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Biomedical subjects

K Singh

Publications and source records attributed to K Singh.

At least 109 records · Page 6Linked to original sources
PubMed2001

Protection against rotavirus diarrhoea in mice by trypsin inhibitor.

To investigate the role of soyabean trypsin inhibitor (TI) during rotavirus (RV) diarrhoea, changes in enzyme activities of six relevant mucosal enzymes (lactase, sucrase, maltase, trehalase, glucoamylase and alkaline phosphatase) were assayed following inoculation of suckling mice with EB rotavirus (serotype 3) along with the TI and compared with the age-matched healthy control mice. The animals were divided into three groups i.e. group 1 (controls), group 2 (RV inoculated) and group 3 (RV + TI inoculated and sacrificed under light anaesthesia on 0, 1, 3, 5, 7 and 10 day post inoculation (dpi). Then intestines were excised and divided into two parts (jejunum and ileum). They were separately homogenized in 0.9% cold normal saline and activities of mucosal enzyme were measured. Alkaline phosphatase and disaccharidases were found to be decreased significantly in RV inoculated animals in both the anatomical portions of small intestine of mice. These enzyme levels were restored with the administration of TI i.e. in group 3 and became comparable to the controls in both intestinal portions. These studies suggest that activity of intestinal enzymes which are important in digestive absorptive functions of small intestine were restored with the addition of TI whengiven to infant mice showing its protective efficacy during rotavirus infection.

Animals↗
PubMed2000

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition.

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 A(2) for the peptide and 625 A(2) for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.

Amino Acid Sequence↗
PubMed2000

Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins.

Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed2000

The J-helix of Escherichia coli DNA polymerase I (Klenow fragment) regulates polymerase and 3'- 5'-exonuclease functions.

To assess the functional importance of the J-helix region of Escherichia coli DNA polymerase I, we performed site-directed mutagenesis of the following five residues: Asn-675, Gln-677, Asn-678, Ile-679, and Pro-680. Of these, the Q677A mutant is polymerase-defective with no change in its exonuclease activity. In contrast, the N678A mutant has unchanged polymerase activity but shows increased mismatch-directed exonuclease activity. Interestingly, mutation of Pro-680 has a Q677A-like effect on polymerase activity and an N678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gln results in a 10-30-fold reduction in k(cat) on homo- and heteropolymeric template-primers, with no significant change in relative DNA binding affinity or K(m)((dNTP)). The mutants P680G and P680Q also showed a nearly complete loss in the processive mode of DNA synthesis. Since the side chain of proline is generally non-reactive, mutation of Pro-680 may be expected to alter the physical form of the J-helix itself. The biochemical properties of P680G/P680Q together with the structural observation that J-helix assumes helical or coiled secondary structure in the polymerase or exonuclease mode-bound DNA complexes suggest that the structural alteration in the J-helix region may be responsible for the controlled shuttling of DNA between the polymerase and the exonuclease sites.

Base Pair Mismatch↗
PubMed2000

A carboxylate triad is essential for the polymerase activity of Escherichia coli DNA polymerase I (Klenow fragment). Presence of two functional triads at the catalytic center.

The catalytic roles of two essential active-site aspartates at positions 705 and 882 of Escherichia coli DNA polymerase I have been well established (Steitz, T. A. (1998) Nature 391, 231-232). We now demonstrate that the participation of at least one additional carboxylate, a glutamate at position 710 or 883, is obligatory for catalysis. This conclusion has been drawn from our investigation of the properties of single (E710D, E710A, E883D, and E883A) and double (E710D/E883D and E710A/E883A) substitutions of residues Glu(710) and Glu(883). While single substitutions of either of the glutamates resulted in some reduction in polymerase activity, the mutant enzyme with simultaneous substitution of both glutamates with alanine exhibited a nearly complete loss of activity. Interestingly, substitution with two aspartates in place of the glutamates resulted in an enzyme species that catalyzed DNA synthesis in a strictly distributive mode. Pyrophosphorolytic activity of the mutant enzymes reflected their polymerase activity profiles, with markedly reduced pyrophosphorolysis by the double mutant enzymes. Moreover, an evaluation of Mg(2+) and salt optima for all mutant enzymes of Glu(710) and Glu(883) revealed significant deviations from that for the wild type, implying a possible role of these glutamates in metal coordination as well as in maintaining the structural integrity of the active site.

