Biomedical subjects

C Bhuvaneswaran

Publications and source records attributed to C Bhuvaneswaran.

At least 19 recordsLinked to original sources

PubMed2000

Lysosome lipid storage disorder in NCTR-BALB/c mice: spleen and lung lysosomes store unesterified cholesterol but differ in their phospholipid composition.

A strain derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jefferson, AR, USA (NCTR-BALB/c) suffers from an autosomal recessive disorder characterized by proliferation of secondary lysosomes with accumulation ofunesterified cholesterol in several tissues. The unesterified cholesterol content of spleens and lungs from the affected mice were elevated 8- and 3-fold respectively over age- and sex-matched controls. Postnuclear supernatants of tissue homogenates were fractionated by sucrose density gradient centrifugation and the fractions were analyzed for unesterified cholesterol, protein and marker enzyme activities for lysosomes (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase), plasma membrane (alkaline phosphodiesterase I), endoplasmic reticulum (glucose-6-phosphatase) and mitochondria (cytochrome oxidase). The enzyme distribution profile showed that lysosomes of affected tissues floated at low density regions (density 1.05-1.08) of the gradient and contained substantial amount of tissue unesterified cholesterol. These low density lysosomes were purified about 17-fold (58% yield) from spleen and about 6-fold (32% yield) from lungs with minimal contamination by other organelles They were mostly intact as judged by high latency for N-acetyl-beta-D-glucosaminidase activity (70-100%). Lysosomes of control tissues were not found at the low density regions. The distribution profiles for other organelles were similar between affected and control tissues. Phospholipid composition of low density lysosomes were distinctly different from their respective tissue homogenates. Spleen and lung lysosomes were enriched in sphingomyelin and phosphatidylcholine respectively. The results suggest that these lysosomes acquire their low densities due to accumulation of unesterified cholesterol, the retention of which may be aided by sphingomyelin and phosphatidylcholine content of the lysosomes.

Animals↗

PubMed1999

Sphingomyelinase activity of livers from control and NCTR-BALB/c mice.

NCTR-BALB/c mice have an autosomal recessive disorder involving storage of sphingomyelin and unesterified cholesterol in their tissues and reduced tissue sphingomyelinase activity. With [N-methyl-14C]sphingomyelin as substrate, Vmax for the enzyme in livers from control and mutant mice were, respectively, 29.6 and 11.6 nmol of substrate hydrolyzed h(-1) mg(-1) protein, and the corresponding Km values were 94.6 and 132.3 microM. The control and mutant enzymes showed similar pH profiles and temperature sensitivities. When the control and mutant liver homogenates were mixed in various proportions, the resulting activities were 70-80% of the theoretical values. Cross and straight addition of total lipid extracts of control and mutant livers had minimal effect on their enzyme activities. The results suggest that the reduced sphingomyelinase activity of mutant liver is not due to the presence of inhibitors or absence of activators in this tissue.

Animals↗

PubMed1997

Activation of acyl-CoA: cholesterol acyltransferase in rat liver microsomes by 25-hydroxycholesterol.

25-Hydroxycholesterol stimulated acyl-CoA:cholesterol acyltransferase (ACAT) activity in rat liver microsomes in vitro with half-maximal stimulation at 16.8 microM oxysterol and a maximal activity that was three times that in its absence. The current study was conducted to determine the effect of 25-hydroxycholesterol on rates and extent of intervesicular cholesterol transfers within microsomes and to determine whether this activation of ACAT could be accounted for on the basis of increased cholesterol availability for the enzyme. Cholesterol transfer kinetics were assessed in systems that either enriched or depleted microsomal cholesterol. Incubation of microsomes at 37 degrees C with phosphatidylcholine:cholesterol liposomes or purified plasma membranes resulted in enrichment of microsomal cholesterol. Incubation of microsomes with just phosphatidylcholine liposomes resulted in depletion of cholesterol. The extent of cholesterol enrichment or depletion depended on incubation time and the initial concentration of cholesterol in donor and acceptor vesicles. The rate and extent of cholesterol transfer from liposomes to microsomes were slightly increased when 25-hydroxycholesterol was present during the transfer process. Irrespective of the treatment, 25-hydroxycholesterol continued to stimulate the ACAT activity of the treated microsomes. Microsomes that were enriched or depleted of cholesterol in the absence of 25-hydroxycholesterol yielded as much enzyme activities when assayed in the presence of 25-hydroxycholesterol as with the systems that contained 25-hydroxycholesterol during both the transfer process and enzyme assays. The results suggest that a major part of the activation of microsomal ACAT by 25-hydroxycholesterol is not ascribable to increased substrate availability for the enzyme.

Animals↗

PubMed1992

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.

Animals↗

PubMed1986

Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.

Animals↗

PubMed1985

Lysosomal accumulation of cholesterol and sphingomyelin: evidence for inhibition of acid sphingomyelinase.

