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Biomedical subjects

A K Mehta

Publications and source records attributed to A K Mehta.

At least 19 recordsLinked to original sources
PubMed2006

Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterial codA gene.

BACKGROUND: Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses. METHODS: SDAP, Farrp, and Swiss-Prot databases were used to study allergenicity of choline oxidase. Digestibility of choline oxidase was assessed in simulated gastric fluid (SGF). Specific immunoglobulin E (IgE) reactivity of native and GM mustard was compared by using enzyme-linked immunosorbent assay (ELISA) and skin tests in respiratory-allergic patients. Allergenicity of GM and native mustard proteins was compared in Balb/c mice. RESULTS: Choline oxidase showed no significant homology with allergenic proteins in SDAP and Farrp databases. Cross-reactive epitope search showed a stretch similar to Hev b 6 having some antigenic properties. Purified choline oxidase showed complete degradation with SGF. Skin prick test of native and GM mustard extract on respiratory allergic patients showed significant correlation (P < 0.05). ELISA with 96 patients' sera showed comparable IgE reactivity. Balb/c mice immunized with native and GM mustard proteins showed low IgE response. Presensitized mice on intravenous challenge with Brassica extract showed no anaphylactic symptoms unlike ovalbumin (OVA) sensitization that showed anaphylactic reaction in mice. Lung histology of OVA-sensitized mice showed narrowing of airway and large eosinophilic infiltration, whereas native and GM Brassica extract showed normal airway. CONCLUSION: Genetically modified mustard with the codA gene possessed allergenicity similar to that of native mustard and no enhancement of IgE binding was observed due to genetic manipulation.

Adolescent↗
PubMed2004

Genetic diversity at 15 fluorescent-labeled short tandem repeat loci in the Patel and other communities of Gujarat, India.

Thirteen tetranucleotide and 2 pentanucleotide repeat units were analyzed in 120 unrelated individuals of Patel and other communities of Gujarat, India. Allele frequency data obtained from the analysis of 15 short tandem repeat markers of the population were found to be satisfying Hardy-Weinberg equilibrium, with marginal deviations. Departures from Hardy-Weinberg equilibrium were observed in Patel communities at locus vWA and for that of the other communities at locus D7S820 and at locus TPOX. The power of discrimination values on an average fall within the range of 0.718 and 0.870, with deviations at locus D3S1358 showing a value of 0.400 for Patels. The value ranged between 0.709 and 0.869, with slight variations among the studied alleles in the other group. Thus, the 15 markers selected for this study were found to be highly suitable in human identification and for providing information on genetic polymorphism of the population of Gujarat.

DNA Fingerprinting↗
PubMed2001

Unsulfated and sulfated neurosteroids differentially modulate the binding characteristics of various radioligands of GABA(A) receptors following chronic ethanol administration.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) inhibited the binding of [(3)H]flunitrazepam (2 nM), [(3)H]muscimol (5 nM) and 4 nM [(35)S]t-butylbicyclophosphorothionate [(35)S]TBPS in the rat cerebellum as well as cerebral cortex. DHEAS-induced inhibition of binding of these radioligands (62% to 100%) was more pronounced as compared to that in the case of DHEA (5% to 31%). DHEAS, unlike DHEA, inhibited [(3)H]flunitrazepam binding significantly to a lesser extent in the cerebellum of ethanol-dependent rats as compared to the control group (I(max):82+/-1vs.92+/-2%, p<0.005). However, DHEA, unlike DHEAS, inhibited [(35)S]TBPS binding to a greater extent in the ethanol-dependent rat cerebellum as compared to the control group (I(max):31+/-2vs.19+/-2%, p<0.005). Furthermore, DHEA was more potent in inhibiting [(35)S]TBPS binding in the cerebellum (IC(50):55+/-5 vs. 74+/-7 microM, p<0.05) and cerebral cortex (IC(50):26+/-4vs.64+/-9 microM, p<0.05) of ethanol-dependent rats as compared to the control group. These observations indicate that unsulfated and sulfated androstane-steroids modulate the GABA(A) receptors in the control as well as the ethanol-dependent rats differentially, and also suggest that the androstane-steroid binding sites associated with the GABA(A) receptors play an important role during ethanol dependence.

Animals↗
PubMed2001

Binding characteristics of the gamma-hydroxybutyric acid receptor antagonist [(3)H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid in the rat brain.