Alanine↗
PubMed2000

p38 mitogen-activated protein kinase pathway protects adult rat ventricular myocytes against beta -adrenergic receptor-stimulated apoptosis. Evidence for Gi-dependent activation.

We have shown that stimulation of beta-adrenergic receptors (beta-AR) by norepinephrine (NE) increases apoptosis in adult rat ventricular myocytes (ARVMs) via a cAMP-dependent mechanism that is antagonized by activation of G(i) protein. The family of mitogen-activated protein kinases (MAPKs) is involved in the regulation of cardiac myocyte growth and apoptosis. Here we show that beta-AR stimulation activates p38 kinase, c-jun N-terminal kinases (JNKs), and extracellular signal-regulated kinase (ERK1/2) in ARVMs. Inhibition of p38 kinase with SB-202190 (10 micrometer) potentiated beta-AR-stimulated apoptosis as measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. SB-202190 at this concentration specifically blocked beta-AR-stimulated activation of p38 kinase and its downstream substrate MAPK-activated protein kinase-2 (MAPKAPK2). Pertussis toxin, an inhibitor of G(i)/G(o) proteins, blocked the activation of p38 kinase and potentiated beta-AR-stimulated apoptosis. Activation of G(i) protein with the muscarinic receptor agonist carbachol protected against beta-AR-stimulated apoptosis. Carbachol also activated p38 kinase, and the protective effect of carbachol was abolished by SB-202190. PD-98059 (10 micrometer), an inhibitor of ERK1/2 pathway, blocked beta-AR-stimulated activation of ERK1/2 but had no effect on apoptosis. These data suggest that 1) beta-AR stimulation activates p38 kinase, JNKs, and ERK1/2; 2) activation of p38 kinase plays a protective role in beta-AR-stimulated apoptosis in cardiac myocytes; and 3) the protective effects of G(i) are mediated via the activation of p38 kinase.

Animals↗
PubMed2000

Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency.

Osteopontin (OPN) is a sialic acid-rich, adhesive, extracellular matrix (ECM) protein with Arg-Gly-Asp cell-binding sequence that interacts with several integrins, including alpha(v)beta(3). Since the ECM is a key regulator of mammary gland morphogenesis, and mammary epithelial cells express OPN at elevated levels, we sought to determine whether this protein plays a role in the postnatal mammary gland development. By generating transgenic mice that express OPN antisense-RNA (AS-OPN mice) in the mammary epithelia we achieved suppression of OPN production in this organ. The pregnant AS-OPN mice displayed a lack of mammary alveolar structures, a drastic reduction in the synthesis of beta-casein, whey acidic milk protein, and lactation deficiency. In agreement with these findings, we uncovered that a mammary cell line, NMuMG, which undergoes both structural and functional differentiation on ECM-coated plates, when transfected with an antisense OPN-cDNA construct, failed to undergo such differentiation. Furthermore, the results of gel-invasion assays demonstrated that these cells manifest elevated matrix metalloproteinase (MMP) activity when OPN expression is significantly reduced. The identity of this proteinase as MMP-2 is confirmed by Western blotting, zymography, and inhibition of its activity by a specific inhibitor, TIMP-2. Taken together, our results demonstrate, for the first time, an essential role of OPN in mammary gland differentiation and that the molecular mechanism(s) of its action, at least in part, involves down-regulation of MMP-2.

Animals↗
PubMed2000

Structure and regulation of opioid receptors.

