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Biomedical subjects

R K Mishra

Publications and source records attributed to R K Mishra.

At least 19 recordsLinked to original sources
PubMed1995

Improved leishmanicidal effect of phosphorotioate antisense oligonucleotides by LDL-mediated delivery.

We have designed antisense oligonucleotides that can interact with lipoproteins in order to use them as vectors to facilitate the uptake by those cells expressing the corresponding receptor. Phosphorothioate (PS) oligonucleotides were linked at the 5' end to a palmityl group giving rise to PSPal conjugates. Such a modification enables the oligonucleotide to form a stable non-covalent complex with low density lipoproteins (LDL) through hydrophobic interactions. The antisense effect of LDL-oligonucleotide complexes was assayed by targeting the mini-exon sequence of Leishmania amazonensis in infected mouse peritoneal macrophages. A 16-mer antisense PSPal oligonucleotide/LDL complex exerted a more pronounced sequence-specific effect than the free oligomer: about 25% and 10% of infected macrophages were cured by a 48 h incubation in the presence of 2.5 microM of the complexed and the free oligomer, respectively. When oxidized LDL was used instead of the native one for complexation, a further 2-fold increase in the antisense effect was observed suggesting that alternative (unregulated) scavenger receptor can be used for more efficient delivery of antisense oligonucleotides into macrophages. In addition, a significant reduction of the parasitic load was observed in those cells that were not fully cured.

Animals↗
PubMed1995

Modulation of N-methyl-D-aspartate (NMDA) antagonist-induced darting behaviour by the peptidomimetic PAMTA.

The N-Methyl-D-Aspartate (NMDA) receptor has attracted much attention in recent years due to its involvement in both the functions and dysfunctions of CNS neurotransmission. The existence of multiple sites by which NMDA receptor channel function can be pharmacologically modified and the interaction between glutamate and other neurotransmitter systems such as dopamine, provide exciting therapeutic avenues for related CNS disorders. In the present study, a novel synthetic analogue of the endogenous brain peptide L-prolyl-L-leucyl glycinamide (PLG) has demonstrated a significant modulatory action on the NMDA receptor. On the basis of radioligand binding studies, the novel synthetic peptide 5-[1(S)-(2(S)-pyrrolidinylcarbonyl)amino-3-methylbutyl]-2- tetrazolylacetamide (PAMTA) has been suggested to act at a polyamine site on the NMDA receptor complex. Scatchard analysis of [3H]MK-801 binding revealed that in the presence of 100 microM PAMTA, a single binding site was obtained with the Kd being increased from 2.5 +/- 0.2 nM to 6.2 +/- 0.1 nM. The ability of PAMTA to inhibit the binding of [3H]MK-801 was sensitive to the presence of both spermidine (polyamine agonist) and arcaine (polyamine antagonist). Analyses of the binding profiles of various NMDA receptor antagonists support PAMTA's interaction with the polyamine site on this receptor complex. Furthermore, we have investigated the behavioural profile of the peptidomimetic PAMTA, by studying its effect on stereotypic behaviours induced by the NMDA receptor antagonist, CPP (3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid). Male Sprague-Dawley rats cannulated bilaterally into the medial prefrontal cortex were injected with PAMTA, CPP, a CPP/PAMTA combination, or a saline control.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene.

The recently described catecholamine-absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high-specific-activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high-specific-activity substituted tetrahydronaphthalene derivative (6-hydroxy-[125I]iodo-[N-(N-2',4'- dinitrophenyl)aminopropyl]-2-amino-1,2,3,4-tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.

Absorption↗
PubMed1994

Distribution of cholecystokinin receptors in the bovine brain: a quantitative autoradiographic study.

