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Biomedical subjects

N Garg

Publications and source records attributed to N Garg.

At least 37 records · Page 2Linked to original sources
PubMed1999

Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis.

The possible role of the central beta-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar "partner" human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SK(KK256.257AA) (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SK(RE248.249AA) and SK(EK281.282AA), referred to as SK(C) and SK(H), respectively] showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SK(KK256.257EE) [SK(D2)]. This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a "burst" of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once "supplied" with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SK(RE24249AA:EK281.282AA) [SK(CH)], SK(EK272.273AA;EK281.282AA) [SK(FH)], and SK(RE248.249AA;EK272.273AA:EK281.282AA+ ++) [SK(CFH)], showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective "single-site" mutants, with the "triple-site" mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK beta-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex.

Amino Acid Sequence↗
PubMed1998

Pressure zone used and the occurrence of mitral regurgitation in Inoue balloon mitral commissurotomy.

Mitral regurgitation (MR) is a known complication of Inoue balloon mitral commissurotomy (BMC) and has been variously ascribed to the presence of severe subvalvular pathology (SVP), preexisting MR, calcification, or oversizing. The pressure zone used--with the low pressure zone (LPZ) the lower half of the spectrum of sizes available out of a single balloon, and the high pressure zone (HPZ) the upper two levels, i.e., within 2 mm of its maximum size--could have a bearing on the occurrence of MR, but has not been studied before. We analysed 251 consecutive patients (mean age 28.6 + 9.7 years), undergoing BMC from October 1993 onwards, with pliable, non-calcific, splittable (bilateral dark zones present) valves with not more than trivial MR (1 + in grades of 1-4). Balloon sizing was done with standard formula using height with stepwise dilatation starting 2 mm below the reference size. Thirty-two patients additionally had severe SVP. Patients were divided into two groups, HPZ-BMC and LPZ-BMC, depending upon the final balloon size needed for a successful result. Incidence of MR (2+ or more) was significantly lower in the LPZ BMC (18%) vs. HPZ BMC (32.2%) (P < 0.05). Moderate to severe MR (3+/4+) was also less in LPZ BMC (2.8%) vs. HPZ BMC (8.2%) (P < 0.05). Amongst patients with severe SVP, 3/15 (20%) developed MR in the LPZ-BMC group (all mild only) as against 8/17 (42%) (P < 0.05) in the HPZ-BMC group with half of them having moderate to severe MR. In 54 patients where the reference size had to be exceeded, no patient (0/8) developed MR as long as the higher size was in the LPZ of the particular balloon used as compared to 17/46 (36.9%) who developed MR when the size used fell in the HPZ. We conclude that the pressure zone used has a strong bearing on the occurrence of MR in Inoue BMC and that a low-pressure strategy could avoid MR.

Adult↗
PubMed1998

Vaccination with trypomastigote surface antigen 1-encoding plasmid DNA confers protection against lethal Trypanosoma cruzi infection.

DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2(b) and H-2(d) mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2(b) mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2Kb-restricted TSA-1515-522 epitope. In H-2(d) mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8(+)-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2(b) and 16 of 18 (89%) H-2(d) mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas' disease.

Animals↗
PubMed1997

Proteins with glycosylphosphatidylinositol (GPI) signal sequences have divergent fates during a GPI deficiency. GPIs are essential for nuclear division in Trypanosoma cruzi.

Glycosylphosphatidylinositols (GPIs) are membrane anchors for cell surface proteins of several major protozoan parasites of humans, including Trypanosoma cruzi, the causative agent of Chagas' disease. To investigate the general role of GPIs in T. cruzi, we generated GPI-deficient parasites by heterologous expression of T. brucei GPI-phospholipase C. Putative protein-GPI intermediates were depleted, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway. Cell surface expression of major GPI-anchored proteins was diminished in GPI-deficient T. cruzi. Four proteins that are normally GPI-anchored in T. cruzi exhibited different fates during the GPI shortage; Ssp-4 and p75 were secreted prematurely, while protease gp50/55 and p60 were degraded intracellularly. These observations demonstrate that secretion and intracellular degradation of GPI-anchored proteins may occur in the same genetic background during a GPI deficiency. We postulate that the interaction between a protein-GPI transamidase and the COOH-terminal GPI signal sequence plays a pivotal role in determining the fate of these proteins. At a nonpermissive GPI deficiency, T. cruzi amastigotes inside mammalian cells replicated their single kinetoplast but failed at mitosis. Hence, in these protozoans, GPIs appear to be essential for nuclear division, but not for mitochondrial duplication.