Significant advances have been made in understanding the structure, function, and regulation of opioid receptors and endogenous opioid peptides since their discovery approximately 25 years ago. This review summarizes recent studies aimed at identifying key amino acids that confer ligand selectivity to the opioid receptors and that are critical constituents of the ligand binding sites. A molecular model of the delta receptor based on the crystal structure of rhodopsin is presented. Agonist-induced down regulation of opioid receptors is discussed, highlighting recent evidence for the involvement of the ubiquitin/proteasome system in this process.

Amino Acid Sequence↗
PubMed2000

Myocardial-directed overexpression of the human beta(1)-adrenergic receptor in transgenic mice.

The beta(1)-adrenergic receptor (AR) is the dominant subtype in non-failing and failing myocardium. beta(1)-AR signaling, by the endogenous neurotransmitter norepinephrine, is central to the regulation of myocardial contractility. In heart failure, the beta(1)-AR undergoes subtype-selective downregulation which may protect against the increased cardiac adrenergic drive associated with this pathophysiological state. To examine the hypothesis that chronically increased beta(1)-AR mediated signaling has adverse myocardial effects, transgenic mice overexpressing the human beta(1)-AR in a cardiac-selective context were produced, utilizing an alpha-myosin heavy chain (MHC) promoter. In these mice, beta(1)-AR protein abundance was approximately 24-46-fold (1-2 pmol/mg protein) that of wild-type mice. Histopathological examination of young (4 months old) and old (approximately 9 months old) transgenic mouse hearts consistently demonstrated large areas of interstitial replacement fibrosis, marked myocyte hypertrophy and myofibrilar disarray. In addition, increased expression of the pre-apoptotic marker, Bax, was observed coincident with regions of fibrosis accompanied by an increased apoptotic index, as measured by TUNEL assay. Older non-transgenic mice exhibited a slight tendency towards a decreased fractional shortening, whereas older beta(1)-AR transgenic mice had a marked reduction in fractional shortening (%FS approximately 30) as determined by echocardiography. Additionally, older beta(1)-AR transgenic mice had an increased left ventricular chamber size. In summary, cardiac-directed overexpression of the human beta(1)-AR in transgenic mice leads to a significant histopathological phenotype with no apparent functional consequence in younger mice and a variable degree of cardiac dysfunction in older animals. This model system may ultimately prove useful for investigating the biological basis of adrenergically-mediated myocardial damage in humans.

Animals↗
PubMed2000

National immunization day to assess nutritional status of underfives in Chandigarh.

National Immunization Days (NIDs) are an additional opportunity to reach a large number of children and can be used to undertake additional activities beside immunization. This study highlights an attempt to assess nutritional status of under five children by using a NID. Seven thousand four hundred and thirteen underfives were selected randomly from urban, rural and slums areas of Chandigarh. Nutritional status was assessed by using weight for age criteria. Prevalence of protein energy malnutrition (PEM) was found to be about 42% while 22.7%, 14.5%, 4.1% and 0.7% children had grade I, II, III and IV PEM respectively. The prevalence of PEM was significantly higher among females (47.6%), in 1-3 years age group (53.80%), in slum area (67%) and children of labour class (60.5%) (p < 0.001). With increase in family size, the prevalence of malnutrition also significantly increased, and decreased with high literacy rate in parents (p < 0.001). The prevalence of PEM in present study (42%) was comparable to a community based survey (51.6%) among pre-school children of an ICDS block of Chandigarh. National immunization days can be successfully used to assess nutritional status of children. This approach can be used by others for screening common childhood problems, preparing normogram for a region or country, administering Vitamin A and educating mothers.

Child, Preschool↗
PubMed2000

Effect of different combinations of MGK-264 or piperonyl butoxide with plant-derived molluscicides on snail reproduction.