Quantitative in vitro receptor autoradiography was used to study the distribution of cholecystokin receptors in the bovine brain. [125I]Bolton-Hunter cholecystokinin octapeptide binding was described in whole hemisphere sagittal and coronal sections using cholecystokinin octapeptide, devazepide and L-365,260 as competitors to identify the subtypes. High levels of cholecystokinin receptors were found in the cortex, where they presented a laminar distribution which varied from area to area. The basal ganglia, the caudate nucleus, nucleus accumbens and putamen presented high to moderate levels of cholecystokinin binding, whereas only very low labelling was found in the globus pallidus. Cholecystokinin binding was present in all portions of the bovine hippocampus; high levels were found in the dentate gyrus, CA1 subfield of Ammon's horn, subiculum and presubiculum. Moderate to high levels were also found in the amygdala, inferior colliculus and olfactory tract, while most of the hypothalamic and thalamic nuclei exhibited very low or no cholecystokinin binding. Low cholecystokinin binding was uniformly distributed across cell layers of the bovine cerebellar cortex. Competition of [125I]Bolton-Hunter cholecystokinin octapeptide binding in the cortex, nucleus accumbens, caudate nucleus, hippocampus, cerebellum and brainstem was much greater in the presence of L-365,260 than devazepide, thereby suggesting that the majority of cholecystokinin receptors in these regions are of the cholecystokinin-B subtype. The results of this study, when compared to distribution profiles in other mammalian species, provide further evidence for species differences in the distribution of cholecystokinin receptors in the brain. The results also support the possible interaction between cholecystokinin and dopaminergic systems in areas of the brain containing dopaminergic terminals, such as the frontal cortex, nucleus accumbens, caudate-putamen and olfactory tubercle.

Animals↗
PubMed1992

Ontogeny of dopamine D2 receptor mRNA in rat brain.

The postnatal development of rat brain dopamine D2 receptor gene expression was investigated in animals 1 day to 1 year old. The level of expression of the striatal D2 mRNA was appreciable at birth (day 1), steadily increased to a maximum at day 28, and showed declines at ages 6 months and one year. The mRNA development profile was similar to that of [3H]spiroperidol binding in striatal membranes except that there was a lack of correlation between mRNA levels and [3H]spiroperidol binding during the early developmental periods. For example, although the mRNA expression at day 1 is about 75% of the 28-day value, the corresponding level of [3H]spiroperidol binding is only 15% of the value observed at day 28. Polymerase chain reaction (PCR) analysis of alternatively spliced forms of D2 receptor mRNA showed that the developmental expression of the two isoforms proceeded in parallel as the ratio of D2L and D2S mRNAs remained more or less constant in different age group of rats. In situ hybridization revealed a differential developmental profile of D2 mRNA for major dopaminergic regions of rat brain such as caudate putamen, nucleus accumbens, olfactory tubercle and substantia nigra.

Animals↗
PubMed1992

Effects of chronic treatment of haloperidol and clozapine on levels of G-protein subunits in rat striatum.

Chronic administration of typical neuroleptic drugs, such as haloperidol, causes the supersensitivity of brain dopamine D2 receptor in striatum and limbic regions, while the atypical neuroleptic clozapine does not. In order to understand the mechanism of their action at a molecular level, studies were carried out to assess the effects of chronic treatment of these drugs on the levels of G-proteins in the rat striatum using the Western blot method. Results of the present study demonstrate that the treatment with haloperidol or clozapine, respectively, down-regulate or up-regulate the levels of G proteins. Quantitative immunoblotting, using site-directed specific antisera, demonstrated that chronic treatment with haloperidol down-regulates Gi alpha, Gs alpha, and beta subunits while chronic treatment with clozapine upregulates Gi alpha, Gs alpha, and beta subunits. Neither of these drugs has any effect on the levels of Go alpha.

Amino Acid Sequence↗
PubMed1992

Quantitative immunological status of males of unexplained infertility in eastern U.P.

126 males of unexplained infertility and 75 fertile males were studied during a period of more than 2 years. Statistically significant increase in the levels of serum IgM was observed in cases of males of unexplained infertility. Level and percentage of cases showing semen IgM was more in study group (134.61 +/- 25.5 mg %, 41.26%) than control (24%, 99.3 +/- 8.3%). Semen IgA was found only in study group in 30.15% cases. Though definite correlation between serum and semen immunoglobulin was not observed, semen immunoglobulin was found to be 1-2 fold lower than serum. Results of the present study indicate towards a possible immunological role in cases of unexplained infertility in males.