The subcellular site of accumulation of non-esterified cholesterol and sphingomyelin in lipid-laden livers and spleens from rats given multiple intravenous injections with liposomes made up of these lipids were investigated by morphological and biochemical techniques. The subcellular fractionation of liver homogenates from cholesterol-sphingomyelin treated rats followed by lipid and enzymatic analyses of the fractions revealed that most of the accumulating lipid was present in very low density lysosomes floating in the post-microsomal supernatant fraction. The low density lysosomes exhibited good latency and had a very much lower relative activity of sphingomyelinase compared with values for N-acetyl-beta-glucosaminidase. Multiple injections of sphingomyelin-cholesterol liposomes resulted in splenomegaly. The spleen homogenates of the treated rats showed a many-fold increase in the concentration of sphingomyelin, of non-esterified cholesterol and in the activity of N-acetyl-beta-glucosaminidase over corresponding values for rats injected with saline. Electron microscopy of liver and spleen sections from treated rats revealed distinctive polymorphic intracellular inclusions bound by a membrane and containing numerous osmiophilic bodies. Structures identical to the storage inclusions seen in fixed liver sections from treated rats were also seen by electron microscopy in the post-microsomal fraction of these livers. The results suggest that sphingomyelin and non-esterified cholesterol accumulate in lysosomes when they occur in cells in excess of that structurally associated with cellular membranes.

Acetylglucosaminidase↗

PubMed1982

Lysosome lipid storage disorder in NCTR-BALB/c mice. III. Isolation and analysis of storage inclusions from liver.

Livers of NCTR-BALB/c mice, affected by excessive accumulation of cholesterol and phospholipid, were fractionated by sucrose density gradient centrifugation. Lysosomes of very low density (rho = 1.05 - 1.08) were found, which by electron microscopy appeared identical to the storage inclusions seen in fixed tissues. These lysosomes could be purified about 10-fold over the original homogenate, and represented 4% of the total protein and 30-40% of the liver acid hydrolase content. The preparations were nearly free of mitochondrial, endoplasmic reticulum, and plasma membrane contamination. The lysosomes were laden with cholesterol and phospholipid. Cholesterol (greater than 97% unesterified) accounted for half of the total lipid, and sphingomyelin accounted for another 20%. Phosphatidylcholine and phosphatidylethanolamine were also present in substantial quantities. All of the excess cholesterol and sphingomyelin of liver could be attributed to the low density lysosomes. Lysosomal acid sphingomyelinase activity, measured with a synthetic substrate, was found to be 10-60% of BALB/c mouse control levels in liver, spleen, and cerebellum, while two other lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased 2-8-fold in the same tissues. These data and the morphologic observations of the preceding paper establish that the disorder affecting NCTR-BALB/c mice is a lysosome storage disease. We propose several possible mechanisms to explain the cholesterol and phospholipid overloading of lysosomes. The specific gene defect remains to be established.

Animals↗

PubMed1982

Lysosome lipid storage disorder in NCTR-BALB/c mice. I. Description of the disease and genetics.

We describe a strain of BALB/c mice, designated NCTR-BALB/c, carrying a new genetic disorder characterized by excessive tissue deposition of cholesterol and phospholipid. The mice exhibit progressive incoordination, grow less rapidly, and die 80-120 days after birth. In comparison with control animals of the same age, organ weights in the affected animals are lower in absolute value but higher relative to body weight, except for the thymus, which is atrophied, and for the lung and testes, whose absolute weights are not changed. Vacuolated cells are found in many tissues, and large foam cells are present in reticuloendothelial system (RES)-rich organs. Compared with those of BALB/c controls, serum lipoproteins migrate more slowly on electrophoresis; the amount of beta-lipoproteins is increased, while alpha-lipoprotein content is decreased. Serum total cholesterol remains normal. The serum activities of aspartate aminotransferase, creatine phosphokinase, and N-acetyl-beta-glucosaminidase are elevated. Free cholesterol levels are increased 8-10-fold in liver, spleen, and thymus, and about 2-fold in other tissues; but esterified cholesterol levels are normal. The phospholipid content of several tissues is increased 50-100%, largely as a result of an increase in sphingomyelin content. Significant increases in phosphatidylcholine occur also in spleen and lung. The disorder is inherited, affecting both sexes equally, and appears to be transmitted as an autosomal recessive mutation.

Animals↗

PubMed1982

Lysosome lipid storage disorder in NCTR-BALB/c mice. II. Morphologic and cytochemical studies.

Electron-microscopic and cytochemical studies were carried out on tissues of NCTR-BALB/c mice. These mice are affected with a neurovisceral genetic disorder involving excessive tissue accumulation of lipid. Distinctive polymorphic intracellular inclusions, bounded by a membrane and containing lamellated bodies, were found in many cells of liver, spleen, lung, kidney, intestine, lymph nodes, and brain. The inclusions transformed reticuloendothelial cells into massive foam cells. Acid phosphatase cytochemical studies performed on sections of liver demonstrated that the inclusions were lysosomes. Fixation of liver in the presence of digitonin produced "spicules" in the inclusions characteristic of digitonin-cholesterol complexes. Clefts of cholesterol crystals were seen in the inclusions in liver, spleen, and lung. We conclude that the NCTR-BALB/c mice are affected by a lysosome lipid storage disease and that cholesterol is a major storage product.

Acid Phosphatase↗

PubMed1980