Animals↗
PubMed1997

Delivery by Trypanosoma cruzi of proteins into the MHC class I antigen processing and presentation pathway.

Class I MHC-restricted T cell responses have been shown to be critical for the development of immune resistance to Trypanosoma cruzi in mice. However, to date, no antigenic targets of this anti-parasite response have been characterized. We have analyzed the characteristics of potential T. cruzi CTL target molecules by expression of the model CTL target molecule chicken OVA in different cellular compartments of T. cruzi. OVA (amino acids 139-385) was expressed as a secretory, cytoplasmic, transmembrane, or glycosylphosphatidylinositol-anchored protein in T. cruzi transfectants. Host cells infected with T. cruzi transfectants that secreted or released OVA, but not those producing cytoplasmic or transmembrane forms of OVA, could process and present OVA peptide via the class I MHC pathway, as indicated by the stimulation of OVA-specific CD8+ T cell hybridomas and the cytolysis of host cells infected with OVA-secreting parasites by OVA-specific CTLs. In addition, infection of mice with OVA-secreting parasites elicited the production of OVA-specific CTLs. These studies demonstrate the ability to target proteins to specific cellular compartments in T. cruzi using either trypanosomal or mammalian signal sequences. Furthermore, these results suggest that proteins secreted or released by T. cruzi in infected cells are a major source of peptides for MHC class I presentation and for the generation of parasite-specific CTL.

Amino Acid Sequence↗
PubMed1997

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes.

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.

Animals↗
PubMed1997

The identification and molecular characterization of Trypanosoma cruzi amastigote surface protein-1, a member of the trans-sialidase gene super-family.

An accumulating body of evidence suggests that T. cruzi-infected host cells are recognized and destroyed by class I major histocompatibility complex (MHC) restricted CD8+ T-cells thus contributing to immune control of the infection [1-6]. However, to date, only a few amastigote proteins which could be the target of this response have been described and gene sequence information is available only for the amastins [7]. In order to identify amastigote proteins which could contribute to immune detection of infected host cells, a panel of monoclonal antibodies specific for amastigote proteins was produced and screened. Three mAbs (IIIC4, VIIC1 and IIID4) were identified which recognized amastigote surface proteins of 78, 26 and 53 kDa, respectively. Screening of an amastigote cDNA expression library with mAb IIIC4 resulted in the isolation of a 2.8 Kb clone. pSI2. The derived amino acid sequence indicates that the pSI2 clone encodes an amastigote surface protein belonging to the T. cruzi trans-sialidase super-family. Based on its preferential expression in the amastigote stage we have named this protein amastigote surface protein-1 (ASP-1). ASP-1 contains the third and fourth Asp block motifs, SxDxGxTW and the fibronectin type III-like domain, VTVxNVxLYNR, thus placing it in family II of the T. cruzi trans-sialidases [8]. ASP-1 is the first trans-sialidase family member shown to be preferentially expressed in the amastigote stage of the T. cruzi life cycle. This expression of ASP-1 on parasites in infected cells and its apparent membrane attachment by a glycosylphosphatidylinositol (GP1)-anchor makes it a prime candidate to enter the class I MHC processing and presentation pathway.

Amino Acid Sequence↗
PubMed1996

Polymer-shielded dye-ligand chromatography of lactate dehydrogenase from porcine muscle in an expanded bed system.