Radioligand binding studies with [(3)H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382), an antagonist of gamma-hydroxybutyric acid (GHB) receptor, revealed specific binding sites in the rat cerebral cortex and hippocampus. However, there was very little binding in the rat cerebellum, heart, kidney, liver, and lung membranes. Binding was rapid and reached equilibrium in about 5 min. Scatchard analysis of saturation isotherms revealed two different populations of binding sites in the rat cerebral cortex (K(d1), 795 nM, B(max1), 25.4 pmol/mg of protein; K(d2), 21 microM; B(max2), 178 pmol/mg of protein) as well as in the rat hippocampus (K(d1), 441 nM; B(max1), 16.2 pmol/mg of protein; K(d2), 9.8 microM; B(max2), 255 pmol/mg of protein). (+/-)Baclofen (500 microM) and gamma-aminobutyric acid (100 microM) inhibited the binding only partially, whereas (+)bicuculline, muscimol, picrotoxinin, and phaclofen did not modify the binding. Interestingly, potassium chloride (100-300 mM) inhibited [(3)H]NCS-382 binding (34-56%), and this inhibitory effect was not affected by picrotoxinin. GHB and NCS-382 completely inhibited the [(3)H]NCS-382 (16 nM) binding in the rat cerebrocortical and hippocampal membranes, and NCS-382 was found to be about 10 times more potent than GHB in this regard. A variety of ligands for other receptors did not modify the [(3)H]NCS-382 binding, thereby suggesting selectivity of this radioligand for the GHB receptor sites in the brain. Based on these observations, [(3)H]NCS-382 seems to be a better radioligand than [(3)H]GHB for investigating the role of the GHB receptors in various pharmacological actions.

Animals↗
PubMed1999

Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain.

A novel anti-beta(3) subunit-specific GABA(A) receptor (GABA(A)R) antibody has been prepared by immunizing a rabbit with a bacterial fusion protein of the large intracellular loop of the beta(3) subunit. The antiserum immunoprecipitated the solubilized GABA(A) receptor. The anti-beta(3) antibody was affinity purified on immobilized beta(3) large intracellular loop peptide. In immunoblots, the purified antibody reacted with a 57 KDa peptide. Immunocytochemistry with the affinity-purified antibody has revealed the localization of the beta(3) subunit in the rat brain. A comparative study with the immunocytochemical distribution of the beta(2) subunit has also been performed. There are areas of the brain and cell types where the distribution of beta(2) and beta(3) overlap (i.e., cerebral cortex, cerebellum,and most layers of the olfactory bulb). There are also clear differences in the expression of beta(3) and beta(2) in other brain areas and cell types. Thus, high beta(3) but low or no beta(2) expression was observed in the corpus striatum and in granule cells of the olfactory bulb. In the hippocampus the expression of beta(3) was considerably higher than that of beta(2), but some hippocampal interneurons showed high expression of beta(2). High beta(2) but little or no expression of beta(3) was observed in thalamic nuclei, substantia nigra, globus pallidus, inferior colliculus and the short axon cells of the olfactory bulb.

Animals↗
PubMed1999

An investigation on the role of nitric oxide in the modulation of the binding characteristics of various radioligands of GABAA receptors by 5alpha-pregnan-3alpha-ol-20-one in the rat brain regions.

Chronic administration of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, diminished the ability of 5alpha-pregnan-3alpha-ol-20-one, a neurosteroid, to potentiate the [3H]muscimol (5 nM) binding in the rat hippocampus but not in the cerebellum or cerebral cortex. This effect of NAME was stereospecific and susceptible to reversal by the pre-treatment of rats with L-arginine. However, chronic administration of L-NAME did not affect the modulation of the [3H]flunitrazepam (2 nM) or [35S]TBPS (4 nM) binding by the neurosteroid in any of the brain regions investigated in this study. These results suggest that nitric oxide may be involved in some of the effects of neurosteroids in hippocampus.

Animals↗
PubMed1999

Prevalence of the GABAA receptor assemblies containing alpha1-subunit in the rat cerebellum and cerebral cortex as determined by immunoprecipitation: lack of modulation by chronic ethanol administration.

The anti-alpha1 antibody elicited higher immunoprecipitation (%) values of the [3H]flunitrazepam and [3H]muscimol binding activity in the rat cerebellum vs. cerebral cortex, whereas immunoprecipitation values for [3H]Ro 15-4513 and [3H]zolpidem were comparable in these brain regions. Chronic ethanol administration neither changed the radioligand binding to the immunoprecipitated pellet nor the percentage immunoprecip-itation values, thereby indicating that chronic ethanol did not result in down-regulation of the GABAA receptor assemblies containing alpha1-subunit.

Affinity Labels↗
PubMed1999

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Alternative Splicing↗
PubMed1999

An update on GABAA receptors.

