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Biomedical subjects

S Raghavan

Publications and source records attributed to S Raghavan.

At least 55 records · Page 3Linked to original sources
PubMed1994

Pathogenesis of Shigella diarrhea: XVII. A mammalian cell membrane glycolipid, Gb3, is required but not sufficient to confer sensitivity to Shiga toxin.

Shiga toxin recognizes a galactose-alpha 1-->4-galactose terminal glycolipid, globotriaosylceramide (Gb3), in sensitive mammalian cells and is translocated by endocytosis to the cytoplasm, where it blocks protein synthesis. To determine if Gb3 is both required and sufficient for toxicity, Gb3 content in cells was altered by blocking key biosynthetic or degradative path enzymes with specific inhibitors. The resulting decrease or increase in cellular Gb3 was associated with a decrease or increase in binding of and response to Shiga toxin. Toxin-resistant Gb3-deficient variants of sensitive cells fused with liposomes containing Gb3 but not globotetraosylceramide (Gb4) became susceptible, whereas fusion of Gb3 liposomes to naturally resistant Gb3-deficient CHO cells increased toxin binding but not cytotoxicity. These data demonstrate that Gb3 is required, but not sufficient, for the action of Shiga toxin and suggest the existence of a toxin translocation mechanism linked to surface glycolipids that is not expressed in CHO cells.

Animals↗
PubMed1993

Gentamicin sulfate attachment and release from anodized Ti-6A1-4V orthopedic materials.

A novel method has been developed to attach, retain, and release antibiotics from titanium based materials. This technique consists of forming porous surface coatings by anodizing and using the surface chemical properties of the oxide coatings to attach antibiotics. Coatings with pores in the size range 0.1-0.5 micron have been formed in acid solutions. The attachment and retainment of gentamicin sulfate, a cationic antibiotic, to the coatings has been investigated using microbiological methods. In vitro test results have shown that the duration of antimicrobial activity on the surface of anodized materials is dependent on the porosity and isoelectric point of the coatings. Using microporous oxide coatings formed in phosphoric acid solutions, it has been found that antimicrobial activity could be retained for more than 2 weeks.

Alloys↗
PubMed1993

Purification and immunological characterization of acid beta-galactosidase from dog liver.

1. Dog liver acid beta-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC. 2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180-190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa. 3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated beta-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat. 4. Western blot analysis of tissue extracts from dogs affected with GM1-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.

Animals↗
PubMed1992

Characterization of 6-hexadecanoylamino-4-methylumbelliferyl-beta-D- galactopyranoside as fluorogenic substrate of galactocerebrosidase for the diagnosis of Krabbe disease.

6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGal) has been shown to be a specific fluorogenic substrate of galactocerebrosidase and to facilitate the simple enzymatic diagnosis of Krabbe disease in human patients and in twitcher mice. HMGal hydrolysis at pH 4.5 is optimally stimulated by sodium taurocholate (0.25%) and oleic acid (0.05%) with a Km of 0.150, 0.04 and 0.03 mM, respectively for control mouse kidney, human fibroblasts and leukocytes. In control samples, the specific activity (nmol/mg prot./h) for HMGal is higher than for the natural substrate, galactocerebroside, and is severely deficient in the twitcher mouse and in patients with Krabbe disease. Comparative investigation of galactocerebrosidase activity in fibroblasts, leukocytes and brain with radioactive and fluorogenic substrates reveals a good agreement between the results of the two methods. Galactocerebroside (Gal-Cer) is a competitive inhibitor of HMGal hydrolysis in mouse kidney homogenates while GM1-ganglioside has no inhibitory effect in the same assay system. The sensitivity and specificity of this fluorogenic substrate for galactocerebrosidase provides a simple and rapid method for the diagnosis of Krabbe disease, and for the purification of this enzyme from normal tissues.

Animals↗
PubMed1991

Late-onset Krabbe disease (globoid cell leukodystrophy): clinical and biochemical features of 15 cases.

The diagnosis of late-onset variants of Krabbe disease (globoid cell leukodystrophy) has been facilitated by the recognition of galactocerebrosidase deficiency as its biochemical hallmark. Fifteen patients, ages 4-73, are presented. Signs included pes cavus, optic disc pallor, progressive spastic tetraparesis, a sensorimotor demyelinating neuropathy and hypodense lesions in the parieto-occipital periventricular white matter. Although intellect was preserved in more than half the cases, significant intrafamilial variability in mental functioning was encountered in 3 families. Bone marrow transplantation was successful in 1 13-year-old girl, but caused the death of 2 teenage twin sisters.

Adolescent↗
PubMed1990

Somatic cell genetic analysis of the galactocerebrosidase gene: lack of complementation in human Krabbe disease/twitcher mouse cell hybrids.

