Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.     

Lethality of tyrosine in mice: its potentiation by decarboxylase inhibitors and reversal byascorbic acid

In Berlin, capital of the German Democratic Republic, 7,386 new cases of gastric cancer (ICD 151) were registered in the period from 1955 to 1969 and in year 1973. 52 percent of patients were male. Percentage of cases 75 years old and more, increased from 21% (1955-1959) to 36% (1965-1969). There were only slight changes in incidence. About 30% of cases were in the operable stages I and II. With rising age, the proportion of the advanced stages III and IV increases. No real progress as made in early diagnosis of stomach cancer as measured by stage distribution during observation period. Patient's delay from first symptom to first visit was shorter than one month in 40%, physician's delay from first visit to correct diagnosis was shorter than one month in 54%. 15% of patients were treated within one month after first symptom and 47% within three months. There was no correlation between duration of history and percentage of early stages. When observation periods 1955 to 1959, 1960-1964, 1965-1969 and 1973 are compared, we find an increase in old patients (75+ years), a decrease of localized stages I + II, and a decrease in resection rate. Therefore, a decrease in survival rates is to be expected. Crude 5-year survival rate was 6.4% (1955-1959) and 6.2% (1960-1964). When we compare data from Berlin with observations in the Region of Erfurt and in Birmingham Region, the situation of detection and treatment of stomach cancer in Berlin seems to be somewhat better. Finally, some suggestions for the improvement of control of stomach cancer are made.     

Characterization of the genes encoding D-amino acid transaminase and glutamate racemase, two D-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208

In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.     

Effects of the E177K mutation in D-amino acid transaminase. Studies on an essential coenzyme anchoring group that contributes to stereochemical fidelity

D-Amino acid transaminase is a bacterial enzyme that uses pyridoxal phosphate (PLP) as a cofactor to catalyze the conversion of D-amino acids into their corresponding alpha-keto acids. This enzyme has already been established as a target for novel antibacterial agents through suicide inactivation by a number of compounds. To improve their potency and specificity, the detailed enzyme mechanism, especially the role of its PLP cofactor, is under investigation. Many PLP-dependent transaminases have a negatively charged amino acid residue forming a salt-bridge with the pyridine nitrogen of its cofactor that promotes its protonation to stabilize the formation of a ketimine intermediate, which is subsequently hydrolyzed in the normal transaminase reaction pathway. However, alanine racemase has a positively charged arginine held rigidly in place by an extensive hydrogen bond network that may destabilize the ketimine intermediate, and make it too short-lived for a transaminase type of hydrolysis to occur. To test this hypothesis, we changed Glu-177 into a titratable, positively charged lysine (E177K). The crystal structure of this mutant shows that the positive charge of the newly introduced lysine side chain points away from the nitrogen of the cofactor, which may be due to electrostatic repulsions not being overcome by a hydrogen bond network such as found in alanine racemase. This mutation makes the active site more accessible, as exemplified by both biochemical and crystallographic data: CD measurements indicated a change in the microenvironment of the protein, some SH groups become more easily titratable, and at pH 9.0 the PMP peak appeared around 315 nm rather than at 330 nm. The ability of this mutant to convert L-alanine into D-alanine increased about 10-fold compared to wild-type and to about the same extent as found with other active site mutants. On the other hand, the specific activity of the E177K mutant decreased more than 1000-fold compared to wild-type. Furthermore, titration with L-alanine resulted in the appearance of an enzyme-substrate quinonoid intermediate absorbing around 500 nm, which is not observed with usual substrates or with the wild-type enzyme in the presence of L-alanine. The results overall indicate the importance of charged amino acid side chains relative to the coenzyme to maintain high catalytic efficiency.     

Punishment of saccharin drinking by amphetamine in rats and its reversal by chlordiazepoxide.

Demonstrates, in a conditioned taste-aversion paradigm, that doses of dextroamphetamine which are intravenously self-administered by rats may punish saccharin drinking. Ss were male Wistar rats. In Exp I (n = 42), single-trial, dose-related aversion was shown. Aversion was not antagonized by chlorpromazine. Exp II (n = 32) demonstrated dose-related acquisition of taste aversion with repeated administration of very low amphetamine doses. In Exp III (n = 18), drug-induced saccharin aversion was reversed by chlordiazepoxide; this action paralleled the action of chlordiazepoxide in numerous other aversive-conditioning situations. Exp IV (n = 30) ruled out the possibility that depression of saccharin drinking was due to a direct pharmacological action of the drug and not to learned saccharin avoidance. Results indicate that the reinforcing action of amphetamine depends on the response with which its effects are correlated. (20 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.     