Effect of sublethal treatment (20% and 60% of LC(50)/24 h) of plant-derived molluscicides, viz. Polianthes tuberosa, Trachyspermum ammi, Allium sativum powder; Azadirachta indica oil; oleoresin of Zingiber officinale; and their active molluscicidal component in combination (1:5) with MGK-264 or piperonyl butoxide on the reproduction of snail Lymnaea acuminata have been studied. It was observed that the combination of plant derived molluscicide and their active molluscicidal components, viz. tigogenin, hecogenin, azadirachtin, allicin, thymol, and [6]-gingerol combination with MGK-264 or piperonyl butoxide caused a significant reduction in fecundity, hatchability, and survival of young snails. Withdrawal of snails to fresh water after the above treatment caused a significant recovery in the fecundity of L. acuminata.

Animals↗
PubMed2000

Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation.

The mechanisms that regulate cardiac myocyte apoptosis are not well understood. To study the role of protein phosphatase 1 (PP1) and 2A (PP2A) in apoptosis, we exposed cultured neonatal rat cardiac myocytes to the phosphatase inhibitor okadaic acid (OA). Exposure (18 h) to 100 nM OA (a concentration which inhibits both PP1 and PP2A) decreased the number of adherent cells, caused genomic DNA fragmentation, and increased the percentage of apoptotic cells. These effects did not occur at a lower concentration of OA (1 nM) which is relatively specific for PP2A. Stimulation of alpha1- or beta-adrenergic receptors with norepinephrine (NE) in the presence of propranolol or prazosin partially blocked OA-induced apoptosis as measured by flow cytometry. Likewise, stimulation of adenylyl cyclase with forskolin reduced OA-induced apoptosis. Conversely, inhibition of protein kinase A with H89 or protein kinase C with chelerethrine potentiated OA-induced apoptosis. OA increased caspase-3 activity, and this effect was reduced by NE. Thus, inhibition of PP1 stimulates apoptosis in NRVM and stimulation of adrenergic receptors protects against OA-induced apoptosis.

Animals↗
PubMed2000

Emerging perspectives on glaucoma: highlights of a roundtable discussion.

PURPOSE: To present emerging perspectives on open-angle glaucoma related to patient compliance, current drug therapy, and treatment options. METHODS: Roundtable discussion. RESULTS: The panel discussion identified the following issues about patient compliance:1. It is a common problem in clinical practice and is often underappreciated by physicians.2. It is difficult for clinicians to detect.3. There are several risk factors that increase the probability of noncompliance. 4. Compliance can be improved through education, communication, improvement of the doctor-patient relationship, compliance aids, and simplifying the treatment regimen. CONCLUSION: Patients' compliance with the therapeutic regimen for open-angle glaucoma is very important to the eventual outcome of their disease treatment. Physicians can improve their patients' compliance by a few simple, cost-effective interventions in the clinical setting. When considering different agents as first-line therapies, once-a-day dosing would be optimal for enhancing compliance.

Communication↗
PubMed2000

Adrenergic regulation of myocardial apoptosis.

Increased sympathetic nerve activity to the myocardium is a central feature in patients with heart failure. Norepinephrine, the primary transmitter of the sympathetic nervous system, signals via binding to alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that beta-AR can stimulate apoptosis. Likewise, in transgenic mice overexpression of beta 1-AR or G alpha s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Whereas beta 1-AR stimulate apoptosis in vitro and in vivo, beta 2-AR may either stimulate or inhibit apoptosis and myocardial failure depending on the level of expression. Receptors coupling to Gi and Gq may also be able to mediate or modulate apoptosis and the development of myocardial failure, suggesting the potential for interactions between the beta-AR system and numerous remodeling stimuli that act through Gi or Gq signaling pathways. It appears likely that the mitogen-activated protein kinase superfamily plays a key role in mediating the actions of adrenergic pathways on myocyte apoptosis. These observations suggest that the adrenergic nervous system plays an important role in the regulation of myocyte apoptosis, and may thus contribute to the development of myocardial failure.

Adrenergic beta-Agonists↗