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Biomedical subjects

V N Reddy

Publications and source records attributed to V N Reddy.

At least 19 recordsLinked to original sources
PubMed1997

Anti-beta-crystallin antibodies (mouse) or sera from humans with age-related cataract are cytotoxic for lens epithelial cells in culture.

Circulating autoantibodies against lens antigens are prevalent in patients with age-related cataract (ARC), but their pathogenic significance is unknown. We hypothesized that these autoantibodies are cytotoxic for lens epithelial cells (LECs). To test this hypothesis. We incubated LECs with mouse polyclonal or monoclonal antibodies against beta-crystallin (anti-beta) in the presence or absence of guinea pig complement. We found that anti-beta in the presence of the complement bound to and killed mouse LECs (MLECs) and human LECs (HLECs). Sera obtained from patients with ARC also were cytotoxic to both HLECs and MLECs in culture. Heat-inactivated human sera were not cytotoxic to LECs in the absence of the complement, but were cytotoxic to both HLECs and MLECs in the presence of additional complement. These results support the hypothesis that autoantibodies against lens antigens are cytotoxic to LECs, and that cell death may involve complement-mediated pathways.

Animals↗
PubMed1997

Peroxide-induced damage in lenses of transgenic mice with deficient and elevated levels of glutathione peroxidase.

Transgenic mice with elevated glutathione peroxidase (GSHPx) activity and gene knockout animals with a deficiency of the enzyme were used to investigate the role of GSHPx in defending the lens against H2O2-induced damage. The effects of peroxide on cultured lenses were determined by using light and transmission electron microscopy to evaluate morphological changes occurring in the epithelium and superficial cortex of the central and equatorial regions of the lens. DNA single-strand breaks in the epithelium were also examined. Following a 30-min exposure to 25 microM H2O2, lenses from normal animals showed distinct changes in the morphology of both the epithelium and superficial cortex. The damage to these cells was extensive in lenses of gene knockout mice in which activity of GSHPx was undetectable. In marked contrast, lenses of transgenic mice, which had 5-fold higher activities of GSHPx, were able to resist the cytotoxic effects. Similar to damage to cell morphology, the extent of DNA strand breaks was significantly lower (40% of control) in H2O2-exposed lenses as compared to normal lenses while DNA damage in gene knockout lenses was 5 times greater than that of GSHPx-rich transgenic lenses. The present studies extend our previous findings on the role of the glutathione redox cycle in the detoxification of peroxide and demonstrate that an increase in GSHPx activity protects the lens against peroxide-induced changes in cell morphology and DNA strand breaks.

Animals↗
PubMed1997

Role of small GTP-binding proteins in lovastatin-induced cataracts.

PURPOSE: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent. METHODS: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays. RESULTS: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes. CONCLUSIONS: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.

Animals↗
PubMed1996

A study of growth factor receptors in human lens epithelial cells and their relationship to fiber differentiation.

Recent studies have demonstrated that several growth factors enhance fiber differentiation in cultured human lens epithelial (HLE) cells in early passages. However, these effects gradually decrease in cells of later passages. The purpose of this investigation is to test the hypothesis that the decreasing effect of growth factors on fiber differentiation in later passages may be due to a decrease or the inactivation of growth factor receptors as a function of serial subcultures. Specimens of HLE cells were obtained from infants. First through to fourth passage cells were treated with 10 ng ml-1 of epidermal growth factor, basic fibroblast growth factor or insulin-like growth factor-I. Fiber differentiation was determined from spontaneous lentoid formation by phase-contrast and transmission electron microscopy. Growth factor binding to the receptor on the cell surface was determined by transmission electron microscopy using the conjugates of colloidal gold and growth factors, and the number of receptors on the cell surface were also quantified by immunocytochemistry. Spontaneous lentoid formation was enhanced by all of the growth factors studied in the first passage. However, in the second and third passage only double layering of cells without characteristic fiber differentiation was observed while in the fourth passage, growth factors had no effect on differentiation. The number of growth factor bindings as well as the number of growth factor receptors gradually decreased with the number of passages. The loss of effect of growth factors on fiber differentiation with increasing number of passages correlated with the decrease in receptor number.

Cell Culture Techniques↗
PubMed1996

Qualitative analysis of biochemical reaction systems.

The qualitative analysis of biochemical reaction systems is presented. A discrete event systems approach is used to represent and analyze bioreaction pathways. The approach is based on Petri nets, which are particularly suited to modeling stoichiometric transformations, i.e. the inter-conversion of metabolites in fixed proportions. The properties and methods for the analysis of Petri nets, along with their interpretation for biochemical systems, are presented. As an example, the combined glycolytic and pentose phosphate pathway of the erythrocyte cell is presented to illustrate the concepts of the methodology.