Dye-affinity chromatography is a widely used technique in protein purification. It has recently been shown that the efficiency of the chromatography process can be significantly improved by pretreatment of the affinity matrix with certain water soluble polymers such as poly(vinyl pyrrolidone). This technique termed as polymer-shielded, dye-affinity chromatography has been successfully used in packed bed mode at a lab scale for the purification of a number of enzymes. The present work deals with the application of polymer-shielded dye-affinity chromatography in an expanded bed system of Streamline-Cibacron Blue 3GA for the isolation of lactate dehydrogenase from a crude porcine muscle extract. The elution conditions were optimised to obtain an efficient process. A higher recovery of the target enzyme (78%) was obtained from the polymer shielded column as compared to the unshielded column (17%), after low ionic strength elution. The purification factor obtained after chromatography on the polymer shielded column was higher (4.1) than that from unshielded column (1.8).

Animals↗
PubMed1995

Photo-osmosis through liquid membrane bilayers. Studies on mixture of bacteriorhodopsin with cytochrome-C, myoglobin, or hemoglobin.

The effect of cytochrome-C, hemoglobin, and myoglobin on photo-osmosis through liquid membrane bilayers generated by bacteriorhodopsin (BR) has been studied. The magnitude of photo-osmotic velocity was found to be much greater when BR was combined with any one of the three pigments than that of BR alone. This has been because of the exclusion of protons and electrons in the illuminated compartment by the action of light, where one acts as the acceptor for the others. The rate of light-induced volume flux of the combined system depends on temperature, intensity, and wavelength of incident light, and the nature and concentration of electron donors and acceptors.

Bacteriorhodopsins↗
PubMed1995

Antiallergic activity of alkyl substituted pyrazolo[3, 4-d]pyrimidine (compound 88-765).

Compound 88-765 (4-amino-6-methylthio-1-(2', 2'-diethoxyethyl)-1 H-pyrazolo[3, 4-d]pyrimidine) has shown potent antiallergic activity in experimental models. The compound inhibited the passive cutaneous anaphylaxis (PCA) reaction in rats in dose-dependent manner (5-100 mg/kg, po) by 47 to 87%. In mice it inhibited PCA by 78% at 50 mg/kg, po. It also inhibited mast cell degranulation of normal and passively sensitised rats induced by compound 48/80 and egg albumin, respectively. These effects of Compound 88-765 were comparable with that of disodium cromoglycate (DSCG). The results suggest that compound 88-765 possesses potent antiallergic activity.

Animals↗
PubMed1994

Interaction of Cibacron blue with polymers: implications for polymer-shielded dye-affinity chromatography of phosphofructokinase from baker's yeast.

Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the dye. The difference spectra for the PVP-dye complex was typical of "electrostatic interaction spectra" at low ionic strength and typical of "hydrophobic interaction spectra" in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One dye molecule was bound by a PVP segment with a molecular mass of 1000-1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One dye molecule was bound by a polymer segment with a molecular mass of about 10,000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded dye-affinity chromatography. This concept was successfully applied to the chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.

Chromatography, Affinity↗
PubMed1992

Respiratory symptoms and ventilatory capacity in metal polishers.

To evaluate the long-term effects of metal dusts on the bronchopulmonary system and the synergistic effect of cigarette smoke, a comparative study of spirometric measurements in 104 polishers and 90 unexposed controls was carried out in 25 brass and steelware polishing industries at Moradabad in northern India. The two groups were comparable in terms of age, height, smoking habit and socio-economic status. A total of 58.6% of the polishers had one or more respiratory symptoms, compared to only 25.5% of the controls (P < 0.05). Chronic cough was present in 21 polishers (20.2%) as compared to 11.1% of the controls. However, this difference was insignificant. Chronic phlegm was nearly three times as frequent among the polishers as among the controls (17.5% vs 4.4%) (P < 0.005). The prevalence of dyspnoea of varying grades was also significantly higher (16.3% as opposed to 4.4%) among the exposed groups. Chronic bronchitis (6.7%) and occupational asthma (4.8%) were found to be confined to polishers. The polishers also experienced acute respiratory symptoms during the work shift. The prevalence of acute respiratory symptoms was recorded for cough in 19 workers (44.1%) followed by dyspnoea in 14 workers (32.5%) and throat irritation in 11 workers (25.5%). Comparison of the mean values of pulmonary function parameters in the polishers and the controls showed significant differences in the smoking and non-smoking groups (P < 0.001). The polishers exhibited significantly greater acute reductions in various lung functions over the work shift, particularly for forced expiratory flow over the 25-75% portion of the spirogram (FEF25-75%) FEF25% and FEF50%, than did the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Occupational morbidity among children employed in brassware industry.

