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Biomedical subjects

S Raghavan

Publications and source records attributed to S Raghavan.

71 records · Page 4Linked to original sources
PubMed1988

Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts.

Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of a twi+/twi X twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression.

Aminopterin↗
PubMed1987

Metabolic activities in human skin fibroblasts preloaded with labeled GM2-ganglioside.

Confluent cultures of human skin fibroblasts were maintained for 10 days with sphingosine labeled [3H]GM2. Labeled medium was then replaced with normal medium and the cells maintained for 42 days with weekly medium changes. Cells were harvested at regular intervals and cells, medium, and trypsin digest supernatant analyzed for [3H]GM2 and its metabolic products. The ganglioside can be membrane associated and removed by trypsin, or membrane incorporated and trypsin insensitive. The membrane incorporated material is apparently transported to the lysosomes slowly by membrane flow, where 80% of the cellular GM2 can be metabolized by day 42. [3H]GM2 as well as its metabolic products in control cells is continuously released into the medium, during which it can also become associated with the cell surface membrane. There is no detectable metabolism of the [3H]GM2 in GM2 gangliosidosis cell lines over the extended post-labeling period, indicating that there is no residual enzyme activity in these cells. Undegraded GM2 is continuously released into the medium and remains associated with the cell surface membrane as well.

Fibroblasts↗
PubMed1986

Optimal assay conditions for enzymatic characterization of homozygous and heterozygous twitcher mouse.

The neurological mouse mutant twitcher is characterized by a genetic deficiency of galactosylceramide beta-galactosidase (galcerase) (EC 3.2.1.46) which also represents lactosylceramide beta-galactosidase I (lactosidase I) activity. The assay conditions for both these activities in several mouse tissues have been optimized to facilitate the enzymatic characterization of homozygous and heterozygous twitcher mice. Galcerase in mouse tissues is optimally activated by 7.0 mg/ml of sodium taurocholate (pure) and 1.5-2.0 mg/ml of oleic acid in this system. When lactosylceramide is used as the substrate, no more than 1 mg/ml of taurocholate is appropriate in the assay, since higher concentrations of this pure bile salt stimulate another enzyme, lactosylceramide beta-galactosidase II (lactosidase II), which is unaffected in twitcher mice. At the optimized condition, lactosidase I in the twitcher mouse amounts to 3-4% of control activity in agreement with the residual galcerase (2%) in this mouse mutant. These assay conditions provide better sensitivity to discriminate heterozygotes from controls until 40 days of age from measurement of this activity in clipped tail samples.

Animals↗
PubMed1986

beta-Glucosidase inhibition in murine peritoneal macrophages by conduritol-B-epoxide: an in vitro model of the Gaucher cell.

Murine peritoneal macrophages were cultured in the presence of conduritol-B-epoxide, a specific covalent inhibitor of beta-glucosidase. The inhibition was found to be dose and time dependent. Upon removal of the inhibitor from the culture medium, beta-glucosidase activity recovered to half maximum by 2.2 days. Treatment of macrophages with this inhibitor for 15 days did not affect cell viability, lysosomal enzyme release to the medium, or levels of intracellular lysosomal enzymes, other than beta-glucosidase activity. This inhibition results in the accumulation of glucocerebroside. In vitro studies on the pathobiology of such macrophages whose beta-glucosidase activity has been reduced may be useful toward understanding the pathogenesis of Gaucher disease.

Animals↗
PubMed1985

GM2-ganglioside metabolism in cultured human skin fibroblasts: unambiguous diagnosis of GM2-gangliosidosis.

