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Biomedical subjects

B Singh

Publications and source records attributed to B Singh.

At least 325 records · Page 18Linked to original sources
PubMed1997

Repeated antidepressant drug treatment, time of death and frequency of handling do not affect [3H]paroxetine binding in rat cortex.

[3H]Paroxetine binding to the serotonin transporter has been shown to be altered in brain tissue from schizophrenic subjects. Some schizophrenic subjects are treated with antidepressant drugs, some of which bind to the serotonin transporter and their time of death is variable. To determine if these confounding factors could affect [3H]paroxetine binding, [3H]paroxetine binding to cortical membrane from rats treated with the antidepressant drugs for 10 or 28 days and in non-treated rats that were killed at different times of the day was measured. Drug treatment, when compared to injection with 0.9% saline and time of death, did not affect [3H]paroxetine binding. Treatment with imipramine [10 days: mean +/- S.D.: 590 +/- 59 fmol/mg protein (P < 0.05); 28 days: 653 +/- 59 fmol/mg protein (P < 0.01)] or mianserin [10 days: 600 +/- 43 (P < 0.05)] caused a significant decrease in the density of [3H]paroxetine binding compared to that in fluoxetine-treated rats (10 days: 820 +/- 211 fmol/mg protein; 28 days: 764 +/- 100 fmol/mg protein). Thus, overall, these data do not suggest changes in [3H]paroxetine binding reported in the human brain tissue would be due to antidepressant drug treatment or time of death.

Animals↗
PubMed1997

Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism.

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.

Animals↗
PubMed1997

The binding of both [3H]nemonapride and [3H]raclopride is increased in schizophrenia.

We performed a postmortem autoradiographic study to compare the density of dopamine D4-like sites in caudate putamen section from age-matched schizophrenia (n = 15) and control (n = 15) populations. The densities of the D4-like sites were estimated by subtracting the density of [3H]raclopride (binding D2 and D3 receptors) from the density of [3H]nemonapride (binding D2, D3, and D4 receptors). The findings revealed that in the schizophrenia population there was a significant increase in the binding of both [3H]nemonapride (2.3-fold) and [3H]raclopride (1.9-fold). In addition, in the schizophrenia population the density of D4-like sites was increased 2.6-fold.

Adult↗
PubMed1997

Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells.

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.

Amino Acid Sequence↗
PubMed1997

Inactivation of lck and loss of TCR-mediated signaling upon persistent engagement with complexes of peptide:MHC molecules.

T cell activation follows recognition of specific peptide:MHC molecule complexes in the context of proper costimulation. The earliest detectable event in T cell activation, within seconds of TCR ligand recognition, is tyrosine phosphorylation of TCR subunits. This causes a cascade of events leading to up-regulation of gene transcription that will drive T cell proliferation and differentiation. Regulation of TCR-mediated signaling upon T cell commitment is unclear. Here, we report that persistent stimulation of T cells, beyond 10 min, correlated with a reversible decrease in tyrosine phosphorylation of T cell lysates that did not affect T cell commitment to proliferation. Loss of Ag-induced tyrosine phosphorylation was not due to lack of Ag presentation, loss of TCR expression, or T cell death, but, rather, it was associated with a lack of TCR subunit tyrosine phosphorylation. We termed this phenomenon TCR desensitization by analogy to the loss of signaling observed in other receptor systems upon persistent engagement with agonist ligands. TCR desensitization correlated with surface reexpression of TCR without concomitant reexpression of coreceptor molecules. Biochemically, TCR desensitization correlated with increased levels of serine-phosphorylated lck, loss of lck kinase activity, and reversible loss of cytosolic lck. Thus, TCR signaling is regulated by desensitization that may be due to serine phosphorylation of lck causing inactivation and loss of this src kinase. This may have important implications by preventing TCR signaling and activation-induced cell death once the T lymphocyte is committed to proliferate.

Amino Acid Sequence↗
PubMed1997

Ultrastructural and immunocytochemical study of the pulmonary intravascular macrophages of Escherichia coli lipopolysaccharide-treated sheep.