Recent advances in molecular biology and complementary information derived from neuropharmacology, biochemistry and behavior have dramatically increased our understanding of various aspects of GABAA receptors. These studies have revealed that the GABAA receptor is derived from various subunits such as alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, pi, and rho1-3. Furthermore, two additional subunits (beta4, gamma4) of GABAA receptors in chick brain, and five isoforms of the rho-subunit in the retina of white perch (Roccus americana) have been identified. Various techniques such as mutation, gene knockout and inhibition of GABAA receptor subunits by antisense oligodeoxynucleotides have been used to establish the physiological/pharmacological significance of the GABAA receptor subunits and their native receptor assemblies in vivo. Radioligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinity chromatography and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of native GABAA receptor assemblies. Partial agonists of GABAA receptors are being developed as anxiolytics which have fewer and less severe side effects as compared to conventional benzodiazepines because of their lower efficacy and better selectivity for the GABAA receptor subtypes. The subunit requirement of various drugs such as anxiolytics, anticonvulsants, general anesthetics, barbiturates, ethanol and neurosteroids, which are known to elicit at least some of their pharmacological effects via the GABAA receptors, have been investigated during the last few years so as to understand their exact mechanism of action. Furthermore, the molecular determinants of clinically important drug-targets have been investigated. These aspects of GABAA receptors have been discussed in detail in this review article.

Animals↗
PubMed1998

Chronic ethanol administration alters the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one on the binding characteristics of various radioligands of GABAA receptors.

In this study, we investigated the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one, a neurosteroid, on the binding characteristics of [3H]flunitrazepam (2 nM), [3H]muscimol (5 nM), and 4 nM [35S]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5alpha-Pregnan-3alpha-ol-20-one potentiated the binding of [3H]flunitrazepam and [3H]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [3H]flunitrazepam as well as [3H]muscimol binding (Emax) in the ethanol-dependent rat cerebellum as compared to control group (p<0. 025). Furthermore, 5alpha-pregnan-3alpha-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [35S]TBPS at lower concentrations (<=100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [35S]TBPS binding (-Emax) by 5alpha-pregnan-3alpha-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the gamma-aminobutyric acidA (GABAA) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.

Animals↗
PubMed1998

Up-regulation of NMDA receptor subunits in rat brain following chronic ethanol treatment.

We investigated the effect of chronic ethanol administration and its withdrawal on the polypeptide levels of NMDA receptor subunits such as NR1, NR2A, and NR2B in the rat cerebral cortex and hippocampus using Western blot analysis technique. Our results indicate that chronic ethanol treatment upregulates NMDA receptor subunits NR1, NR2A, and NR2B ( approximately 35%). At 48 h of last dose of ethanol administration, the protein content returned to almost control level, thereby demonstrating the reversibility of the changes.

Alcoholism↗
PubMed1997

GABAA receptor subunit expression changes in the rat cerebellum and cerebral cortex during aging.

Significant aging-related decreased expression of various GABAAR subunit mRNAs (alpha 1, gamma 2, beta 2, beta 3 and sigma) was found in both cerebellum and cerebral cortex using quantitative dot blot and in situ hybridization techniques. Contrary to the other subunits, the alpha 6 mRNA expression was significantly increased in the aged cerebellum. Parallel age-related changes in protein expression for gamma 2 and beta 2/3 (decrease) and alpha 6 (increase) were revealed in cerebellum by quantitative immunocytochemistry. However, no significant changes in alpha 1 protein expression nor in the number or affinity of [3H]zolpidem binding sites were detected in cerebellum even though alpha 1 mRNA expression was significantly decreased in the aged rat. Age-related increased expression of alpha 6 mRNA and protein in the cerebellum was accompanied by no significant changes in the number of diazepam-insensitive [3H]Ro15-4513 binding sites. In the cerebral cortex, no changes in the protein expression of the main GABAA receptor subunits (alpha 1, gamma 2 and beta 2/3) were observed which contrasted with the age-related decreased expression of the corresponding mRNAs. No significant changes in the number or affinity of [3H]zolpidem binding sites were observed in the cerebral cortex. Thus, age-related changes in the mRNA expression of a particular subunit does not necessarily lead to similar changes in protein or assembly into mature GABAA receptors. The results reveal the existence of complex regulatory mechanisms of GABAA receptor expression, at the transcriptional, translational and post-translational and/or assembly levels, which vary with the subunit and brain area.

Aging↗
PubMed1996

The alpha 4 subunit of the GABAA receptors from rat brain and retina.

A novel anti-alpha 4 antibody has been used for the purification and characterization of the alpha 4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the alpha 4 subunit peptide in rat brain and retina. The anti-alpha 4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10-28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-alpha 4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-alpha 6 antibody but not by the anti-alpha 4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-alpha 4 revealed molecular colocalization of alpha 4 and gamma 2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the alpha 4 and gamma 2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of alpha 4, gamma 2 and beta 2 subunits. Immunocytochemistry has revealed the abundance of alpha 4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The alpha 4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, alpha 4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer.