The metabolism of GM2-ganglioside was studied in situ using cultured skin fibroblasts from normal individuals and patients with different forms of GM2-gangliosidosis. [3H]Sphingosine-labeled GM2 was provided in the culture medium to confluent cells in 6-cm petri dishes. After 10 days, the cells were washed free of radioactivity and harvested by trypsinization. The cellular lipids were extracted and analyzed for radioactivity in GM2 and its metabolic products. In fibroblasts from healthy subjects, 50-60% of the total cellular radioactivity was found in the neutral glycosphingolipids, ceramide, sphingomyelin and fatty acids. Degradation of the labeled GM2 progressed rapidly via GM3, ceramide dihexoside and ceramide monohexoside with a build-up of radioactivity mainly in the ceramide pool of the cell. The labeled ceramide is also reutilized for the synthesis of ceramide trihexoside, globoside and sphingomyelin or is converted to fatty acid and incorporated in ester linkages. In contrast, cells from patients with GM2-gangliosidosis representing Tay-Sachs, Sandhoff and AB variant forms of the disease did not metabolize the ingested labeled GM2-like controls. Nearly all of the radioactivity was present in the ganglioside fraction in the lipid extracts from these cells and consisted of unhydrolyzed GM2. High-performance liquid chromatographic analysis of monosialogangliosides from cells grown without added labeled GM2 in the medium indicated accumulation of endogenously synthesized GM2 in cell lines from all patients with GM2 gangliosidosis compared to healthy controls. This approach provides a reliable tool for pre- and post-natal diagnosis of all forms of GM2-gangliosidosis without ambiguity.

Cells, Cultured↗
PubMed1982

Nonuniform deficiency of hexosaminidase A in tissues and fluids of two unrelated individuals.

Serum samples from two unrelated, clinically normal individuals lacked detectable hexosaminidase A by heat inactivation and electrophoretic analysis. In contrast, 15 and 17% of the hexosaminidase in their leukocytes and 23 and 26% of the hexosaminidase of their cultured fibroblasts had the heat stability and electrophoretic properties of the A form of this enzyme. An in vitro measurement of fibroblasts GM2 ganglioside-beta-galactosaminidase was in the range expected for Tay-Sachs disease (TSD) heterozygotes (2.5 and 3.1 versus a normal mean of 3.7). In contrast, fibroblasts from a patient with TSD, analyzed in an identical fashion, contained no detectable activity. Ten days after addition of labeled GM2 ganglioside to the medium of the cultured fibroblasts, 43 and 59% of the radioactivity taken up by the cells of these patients remained as unhydrolyzed ganglioside as compared with 94% in TSD fibroblasts and 42% in control cells. An analysis of sphingolipid composition by high performance liquid chromatography although the endogenous level of GM2 was elevated in TSD fibroblasts (0.39 nmoles/mg protein) there was no increase in the cells of these patients (0 and 0.12 versus control of 0.17 nmoles/mg protein). Finally, the synthesis of hexosaminidase was examined by an electrophoretic analysis of immunoprecipitates of the enzyme precursors that had been radiolabeled by culturing fibroblasts in medium containing [3H]-leucine. These studies revealed a normal pattern of biosynthesis, processing and secretion of the alpha and beta chains. The ratio of the alpha chain to the beta chain, however, was in the range expected for TSD heterozygotes.

Adult↗
PubMed1981

Reduction of postprandial plasma glucose by Bengal gram dal (Cicer arietinum) and rajmah (Phaseolus vulgaris).

Postprandial plasma glucose levels were measured in six healthy subjects at 0, 15, 30, 45, 60, 90, and 120 min after taking 50 g of carbohydrate in the form of wheat, rice, bengal gram dal (channa dal), and rajmah (red kidney beans) and compared with the plasma glucose values obtained after taking 50 g dextrose. Bangal gram dal and rajmah, when compared with dextrose, were found to be more effective in reducing postprandial plasma glucose levels than wheat and rice. Moreover, the mean peak rise in plasma glucose was decreased by 82.1% with bengal gram dal, 67% with rajmah, while wheat and rice showed reduction only by 25 and 16%, respectively, when compared with dextrose. This study suggests a reappraisal of the diet for diabetics.