BACKGROUND: Pulmonary intravascular macrophages (PIMs) of sheep, cattle, goats, and horses have a novel heparin-sensitive chain of globules, called a surface coat, on their plasma membrane. The globules are arranged at a distance of 32-39 nm from the plasma membrane of PIMs. Intravascular nonbiological tracer particles complex with these globules prior to their endocytosis by the PIMs. METHODS: We conducted a preliminary in vivo time-course study in sheep to investigate responses of the coat globules to a single dose of Escherichia coli lipopolysaccharide (E. coli LPS). Six sheep (6-9 months of age) were used in this study, and five of them were intravenously injected with E. coli (1 microgram/kg body weight) and euthanised at 3, 8, 10, 30, and 180 min (n = 1 each) after treatment. One sheep injected with saline solution served as the control. Acid phosphatase (AcPase) cytochemistry and immunocytochemistry using a polyclonal antibody were employed to localize secretory activity and E. coli LPS respectively in the PIMs. RESULTS: The surface coat of PIMs disappeared rapidly following the LPS administration. Escherichia coli LPS micelles and coat globules were colocalized as a complex in the endosomes of PIMs. At 8-10 min following the treatment, endosomal and the other membranes were disrupted, and the LPS was identified in cytoplasm and nuclear matrix of PIMs simultaneously with the development of pulmonary interstitial edema. Progression of AcPase reactivity along the nucleus-Golgi complex axis coupled with intense buildup of coated transport vesicles within 30 min of the LPS injection suggested enhanced biosynthetic activity in the PIMs. CONCLUSIONS: This study provides initial data on the sensitivity of the coat globules and their possible role in the endocytosis of E. coli LPS by the PIMs. Rapid biosynthetic activation of PIMs concurrent with loss of the coat and treatment with the LPS probably results in the secretion of inflammatory substances and contributes to the enhanced susceptibility of sheep to endotoxin-induced lung pathology.

Acid Phosphatase↗
PubMed1997

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes.

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 microg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6-8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[35S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes.

Animals↗
PubMed1997

Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences.

Heat shock proteins (HSP) in the size range of M(r) 60,000 are major targets of the immune response in vivo. The leishmania heat-inducible proteins of M(r) 65-67,000 are expressed at relatively high levels in infected macrophages (Infection and Immunity 1993, 61, 3265-3272) and may be important targets of the host response. To facilitate further studies concerned with these proteins, the HSP60 gene of Leishmania major was cloned, sequenced, and expressed. A lambdaEMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was characterized. Sequence analysis revealed an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M(r) 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from Trypanosoma cruzi. Less extensive amino acid sequence similarity (48%) was observed between that leishmania HSP60 and the corresponding human protein. Notably, significant regions of sequence dissimilarity between the leishmania and human proteins were identified principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was subcloned into the pET-3a vector and expressed in Escherichia coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from Colombia for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. This recognition had specificity in that these same sera showed little or no reactivity with either recombinant mycobacterial HSP65 or recombinant human HSP60. These findings indicate that patients with tegumentary forms of leishmaniasis have humoral responses to leishmania HSP60. Further studies of this protein will clarify its importance as a target of the immune response and as a potential antigen for serodiagnosis.

Amino Acid Sequence↗
PubMed1997

Modulation and detection of IDDM by membrane associated antigens from the islet beta cell line NIT-1.

We have utilized the NOD islet beta-cell line NIT-1 to monitor beta-cell specific autoantibodies and to investigate the modulation of IDDM in NOD mice by NIT-1 membrane associated antigens. The sera from diabetic but not from pre-diabetic or protected NOD mice strongly stained NIT-1 cells in FACS analysis. The cell surface antigens on NIT-1 cells were trypsin-sensitive. NIT-1 cells could not be stained by anti-mouse GAD67 antibody; however, we could demonstrate the presence of GAD65 and GAD67 mRNA by RT-PCR. Longitudinal analysis of anti-NIT-1 antibodies showed that these antibodies were present in the neonates but disappeared after weaning. Sonicated NIT-1 cell membrane preparations protected NOD mice from diabetes when injected intravenously in 5 week old mice. The protection was associated with reduced cytotoxic activity and elevated Th2-like responses as indicated by IgG1 antibodies against the NIT-1 cells. Subcutaneous injection of sonicated NIT-1 membranes or the injection of control red blood cell membranes failed to induce protection. We conclude that NIT-1 cell membranes do not express GAD but contain other antigens that are important in the development and prevention of IDDM. These antigens could be useful for the diagnosis of diabetes by monitoring autoantibody levels and for the modulation of IDDM by immunotherapy.

Animals↗
PubMed1997

BCG vaccination prevents insulin-dependent diabetes mellitus (IDDM) in NOD mice after disease acceleration with cyclophosphamide.