Animals↗
PubMed1995

Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS.

The binding of [3H]Ro 15-4513, [3H]flunitrazepam and [3H]flumazenil to rat CNS membranes was studied at 2 degrees C, 22 degrees C and 37 degrees C using ligand concentrations ranging from approximately 0.06 nM to 10 microM. Analysis of the binding saturation data suggested the existence of high-affinity sites (Kd < 10 nM) and low-affinity sites (Kd > 100 nM) for each ligand. When binding was performed using very low ligand concentrations a benzodiazepine site with an exceptionally high affinity for Ro 15-4513 (Kd approximately 0.1 nM) was evident in all major regions of the CNS except the cerebellum. This site was most prevalent in the hippocampus, medulla and spinal cord where it accounted for approximately 70% of the specific binding when [3H]Ro 15-4513 was approximately 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depending on the incubation temperature and CNS region. The affinity for the very high-affinity site was decreased approximately 3-fold as temperature was increased from 2 degrees C to 37 degrees C (Kd approximately 0.1 nM and approximately 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites (Kd approximately 2.6 nM at 2 degrees C and approximately 8 nM at 37 degrees C). Flumazenil, flunitrazepam, and diazepam did not differentiate the very high-affinity [3H]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC50 approximately 5 microM). GABA at 100 microM had little effect on the Kd value for the very high-affinity site (GABA shift: approximately 0.8 to 1.0), suggesting that Ro 15-4513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of BZ sites on different subtypes of GABAA receptors.

Affinity Labels↗
PubMed1995

Interaction of abecarnil, bretazenil, and RO 19-8022 with diazepam-sensitive and -insensitive benzodiazepine sites in the rat cerebellum and cerebral cortex.

Abecarnil, bretazenil, and Ro 19-8022 inhibited the binding of [3H]Ro 15-4513 to diazepamsensitive and -insensitive sites in the rat cerebellum and cerebral cortex, but all three had a much higher affinity for the diazepam-sensitive sites in both tissues. The GABA-shift for bretazenil and Ro 19-8022 was low ( < 2) for all sites studied, consistent with their partial agonistic profile. The GABA-shift for abecarnil was appreciably higher for diazepam-sensitive binding in the cerebellum than in the cerebral cortex (1.97 vs 1.18). Furthermore, the GABA-shift for abecarnil was markedly different for the diazepam-sensitive and -insensitive sites in the cerebellum (1.97 vs 0.71). All three compounds inhibited [3H]Ro 15-4513 binding to the diazepam-sensitive sites with a slope factor > 1, suggestive of positive cooperativity in their binding to GABAA receptors. Abecarnil only partially inhibited diazepam-insensitive binding of [3H]Ro 15-4513 in the cerebellum, indicating that this site can be differentiated into abecarnil-sensitive and -insensitive components.

Animals↗
PubMed1994

Ethanol enhancement of GABA-induced 36Cl- influx does not involve changes in Ca2+.

The effect of changes in intracellular Ca2+ on ethanol enhancement of GABA-mediated 36Cl- influx was investigated in mammalian cortical neurons in culture. Ethanol potentiated the effect of submaximal concentrations of GABA on the 36Cl- influx, and at 50 mM ethanol directly activated 36Cl- influx in these neurons. Pretreatment of the neurons with dantrolene (an agent that depletes intracellular Ca2+) and the Ca2+ ionophore, A 23187, did not alter the effect of GABA or ethanol enhancement of GABA-mediated 36Cl- influx. Together, these results suggest that changes in intracellular Ca2+ are not involved in ethanol modulation of GABAAergic responses in cortical neurons.

Animals↗
PubMed1993

Ethanol- and diazepam-withdrawal hyperlocomotion is not due to 5-HT3 receptor stimulation.

The effect of 5-HT3 receptor antagonists such as ondansetron, ICS 205-930, MDL 72222, metoclopramide, and zacopride was investigated on the ethanol as well as diazepam withdrawal phenomena in the present study. There was a significant increase in locomotor activity in the ethanol- as well as diazepam-withdrawn rats. The treatment of rats with 5-HT3 receptor antagonists during withdrawal phase did not modify the effect. The ethanol-withdrawn rats were more sensitive to pentylenetetrazole (PTZ)-induced convulsions as compared to control animals. 5-HT3 receptor antagonists did not attenuate the increased sensitivity of ethanol-withdrawn rats to PTZ. These observations indicated that 5-HT3 receptor antagonists are ineffective in attenuating hyperlocomotor activity following abrupt termination of chronic administration of ethanol or diazepam, and increased sensitivity to PTZ in the ethanol-withdrawn rats.

Animals↗