From good substrates to good inhibitors: design of inhibitors for serine and thiol proteases

Serine and thiol proteases react with peptide substrates to form an acyl-enzyme. We have synthesized inhibitors which are pseudo-substrates and react with the proteases to generate acyl-enzymes which hydrolize slowly. This is achieved by incorporating an electron-donating group near the carbonyl group of inhibitors I [Ac-Phe--C(O)NH--NH--C(O)X] and II [benzyl-O-C(O)-psiAla-Leu-ArgOMe]. The acyl-enzymes derived from the reaction of I with papain and II with chymotrypsin hydrolyze with t1/2 of 12 and 1 h, respectively. The increased electron density on the carbonyl group of the inhibitor also reduces the rate of acyl-enzyme formation. Components were incorporated into the inhibitor which interact with the leaving group binding site (S' subsite) and which accelerate the rate of reaction of inhibitor with enzyme. For inhibitor I, X = NH(CH3), k(on) < 0.13 M(-1) s(-1) for the reaction papain, but if X = psiLeu(CH3)2,k(on) =10(5) M(-1) s(-1). Similar results were obtained with II and chymotrypsin. Concomitant with acyl-enzyme formation, X is released and a slowly hydrolyzing acyl-enzyme remains.     

Taxol-induced flexibility of microtubules and its reversal by MAP-2 and Tau

When microtubules, ordinarily quite rigid structures, are treated in vitro with the anti-tumor drug taxol, they rapidly develop a wavy appearance and become strikingly flexible. A quantitative measure of their flexibility, the reciprocal statistical length, lambda, increases by an order of magnitude when taxol is bound. Subsequent addition of either of the microtubule-associated proteins MAP-2 or tau causes the flexibility to disappear. It can be restored again by removing the microtubule-associated protein. These results show that taxol changes microtubular structure substantially, probably by weakening the interactions between protofilaments, and that microtubule-associated proteins reverse these effects, possibly by bridging protofilaments. This structural change and the accompanying flexibility may contribute importantly to taxol's cytotoxic activity.     

Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.     

Inhibition of platelet aggregation by acetylsalicylic acid and other inhibitors

Effects on platelet aggregation were examined of acetylsalicylic acid (ASA), indomethacin and a number of other agents including dipyridamole, phenylbutazone and sulfinpyrazone under standardized conditions. The Born turbidometric method of measuring platelet aggregation was used with collagen as the stimulus for aggregation. ASA and indomethacin were shown to be among the most potent inhibitors of aggregation, being active at minimal effective concentrations of 1-3 mug/ml using a 10 min time of pre-incubation with the platelet-rich plasma (degree of aggregation inhibition was time dependent). Most of the other agents tested were also active in vitro and both prostaglandin E1 and adenosine were more potent than ASA or indomethacin. However, these agents were shown not to exert significant inhibitory effects when administered orally to rats (dose 10 and 30 mg/kg). ASA proved to be effective in doses as low as 3 mg/kg, and indomethacin in doses as low as 1 mg/kg orally. The inhibitory effects of ASA on aggregation remained for several days after a single oral dose, whereas the effects of indomethacin disappeared within 24 h.     

Unfolding and inactivation of Penaeus penicillatus acid phosphatase during denaturation by guanidine hydrochloride

Neuromuscular terminals of a single motoneuron to four muscles (CPV7a, GM5a, CV2, and CV3) in the stomach of the blue crab Callinectes sapidus showed structural evidence for the exocytotic release of dense-core vesicles exclusively at synapses. The primary evidence was the appearance of dense cores in the synaptic cleft, accompanied by indentations of the presynaptic or postsynaptic membrane. In their simplest form, these consisted of an omega-shaped figure of the presynaptic membrane enclosing one dense core, denoting release of a single dense-core vesicle. A larger indentation of the presynaptic membrane enclosing several dense cores denoted multiple release. A more complex form of multiple release was where the presynaptic membrane was normal, but the postsynaptic membrane elaborated into a sac projecting into the granular sarcoplasm and filled with dense cores. The postsynaptic sac in some instances was compressed into a thin, fingerlike extension, which lacked dense cores and, at its distal end, separated into small cisternae, suggesting a mechanism for membrane recycling. Profiles depicting single and multiple releases of dense-core vesicles were found more frequently at neuromuscular terminals that release relatively large amounts of transmitter with a single stimulus, such as CV2 and CV3, compared to those releasing smaller amounts, such as CPV7a and GM5a. The disparity in release sites among the four muscles of this single motor unit and the fact that many of the multiple-release figures were closely adjacent to the active zones for transmitter release suggest a possible modulatory role for dense-core vesicles in synaptic transmission. Such modulation may be long lasting, as implied by the postsynaptic sacs, which may permit prolonged release of the contents of their dense cores into the synaptic cleft. This is in keeping with the functional role of these stomach muscles, which is to be continuously active for long periods of time.     