Fifty seven male children between ages of 10-16 years engaged in the brassware industry at Moradabad in Northern India were studied for occupational morbidity. The finding were compared with those obtained in the children (n = 29) engaged in other ancillary units which did not involve exposure to the metal fumes and dust in their work environment. The study showed a high prevalence of respiratory morbidity in the children engaged in the main units in comparison to those employed in the ancillary units (40.3 vs 27.6%; p less than 0.05). This was associated with significantly higher prevalence of pulmonary impairment in the former group (21.0%) particularly demonstrating restrictive ventilatory abnormality (10.5%) followed by bronchial obstruction (7.0%). The high respiratory morbidity may be attributed to chronic exposure to the fumes and dust of the metals such as nickel, chromium and cadmium. The children employed in the ancillary as well as in the main units showed high prevalences of musculo-skeletal disorders (27.6 and 22.8%) which may be caused by sustained faulty posture adopted during work and physical stress.

Adolescent↗
PubMed1992

Blood chromium and nickel in relation to respiratory symptoms among industrial workers.

Seventy-eight workers exposed to fumes and dust of nickel and chromium in their occupation in the glass industry were studied for respiratory symptoms in relation to nickel and chromium concentrations in their blood. A significant (p less than 0.01) association was observed between respiratory symptoms and elevated blood nickel and chromium. An interaction between nickel and chromium was found in relation to the prevalence of respiratory symptoms.

Adolescent↗
PubMed1991

A cross-sectional study of pulmonary function among workers exposed to multimetals in the glass bangle industry.

Spirometric lung functions were evaluated in 220 asymptomatic glass bangle workers exposed to the salts of various heavy metals, such as arsenic, lead, zinc, copper, manganese, cobalt, cadmium, and selenium, which are used as coloring agents in the manufacture of glass bangles. The mean values of various spirometric variables (viz. FVC, FEV1, IMBC, and PEFR) were within normal range (more than 80% of the predicted values) in both smoking and nonsmoking glass bangle workers and did not reveal any significant differences in comparison with those observed in the unexposed controls. However, variables such as FEV1/FVC% ratio, FEF25-75 and FEF75-85 were reduced significantly (p less than 0.001) in the exposed group. The reduction was more marked in the smoking glass bangle workers indicating additive effect of cigarette smoking on the small airways. The exposed group showed a significantly higher prevalence (16.3%) of respiratory impairment in comparison to that observed in the controls (7.9%) as a result of exposure to various metals in the work environment (p less than 0.01). The respiratory impairment observed in the exposed group indicated primarily restrictive pattern of pulmonary abnormality (10.4%), while the controls revealed only 1.1% prevalence of this disease (p less than 0.001). The effect of the duration of exposure on the prevalence of respiratory impairment in the glass bangle industry revealed significantly higher prevalence (p less than 0.05) in those who worked for more than 10 years (23.0%) than in those who worked for less than 10 years (10.8%) thereby indicating that the duration of exposure is directly related to the prevalence of respiratory impairment.

Adult↗
PubMed1991

Production of Tsr factor by Rhizobium meliloti.

The root exudates of alfalfa (Medicago sativa) and mungbean (Vigna radiata) induced the Tsr (thick and short roots) factor production in Rhizobium meliloti. The factor caused a 30-40% reduction of root length in alfalfa seedlings. Pea root exudate had no Tsr induction activity. The flavonoid naringenin could replace the roots in inducing Tsr production. Naringenin-induced Tsr factor caused 70% shortening of main roots. The Tsr inducing property of naringenin was specific since quercetin and syringaldehyde had no such effect.

Bacterial Proteins↗