Humans↗
PubMed1991

Localization of cholecystokinin binding sites in canine brain using quantitative autoradiography.

1. CCK receptors have been characterized and localized in various mammalian species and significant species-specific differences in their distribution have been identified. In the present study, we report the first autoradiographic localization of CCK binding sites in the canine brain. 2. High densities of [125I]BH-CCK-8 binding sites were found in the cortex, cerebellum, hippocampus, caudate nucleus, olfactory bulb and nucleus accumbens. Moderate densities were present in the putamen, amygdala, and substantia gelatinosa. Low binding densities were observed in the globus pallidus, inferior colliculus, hypothalamus and thalamus. 3. Although the distribution profile of CCK binding sites in canine brain is similar to those previously reported in the rodent, primate and human brain, notable differences were observed in the hippocampus, cortex and cerebellum.

Animals↗
PubMed1991

Mortality events amongst non insulin dependent diabetes mellitus patients in Orissa.

In a study over one year, it was observed that mortality amongst hospitalised patients with non insulin dependent diabetes mellitus (NIBDM) was nearly 20%. Those dying within 24 hr were classified as group A, between one day and one week as B, between one week and one month as C, and those after one month as D. There were 31 patients each in groups A and B, 14 in C, and 4 in D. The mean age at death was 61 years in the first three groups. The prevalence of cerebro-vascular accident as a terminal event was similar i.e. 32.2, 35.5 and 35.7 per cent in groups A, B and C respectively; 48% of patients in group A suffered from ischaemic heart disease. Diabetic ketoacidosis was equally prevalent amongst groups A, B and C. Infection was significantly more common in group B (45.2%) than A (P less than 0.05). Nephropathy was observed in 57% of patients in group C as compared to 22.5% in A (P less than 0.02). Cerebrovascular accident and infection were the major causes of mortality in groups B and C (80.7% and 71.4%), whereas ischaemic heart disease and cerebrovascular accident accounted for 80% of deaths in group A.

Cause of Death↗
PubMed1990

Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region.

Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter of bacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid. Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions. Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1. Sequence comparison reveals a high G/C content upstream to the -35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.

Base Sequence↗
PubMed1990

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: effects of agonist and antagonist pretreatment.

The bovine striatal dopamine D1 receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [3H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a Kd of 0.95 and 1.1 nM and a Bmax of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [3H]SCH-23390 binding exhibited a clear D1 dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent than cis-flupentixol in displacing [3H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[3H]SCH-23390 competition studies revealed the presence of a high-affinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40-80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obtained from the agonist-treated membrane. In both the preparations, 100 microM guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Effect of haloperidol on expression of dopamine D2 receptor mRNAs in rat brain.

Chronic administration of the neuroleptic drug haloperidol previously has been shown to increase the density of striatal dopamine D2 receptor, which is believed to be the underlying factor in neuroleptic-induced tardive dyskinesia. To search for the mechanism of receptor upregulation, the expression of the isoforms of dopamine D2 receptor mRNA in rat striatum was analyzed by Northern, solution, and in situ hybridizations in haloperidol-treated rats (1-35 days). Northern blot analysis of poly(A)+ RNA hybridized with a probe common for both isoforms as well as an insert-specific probe for the long isoform of the receptor revealed no significant difference in hybridization signal between the control and any of the haloperidol-treated groups of rats. The receptor density, however, was increased by 30-40% in animals receiving haloperidol for 7-35 days. Solution hybridization with an antisense riboprobe specific for a consensus sequence as well as in situ hybridization with a consensus oligonucleotide probe similarly failed to detect any increase in the expression of receptor mRNA following haloperidol treatment. The results suggest that post-transcriptional mechanisms may be responsible for regulating the haloperidol-induced increase in dopamine D2 receptors.

Animals↗
PubMed1990

Modulation of high-affinity CNS dopamine D2 receptor by L-pro-L-leu-glycinamide (PLG) analogue 3(R)-(N-L-prolylamino)-2-oxo-1-pyrrolidineacetamide.