Conversion of cortisol to cortisone by the human uterus and its reversal in pregnancy

Inter-conversion of cortisol (F) and cortisone (E) was investigated by incubating minced tissue with tritiated cortisol or cortisone and then separating the products by Sephadex LH-20 column chromatography. In non-pregnant subjects conversion of F to E predominated (43.4+/-3.4% vs 0.1+/-0.4% for E to F). In early pregnancy F leads to E decreased and E leads to F rose while at term E leads to F (46.3+/-9.1%) exceeded F leads to E (15.1+/-6.8%). These results were in accord with those obtained by assaying the endogenous concentrations. In non-pregnant subjects the F/E ratio (1.1+/-0.6) was lower than that found in serum (6.3+/-2.2) while at term the uterine F/E (9.0+/-1.8) was similar to that of serum (8.8+/-2.0). These changes resulted in an 8-fold increase in uterine F compared with a 3-fold increase in serum F, while uterine E fell to 1/2 and serum E doubled. Thus, during pregnancy there is a dramatic reversal of the reaction in the uterus in favour of the active hormone. It seems possible that the increase in cortisol thus brought about may play an anti-immune role in uterine wall, the single tissue apart from blood in direct contact with fetal tissue.     

Screening of potential transport systems for methyl mercury uptake in rat erythrocytes at 5 degrees by use of inhibitors and substrates

This report is devoted to the characterization of the apolipoprotein E (ApoE) in Microcebus murinus. Only one allele homologous to the human ApoE4 allele was evidenced. The distribution of the corresponding ApoE protein in the brain was found in association with the pathological proteins characteristic of Alzheimer's disease (AD). Immunocytochemistry revealed brain deposits of ApoE in: (1) the cortical amyloid plaques; (2) the neurones of the various cortical lobes, the hippocampus and the brainstem; (3) the glial cells, astrocytes of the cortical parenchym and oligodendrocytes of the corpus callosum; and (4) the vessel walls. Most ApoE, beta-amyloid protein, abnormally phosphorylated Tau proteins and gliofilament acid proteins were seen in the same cortical areas. These findings for ApoE report the view that Microcebus murinus, in captivity, presents a pathological profile very similar to that observed in AD.     

Medical therapy of chronic heart failure. Role of ACE inhibitors and beta-blockers

Heart failure has long been considered to have a progressive downhill course leading inexorably to an early demise. This course often occurs silently, in the absence of any obvious cardiac insults. The reason for this is a combination of cell loss, myocyte dysfunction, impaired energetics, and pathologic remodeling of the chamber. Improved clinical outcome should result from strategies that reduce the biologic signals responsible for myocyte growth, dysfunction, and loss and chamber remodeling. Clinicians should no longer attempt to treat chronic heart failure with pharmacologic growth and remodeling process. In time, it may be possible for the clinician to view the treatment of heart failure largely as a matter of improving the biologic function of the myocardium.     

Role of butyric acid and its derivatives in the treatment of colorectal cancer and hemoglobinopathies

Butyric acid, a short chain fatty acid (SCFA), is a natural component of the animal metabolism. Physiological concentrations induce multiple and reversible biological effects. They concern regulatory mechanisms of gene expression conducing to promote markers of cell differentiation, apoptosis and cell growth control. The described hyperacetylation of histones and the induction of several immune or non-immune cell-activating mediators are consistent with the pleiotropic stimulatory effect of the agent. Butyric acid is considered as a biological response modifier (BRM) and is an interesting tool for biological studies. The history of butyric acid as a putative medication in human health is spanning since 60 years and is confusing in part because of conflicting data between exciting experimental results and clinical trials. In light of minimal impact of systemic therapy and the short half-life of the saline molecule used, it is evident that continuous infusions of butyrate are required to improve the efficacy of the treatment. Butyric acid has been viewed with skepticism because of less convenient for long-term chronic therapy. New experimental data from several studies conduced within the past decade with butyric derivatives, delivery systems, and long-acting prodrugs, have demonstrated the practical value of the therapeutic concept. To support issues regarding clinical development, it was of interest to evaluate the recent information, showing butyric acid currently considered as therapeutic purposes in the treatment of colorectal cancer and hemoglobinopathies.     

Characterization of phospholipase A inhibition of stereospecific opiate binding and its reversal by bovine serum albumin

Opiate receptor binding is inhibited by phospholipase A from some sources, whereas the enzyme from other sources is inactive even at much higher concentrations. Evidence is presented that an active enzymatic site is required for inhibition and that the degree of inhibition seems to correlate with the extent of phospholipolysis. The inhibition is reversed almost completely by treatment with 0.5 to 1% bovine serum albumin, even up to 90% inhibition by phospholipase. As more enzyme is added or incubation time is increased, the extent of reversal diminishes. Based on our evidence, the most likely explanation of these results is that the inhibition of opiate binding activity by phospholipase A is due to the toxicity of the products of phospholipolysis and that bovine serum albumin reverses the inhibition by removing these products from the membranes, thereby restoring the active conformation of the receptors.