We have previously shown that immunotherapy with complete Freund's adjuvant (CFA) or BCG is highly effective in the prevention of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). The present study was undertaken to determine the effect of BCG vaccination on the progression of Cy-accelerated diabetes in NOD mice and to understand the mechanism of BCG immunotherapy. The time course of Cy and BCG administration showed that the progression of Cy-induced diabetes can only be blocked when BCG vaccination is given within 3 days of Cy administration. Mice given BCG 3 days before (-3 days) or 7 days after Cy treatment were not protected. BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice. Cy treatment reduced the endogenous production of anti-GAD67 antibody, whereas BCG vaccination 1 day after Cy treatment restored the production of anti-GAD67 antibody of IgG1 isotype. The comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase IL-4 production and change the IL-4/IFN-gamma ratio in both serum and supernatant of spleen cell cultures. We found that BCG-induced protection was associated with increased splenic CD4+CD45 RB(high) T cells. Taken together, our results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM. However, BCG immunotherapy has a narrow window of up to 3 days after Cy treatment to block the progression of Cy-induced diabetes and to allow for the induction of regulatory cells which may effectively downregulate the diabetogenic cells. In summary, our results suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment.

Animals↗
PubMed1997

3,4,5-Trihydroxy benzoic acid (gallic acid), the hepatoprotective principle in the fruits of Terminalia belerica-bioassay guided activity.

Compound I isolated from fraction TB5 of Terminalia belerica and finally identified as 3,4,5-trihydroxy benzoic acid (gallic acid) was evaluated for its hepatoprotective activity against carbon tetrachloride (CCl4)-induced physiological and biochemical alterations in the liver. The main parameters studied were hexobarbitone-induced sleep, zoxazolamine induced paralysis, serum levels of transaminases and bilirubin. The hepatic markers assessed were lipid peroxidation, drug metabolising enzymes, glucose-6-phosphatase and triglycerides. Administration of Compound I led to significant reversal of majority of the altered parameters. Our results confirm the presence of hepatoprotective activity in altered parameters. Our results confirm the presence of hepatoprotective activity in Compound I.

Animals↗
PubMed1997

Mos and the cell cycle.

The mos proto-oncogene-encoded serine/threonine protein kinase plays a key cell cycle-regulatory role during meiosis. The Mos protein is required for the activation and stabilisation of M phase-promoting factor MPF. As a component of a large multiprotein complex known as the cytostatic factor (CSF), Mos is involved in causing metaphase II arrest of eggs in vertebrates. Upon expression in somatic cells, Mos causes cell cycle perturbations resulting in cytotoxicity and neoplastic transformation. All the known biological activities of Mos are mediated through activation of the mitogen activated protein (MAP) kinase pathway. Here we discuss the interrelationship between Mos and other cell cycle regulators.

Animals↗
PubMed1997

Functional suitability of commercially milled rice bran in India for use in different food products.

The effect of blending of commercially available full fat and defatted rice brans in India from modern multistage rice mills with parboiling/stabilizing facilities in different food products in comparison to those obtained from laboratory milling of rice is reported. Bread volume and cookie spread decreased but muffin volume increased with the addition of different types of bran to wheat flour, however, the cookie spread factor was not affected by addition of full fat rice bran. The yields of the extrudate were increased by the blending of full fat rice bran but were decreased by the addition of defatted rice bran. Rice brans could be added to different food products to the extent of 5-10%. However, the full fat rice bran could not be used for production of extruded snack food.

Bread↗
PubMed1997

Molecular methods for diagnosis and epidemiological studies of parasitic infections.

Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. In order to overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed over the past 12 years. The parasites which have been studied with these techniques include the human Plasmodia, Leishmania, the trypanosomes, Toxoplasma gondii, Entamoeba histolytica, Giardia, Trichomonas vaginalis, Cryptosporidium parvum, Taenia, Echinococcus, Brugia malayi, Wuchereria bancrofti, Loa loa and Onchocerca volvulus. Early methods, which involved hybridisation of specific probes (radiolabelled and non-radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis have also proved valuable for epidemiological studies of parasites. The general principles and development of DNA-based methods for diagnosis and epidemiological studies will be described, with particular reference to malaria. These methods will probably not replace current methods for routine diagnosis of parasitic infections in developing countries where parasitic diseases are endemic, due to high costs. However, they will be extremely useful for genotyping parasite strains and vectors, and for accurate parasite detection in both humans and vectors during epidemiological studies.

Animals↗
PubMed1997

Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction.

Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we report a method for extracting DNA from dried blood spots on filter paper which is capable of detecting one Plasmodium falciparum and two Plasmodium vivax parasites/microliter of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.

Animals↗
PubMed1997

Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein.

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68-86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68-86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44-62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68-86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Acylation↗