Studies on bicuculline binding sites on neuronal membrane using fluorescent antibody technique: Comparative binding of gaba and bicuculline

Highly purified fluorescent labelled anti-bicuculline antibodies were used to mark bicuculline binding sites in cerebral cortex of monkey brain. Specific binding of bicuculline could be demonstrated in the synaptosomal fraction, when bicuculline was added both Image and Image . Addition of γ-aminobutyric acid (GABA) to the bicucullinised membrane led to a decrease in fluorescence indicating same receptor loci and establishing GABA-bicuculline antagonism at a molecular level.

Effect of some "strong" excitants of central neurones on the uptake of L-glutamate and L-aspartate by synaptosomes

"Strong" excitants of central neurones such as β N-oxalyl L α,β-diaminopropionic acid (ODAP), N-methyl-D-aspartic acid (NMDA) and kainic acid (KA) were found to inhibit the high affinity uptake of glutamate and aspartate in synaptosomes isolated from young rat brain. The potency of these "strong" excitants as convulsants appear to parallel their ability to inhibit glutamate uptake by synaptosomes. The data suggest the possibility that the convulsive effect of these "strong" excitants could be mediated by glutamate/aspartate.

Effect of beta-n-oxalyl-L-alpha, beta-diaminopropionic acid on glutamate uptake by synaptosomes

THE unusual amino acid beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), isolated from the seeds of Lathyrus sativus is a potent neurotoxin1−3. It produces biochemical changes in the brain typical of an excitant amino acid and is implicated in the aetiology of human neurolathyrism caused by eating the seeds of L. sativus 4−6. It may act as a glutamate antagonist: ODAP inhibits glutamate oxidation7 possibly by inhibiting glutamate uptake in bovine brain mitochondria; it also acts as a competitive inhibitor of glutamate uptake in certain strains of yeast8, and a similar process might occur at the synaptic level. Any effect of ODAP on glutamate uptake at synapses is significant in view of the neurotransmitter function of glutamate, which seems to be neuroexcitory as well as neurotoxic9−12. But Balcar and Johnston13 have shown with rat brain slices that ODAP does not inhibit the glutamate uptake by the high affinity system.

Regulation of arginine decarboxylase and putrescine levels in Cucumis sativus cotyledons

Arginine decarboxylase (arginine carboxy-lyase EC 4.1.1.19) of Cucumis sativus cotyledons, has a pH optimum of 8.3 and a temperature optimum of 40°. Among the various plant hormones administered to excised cotyledons in culture, benzyladenine and its riboside were most effective in increasing the arginine decarboxylase activity and putrescine content. The enzyme activity and putrescine content were significantly increased on acid feeding of the cotyledons and decreased by KCl treatment. The KCl effect could be only partially reversed by benzyladenine. Abscisic acid inhibited cotyledon growth and also reduced arginine decarboxylase and putrescine levels. This effect was overcome by cytokinins. The half life of the enzyme using cycloheximide was 3.7 hr. Dibutyryl cyclic AMP and 5′-AMP also marginally stimulated the enzyme and putrescine levels. Mixing experiments indicate that there is neither a non-dialysable activator nor inhibitor of the enzyme.

Putrescine-Sensitive (Artifactual) and Insensitive (Biosynthetic) S-Adenosyl-L-Methionine Decarboxylase Activities of Lathyrus sativus Seedlings

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+ -dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.

X-ray studies of crystalline complexes involving amino acids. II. The crystal structure of L-arginine L-glutamate

Abstract is not available.

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms

A CD8(+) T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: Implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of pepti-domimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of ail anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8(+) CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization. (C) 2007 Elsevier Inc. All rights reserved.

Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

Background: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. Methodology and Results: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of similar to 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size similar to 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. Conclusion: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

Analysis of connectivity map: Control to glutamate injured and phenobarbital treated neuronal network

We study the responses of a cultured neural network when it is exposed to epileptogenesis glutamate injury causing epilepsy and subsequent treatment with phenobarbital by constructing connectivity map of neurons using correlation matrix. This study is particularly useful in understanding the pharmaceutical drug induced changes in the neuronal network properties with insights into changes at the systems biology level. (C) 2010 American Institute of Physics. [doi:10.1063/1.3398025]

Enzyme catalysed non-oxidative decarboxylation of aromatic acids II. Identification of active site residues of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the mechanism of decarboxylation by 2,3-dihydroxybenzoic acid decarboxylase, chemical modification studies were carried out. Specific modification of the amino acid residues with diethylpyrocarbonate, N-bromosuccinimide and N-ethylmaleiimide revealed that at least one residue each of histidine, tryptophan and cysteine were essential for the activity. Various substrate analogs which were potential inhibitors significantly protected the enzyme against inactivation. The modification of residues at low concentration of the reagents and the protection experiments suggested that these amino acid residues might be present at the active site. Studies also suggested that the carboxyl and ortho-hydroxyl groups of the substrate are essential for interaction with the enzyme.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. (C) 2010 Elsevier B.V. All rights reserved.

Passive immunization with an antibody to the beta-subunit of ovine luteinizing hormone as a method of early abortion--a feasibility study in monkeys (Macaca radiata)

Antiserum to the beta-subunit of ovine luteinizing hormone (oLH-beta) raised in monkeys (Macaca radiata) has been tested by a variety of criteria both in vivo and in vitro to establish its ability to neutralize oLH, hLH, and human chorionic gonadotropin (hCG). Passive administration of this antiserum caused inhibition of ovulation and termination of pregnancy in recipient monkeys as indicated by premature vaginal bleeding and a significant reduction in serum progesterone and estrogen levels. The results suggest that antiserum raised in monkeys against oLH-beta can neutralize monkey LH as well as monkey CG.

Chemical approaches to penicillin allergy—V. Nature of reaction between rabbit antigen (CRP-penicillin conjugate) and rabbit antipenicillin antibody

The availability of an electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) and rabbit antipenicillin antibody afforded an idealin vitro system to calculate the thermodynamic parameters of the binding of14C benzyl penicillin CRP conjugate (antigen) to the purified rabbit antipenicillin antibody. The thermodynamic parameters of this antigen-antibody reaction has been studied by radio-active assay method by using millipore filter. Equilibrium constant (K) of this reaction has been found to be 2·853×109M−2 and corresponding free energy (ΔG) at 4°C and 37°C has been calculated to be −12·02 and −13·5 kcal/mole, enthalpy (ΔH) and entropy (ΔS) has been found to be 361 kcal/mole and +30 eu/mole respectively. Competitive binding studies of CRP-analogue conjugates with the divalent rabbit antibody has been carried out in the presence of14C-penicilloyl CRP. It was found that 7-deoxy penicillin-CRP complex and 6-amino penicilloyl CRP conjugate binds to the antibody with energies stronger than that with the14C-penicilloyl CRP. All the other analogue conjugates are much weaker in interfering with the binding of the penicilloyl CRP with the antibody. The conjugate of methicillin,o-nitro benzyl penicillin and ticarcillin with CRP do not materially interfere in the process.