1. In our previous studies, we reported that one of the conformationally constrained analogues of PLG, 3(R)-(N-L-prolylamino)-2-oxo-1-pyrrolidineacetamide (PAOPA), was found to be extremely potent in enhancing the [3H]ADTN binding when membranes were preincubated with this compound. The PAOPA was without effect when directly added to assay tubes. In this study, the compound PAOPA was examined on agonist binding to high affinity state of the dopamine D2 receptor. 2. The potent analogue PAOPA was able to prevent the GTP-induced conversion of high affinity state of dopamine D2 receptor to low affinity state. 3. PLG analogue PAOPA modulates the affinity states of the dopamine D2 receptor possibly by affecting its interaction with the G-protein(s).

Animals↗
PubMed1990

Synthesis and biological evaluation of analogues of Pro-Leu-Gly-NH2 modified at the leucyl residue.

A series of analogues of Pro-Leu-Gly-NH2 (PLG) in which the leucine residue has been replaced with the aliphatic amino acids L-isoleucine, L-2-aminohexanoic acid (Ahx), L-2-aminopentanoic acid, and L-2-aminobutanoic acid and the aromatic amino acids L-phenylalanine, L-phenylglycine, L- and D-2-amino-4-phenylbutanoic acid, L-O-methyltyrosine, and L-4-nitrophenylalanine have been synthesized. These analogues were tested for their ability to enhance the binding of the dopamine receptor agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to striatal dopamine receptors. Two of the above analogues, Pro-Ahx-Gly-NH2 (3) and Pro-Phe-Gly-NH2 (6), showed significant activity in this assay system. Pro-Ahx-Gly-NH2 produced a 16% enhancement of ADTN binding at 0.1 microM, while Pro-Phe-Gly-NH2 enhanced the binding of ADTN by 31% at a concentration of 1 microM.

Animals↗
PubMed1990

The effect of dopamine D1 and D2 receptor agonists on inositol phosphate turnover in rat striatal slices.

There is a lot of controversy in the literature about the role of dopamine D1 and D2 receptor stimulation on the turnover of phosphoinositols, and phosphoinositols are one of the important second messenger. In order to resolve this controversy, the effect of dopamine receptor stimulation on turnover of phosphoinositols was studied by estimation of the accumulation of individual labelled inositols in rat striatal slices which were prelabelled with [3H]myoinositol. Incubation of the prelabelled striatal slices with 1 microM of quinpirole, D2 specific agonist or with 1 microM of SKF-38393, D1 specific agonist, did not affect the accumulation of basal level of either inositol monophosphate, or inositol biphosphate, or inositol triphosphate. In addition, in conclusion of D1 specific antagonist cis-flupentixol or D2 specific antagonist sulpiride did not affect the basal levels of inositol phosphates. The activity of enzyme phospholipase-C which produces these inositol phosphates was also measured in rat striatal membrane. Incubation of rat striatal membrane with either agonist quinpirole or SKF-38393 did not change the basal level of phospholipase C. Our data thus indicate that occupation of dopamine receptors did not affect the inositol phosphate system in rat striatum.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Chemical modification reveals involvement of tyrosine in ligand binding to dopamine D1 and D2 receptors.

The effect of tyrosine-alkylating agents on the ligand-binding properties of bovine striatal dopamine D1 and D2 receptors was investigated. The tyrosine-alkylating agents, p-nitrobenzenesulphonylfluoride (pNBSF) and tetranitromethane (TNM) caused a time-and dose-dependent loss of the binding of [3H]SCH-23390 and [3H]spiroperidol, ligands specific for dopamine D1 and D2 receptors, respectively. The two dopamine receptors, however, showed a differential sensitivity to inactivation by these agents. The mechanism of inhibition of the two receptors appears to be complex as treatment of membranes with pNBSF and TNM resulted in a decrease of both the Kd and the Bmax of ligand binding. Spiroperidol almost completely protected the TNM-induced inhibition of [3H]spiroperidol binding to dopamine D2 receptors whereas SCH-23390 afforded only partial protection of the [3H]SCH-23390 binding by TNM suggesting that more than one tyrosine groups may be involved in the D1 receptor binding activity.

Alkylating Agents↗
PubMed1989

DNA duplex with the potential to change handedness after every half a turn.

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.

Base Sequence↗