tetramethyl-p-phenylenediamine or externally added cytochrome c mediating between ascorbate and cytochrome c in rotenone- and antimycin-inhibited mitochondria. Oxygen pulse experiments with ferrocytochrome c as substrate show fully uncoupler-sensitive redox-linked proton ejection with a stoichiometry of 0.78 +/- 0.14. With murexide to measure Ca2+ uptake during oxidation of ferrocyanide, we found a stoichiometry of two positive charges taken up/electron transferred, confirming earlier findings. These results provide strong evidence that cytochrome c oxidase functions as a redox-linked proton pump with a stoichiometry of one H+ ejected and two charges translocated/electron transferred. The thermodynamic consequences of the proton pump are discussed and a maximal P/O ratio of 1 1/3 for 'site 3' is predicted in agreement with state 4 redox potentials and phosphate potential.


Accumulation of pyruvate by isolated rat liver mitochondria. 1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.


Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values. The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.


The effect of intracellular pH on the rate of hexose uptake in Chlorella. The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.


Induction of a chloride conductance in gastric vesicles by limited trypsin or chymotrypsin digestion or ageing. Transport activity of the hog gastric (H+ + K+)-ATPase system was measured either as the formation of proton gradient using the dye probe acridine orange or as the formation of a proton diffusion potential using the cyanine dye 3,3'-diethyloxdicarbocyanine iodide in the presence of the protonophore tetrachlorosalicylanilide. The development of these gradients has been compared in K+ media in the presence of either Cl- or SO4-2 as the anionic species. This comparison of proton diffusion potential formation to proton gradient formation has been used to demonstrate that a Cl- conductance in this vesicular system results from limited enzymic digestion with either trypsin or alpha-chymotrypsin from the ageing process itself. The possible significance of this finding is discussed.


Purification and comparison of several catalytic parameters of the gamma-glutamyltranspeptidase of rat mammary adenocarcinoma (13762) and of normal rat mammary gland. A method for the purification of a membrane-bound glycoprotein, gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2), from a transplantable rat mammary tumor (13762 MT) is described. The properties of the tumor enzyme were compared with those of gamma-glutamyltranspeptidase similarly isolated from mammary tissue of nonpregnant multiparous rats. Evidence has been presented elsewhere that the mammary and tumor enzymes exist as groups of species differing in isoelectric point and that the tumor enzyme contains more of the those species with lower isoelectric points. In this study the normal and tumor enzyme preparations are found to be identical or very similar in regards to the effect of papain on molecular size, the ratios of the enzymatic activities as measured with various amino acids, the Km for gamma-glutamyl-p-nitroanilide, and the Ki for inhibition by glutathione. Neuraminidase treatment had no effect on these catalytic properties. The properties observed were generally similar to those previously reported for highly purified rat kidney preparations.


An essential carboxylic acid group in human prostate acid phosphatase. Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and L-(+)-tartrate, but not in the presence of non-inhibitory D-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of D- and of L-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.


The solubilisation of the membrane-bound D-alanyl-D-alanine carboxypeptidase of Bacillus coagulans NCIB 9365. Protoplast membranes and the particulate D,D-carboxypeptidase of Bacillus coagulans NCIB 9365 were extremely resistant to disruption by either detergents or urea. A combination of urea and the non-ionic detergent Genapol X-100 was required to achieve a significant solubilisation of membrane protein and D,D-carboxypeptidase in an active form; the pH optimum for this treatment was pH 7.5. Solubilisation of the enzyme was accompanied by a two-fold enhancement of activity. Kinetic results indicated that the enhancement may be due to an alteration in the conformation of the enzyme following disruption of membrane structure.


The purification of glutamine synthetase from Azotobacter and other procaryotes by blue sepharose chromatography. We report the facile purification of glutamine synthetase (L-glutamate: ammonia ligase (adenosine 5'-diphosphate-forming), EC 6.3.1.2) in both the adenylylated and unadenylylated form, from Azotobacter vinelandii ATCC 12837. A general affinity column, which used as an affinity ligand Reactive blue 2 dye (Cibacron blue) covalently linked to Agarose, was employed as an efficient first step of purification. Further purification to electrophoretic homogeneity employed DEAE-cellulose chromatography and an additional Affigel chromatographic step. The method was used successfully to prepare glutamine synthetase from Escherichia coli, Rhodopseudomonas sphaeroides and Anabaena sp. strain CA.


Antigen-antibody interactions and the anomalous kinetics of arylsulfatase A. Antibodies against homogeneous rabbit liver arylsulfatase A (aryl-sulfatase sulfohydrolase, EC 3.1.6.1) were produced in a goat and the effects of these antibodies on the kinetic parameters of the enzyme have been studied. The results indicate that the binding of antibody to the enzyme does not alter the enzyme active site, since Km and -ki values are unaffected. However, a small reduction in the enzyme activity was observed as the result of a reduction of V in the enzyme-antibody complex. The binding of antibodies led to a change in the pH-rate profile, giving one broad pH optimum shifted toward higher pH value. The enzyme-antibody complex still showed the characteristic arylsulfatase A anomalous kinetics at pH 5.5, but the inactivation was significantly slower than for the native enzyme. As calculated from quantitative immuno-precipitation data, the native enzyme bound 5--7 molecules of IgG. The number of IgG molecules which bound to the turnover-modified enzyme was reduced to 3--4. The loss of antigenic determinants from the turnover-modified enzyme indicates that significant conformational changes occur during the turnover-induced modification, or that a covalent modification of residues present at the antigenic sites has occurred, or both.


Collagenase activity in cultures of rat prostate carcinoma. A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.


AMP deaminase from baker's yeast. Purification and some regulatory properties. AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified  to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.


Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production. Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.


Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme. Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.


Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase. The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.