Adult↗
PubMedDate not supplied

Perinatal outcome in relation to mode of delivery in meconium stained amniotic fluid.

Meconium staining of the amniotic fluid is a common complication during labour. When facilities like electronic monitoring, foetal blood sampling are not available, it is difficult to decide whether labour should be allowed to continue or caesarean section should be done. Even when caesarean section is done, meconium aspiration syndrome (MAS) can still occur and considerable morbidity and mortality may result in the newborn. Amino infusion is being considered as useful in decreasing MAS and its sequelae. Before resorting to amino infusion, we decided to analyse the perinatal outcome in meconium stained liquor to compare whether early caesarean section offered any advantage. This is a retrospective study of 150 labours complicated by thick meconium stained liquor, during a 12 month period (1992-93).

Asphyxia Neonatorum↗
PubMedDate not supplied

Biological monitoring among benzene-exposed workers in Bangalore city, India.

Environmental and biological monitoring was carried out in the winter season of 2004 for 30 gasoline station workers (study subjects) and 30 office workers (controls) of Bangalore city, India. Personal air sampling was carried out in the breathing zone of workers using an Anasorb CSC sorbent tube (SKC 226-01) fitted to the low-flow personal samplers (PCXR4 and pocket pump Model No. 210-1002) at a flow rate of 200 ml min(-1) during the shift work of 8 h. The benzene content adsorbed in the sorbent tube (SKC 226-01) was desorbed with 1 ml of benzene-free carbon disulfide on a developing vibrator and later analysed by Trace GC fitted with MXT-624 column and flame ionization detector. The mean time weighted average benzene concentration found among study and controls was 1.10+/-1.08 and 0.070+/-0.035 mg m(-3), respectively. Biological monitoring for benzene exposure was performed by measuring trans,trans muconic acid (t,t-MA) in the end shift urine samples using HPLC-UV technique. End-shift urine samples (1 ml) were adjusted to pH 7-9 with phosphate buffer pH 7.4 passed through the preconditioned Q-SAX anion-exchange cartridge and the (t,t-MA) is extracted with 10% acetic acid and later analysed by HPLC-UV detection The mean t,t-MA found among study and controls were 563.16+/-281.81 and 266.88+/-110.65 microg g(-1) creatinine. About 50% of the study subjects (15) have higher t,t-MA values than the biological exposure index of the American Conference of Government Industrial Hygienist (ACGIH). Correlation is significant at 5% level (p<0.05) between personal air benzene concentration and urinary t,t-MA in the study group. Based on these findings, the t,t-MA can be used as a biomarker for benzene exposure.

Benzene↗
PubMedDate not supplied

Antigen-specific in vitro suppression of murine Helicobacter pylori-reactive immunopathological T cells by CD4CD25 regulatory T cells.

A Helicobacter pylori-specific in vitro coculture system was established and used to study the role of CD4+CD25+ regulatory T cells (Treg) in gastritis development in mice with H. pylori infection. Effects of therapeutic immunization against H. pylori infection on the Treg function were also studied to better understand the mechanisms leading to postimmunization gastritis in these mice. Depletion of Treg led to extensive proliferation to H. pylori antigens of CD4+ T cells isolated from either naïve, H. pylori-infected or H. pylori-immunized mice. Using the Treg-depleted CD4+ T cells from immunized mice as effector cells, we compared the suppressive efficacy of Treg isolated from naïve, infected or immunized mice and found that Treg from naïve mice, and slightly less efficiently from infected mice, suppressed the CD25- effector T-cell response and in most cases were distinctly more efficacious than Treg isolated from immunized mice. The suppressive efficacy of Treg isolated from the differently treated mice correlated closely with production of interleukin-5 (IL-5) by the Treg and suppression of interferon-gamma and IL-2 production by the CD25- effector T cells. Our study is the first to demonstrate in H. pylori-induced chronic infection, antigen-specific Treg with differential efficacy in suppressing H. pylori proinflammatory T effector cells.

Animals↗