The inherited deficiency of galactosylceramide beta-galactosidase (E.C. 3.2.1.46: galactocerebrosidase) activity results in globoid cell leukodystrophy in humans (Krabbe disease) and in mice (twitcher mutant). To determine whether Krabbe patients' cells complement twitcher cells to produce, in hybrid combination, greater than deficient levels of galactocerebrosidase activity, five separate crosses were made between an established twitcher mouse cell line and five cell strains from unrelated Krabbe disease patients. A total of 57 twitcher mouse/Krabbe somatic cell hybrid lines developed from all of these crosses were deficient in galactocerebrosidase activity despite the presence of human chromosomes 14 or 17, which have been previously implicated as bearing the galactocerebrosidase gene. A control cross between twitcher mouse/positive control human fibroblasts resulted in 14 of 21 independent hybrid lines that expressed higher than deficient levels of galactocerebrosidase activity. The lack of complementation between Krabbe disease patient and twitcher mutant mouse cells provides further evidence that the twitcher mouse is an authentic murine model for Krabbe disease and supports the hypothesis that the mutations in both species are within the structural gene for the galactocerebrosidase enzyme.

Animals↗
PubMed1989

New insights into lactase and glycosylceramidase activities of rat lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase, a small intestinal disaccharidase, has been considered mainly an enzyme important only for the hydrolysis of lactose. After weaning in most mammals lactase-specific activity falls markedly, and, functionally, adult mammals are considered to be lactase deficient. However, the persistence of low levels of lactase activity in adulthood has never been explained. In addition, it has been suggested that lactase-phlorizin hydrolase is associated with glycosylceramidase activity when the enzyme is prepared by column chromatography, but it is unclear whether this represents copurified activities or two catalytic sites on one peptide. The developmental patterns of lactase-phlorizin hydrolase and other disaccharidases were investigated in homogenates of total rat small intestine; lactase and several glycosylceramidases were measured in immunoprecipitates from these homogenates using a monoclonal antibody. The developmental pattern of total lactase activity showed a steady 2.3-fold increase to adult levels (specific activity decreased eightfold), whereas total phlorizin-hydrolase activity increased 10.7-fold (specific activity decreased threefold). As expected, levels of both total and specific sucrase and maltase activities increased during development. In lactating rats total lactase activity showed a significant increase compared with adult males. The developmental pattern of the enzyme activities for the glycolipid substrates was similar to that found for lactase, and the immunoprecipitated enzyme showed a 40- to 55-fold higher affinity for the glycolipids than for lactose. Galactosyl- and lactosylceramide inhibited lactose hydrolysis by 38%, without a competitive pattern, suggesting two different active sites for lactose and glycolipid hydrolysis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Galactocerebrosidase activity in somatic cell hybrids derived from twitcher mouse/control human fibroblasts is associated with human chromosome 17.

Somatic cell hybrids derived from twitcher mouse cells and from control human fibroblasts were selected by two different methods. One method utilized 6-thioguanine-resistant twitcher cells as a parental line and the other used neomycin-resistant control human fibroblasts as a parental line so that hybrid lines could be selected in either HAT or in G-418 medium, respectively. The hybrid lines were analyzed for galactocerebrosidase activity. Since the twitcher cell lines are deficient in galactocerebrosidase activity, the presence of this activity in these hybrid lines depends upon the presence of human chromosome contents. Both galactocerebrosidase-positive and -deficient hybrid lines were analyzed for their human chromosome contents by the use of isozyme markers. In hybrids derived from both selection methods the expression of galactocerebrosidase activity was associated with the presence of human chromosome 17 marker isozymes. This was confirmed cytogenetically by means of trypsin-banded Giemsa staining of intact human chromosome 17 in three galactocerebrosidase-positive hybrid lines.

Animals↗
PubMed1988

Characterization of alpha-mannosidase in feline mannosidosis.

Acidic alpha-mannosidase deficiency has been identified in a family of Blue Persian cats. Characterization of the residual activity revealed that the Km for the substrate, 4-methylumbelliferyl-alpha-D-mannoside, increased approximately three-fold with a severe deficiency in Vmax (1-2%) in homogenates of liver and brain of affected cats compared with controls. The residual activity at pH 4.0 in liver homogenates from affected cats is very thermolabile at 51 degrees C while the control activity is stable at this temperature for 1 h. Subcellular fractionation of liver was performed from a control and diseased cat in order to compare the properties of the different alpha-mannosidases localized in these fractions. The residual activity present in the lysosomal fraction from diseased cat liver showed altered pH optimum, two-fold increase in Km with a severely reduced Vmax and increased thermolability compared with the activity in the lysosomal fraction from control liver. The thermal inactivation pattern and Km of the residual activity in the lysosomal fraction is different from the non-lysosomal alpha-mannosidase in the liver of the affected cat. This suggests that the residual activity in the lysosomal fraction of the liver from the affected cat is not due to contamination of non-lysosomal alpha-mannosidase in this fraction. Whether this residual activity represents the properties of the mutant enzyme or yet another minor normal component of lysosomes different from the major inactive mutant or absent lysosomal enzyme remains to be elucidated.

Animals↗
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↗