Animals↗
PubMed1995

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen.

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Simultaneous measurement of reduced and oxidized glutathione in human aqueous humor and cataracts by electrochemical detection.

A sensitive, electrochemical method was employed for the simultaneous measurement of reduced and oxidized glutathione in lens cortex, nucleus and capsule epithelia of rabbit lenses, normal human lenses and human cataracts. In addition, aqueous humor from cataract patients was also analyzed. The level of GSSG in the nucleus of human cataracts was significantly higher than that in the nucleus of normal eye bank lenses. The capsule epithelium of intracapsular extracted cataracts possessed high levels of reduced glutathione, despite the fact that much of the glutathione in the cortex and nucleus of the lenses was depleted. Levels of GSH in the aqueous humor of cataract patients were several times higher than those reported for normal aqueous humor. Electrochemical detection proved to be a useful technique for analysis of reduced and oxidized glutathione in lens and aqueous humor, especially when sample size is small, such as for capsule epithelium.

Aged↗
PubMed1995

Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture.

PURPOSE: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures. METHODS: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin. RESULTS: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin. CONCLUSIONS: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures.

Cell Differentiation↗
PubMed1994

Studies on congenital hereditary cataract and microphthalmia of the miniature schnauzer dog.

Hereditary cataract in dogs occurs as an autosomal recessive trait. The opacity is primarily in the lens nucleus and posterior cortex. The affected animals also have other ocular abnormalities such as microphthalmia. To understand the genetic basis of this disorder, we have analyzed leukocyte DNA from affected and normal dogs for possible mutations in the homeobox containing gene and myotonic dystrophy locus. The results show that there are no signs of microdeletion, insertion, point mutation and rearrangements in these loci. Although these observations cannot completely rule out the possibility of point mutations, they suggest that the above loci are unlikely to be associated with the disease.

Animals↗
PubMed1994

A physiological level of ascorbate inhibits galactose cataract in guinea pigs by decreasing polyol accumulation in the lens epithelium: a dehydroascorbate-linked mechanism.

It was reported previously that dietary ascorbate (ASC) delays the development of galactose-induced cataract in guinea pigs compared to the rate which is observed in ASC-deficient animals. Experiments were conducted to explore the possible mechanism of this phenomenon. Guinea pigs were fed for a period of up to 4 weeks either a normal diet (1 g ASC/kg diet) or a scorbutic diet (< 0.04 g ASC/kg diet) combined with 10% galactose in the drinking water. After 2 weeks, levels of ASC in animals on the scorbutic diet decreased by 95% in the aqueous humor and by 78% in the lens. Slit lamp examination showed that galactose-induced vacuoles in the lens equator formed at a significantly faster rate in the scorbutic animals. However, examination of biochemical parameters in whole lenses of the two groups of animals after 2 weeks showed no significant differences with regard to accumulation of galactose and galactitol, decreases in the levels of myoinositol, taurine and GSH or changes in cation concentrations. In order to examine possible regional changes in the lenses, various parameters were studied in the lens capsule-epithelium. On day 4, the capsule epithelia of scorbutic animals on a galactose diet had a content of galactitol two-and-a-half times higher than that of normal galactose-fed animals. Scorbutic conditions also intensified the loss of Na(+)-K+ ATPase activity in the lens capsule-epithelium caused by galactose feeding. Oxidized glutathione was not detectable in the lens capsule epithelia of any of the animals studied. Hexose monophosphate shunt activity was elevated in lenses of normal galactose-fed animals during the first hour of culture after death whereas lenses of scorbutic galactose-fed animals were not. Consistent with the in vivo findings, galactitol accumulation in dog lens epithelial cells exposed to 30 mM galactose was significantly inhibited by the presence of either ASC or dehydroascorbate (DHA) in the medium. Hexose monophosphate shunt activity in the cells was stimulated to two-and-a-half times its initial level by either 1 mM DHA or 30 mM galactose and slightly more than three-fold by a combination of the two challenges. The results suggest that decreased polyol accumulation in the lens epithelium of the normal galactose-fed guinea pig, which has a high level of ASC in the aqueous humor, accounts for the delay in onset of cataract compared to that for the ASC-deficient animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The effect of ganglioside on oxidation-induced permeability changes in lens and in epithelial cells of lens and retina.

Several recent reports have indicated that ganglioside treatment both in vivo and in vitro has a protective effect on the loss of membrane permeability resulting from inhibition of transport enzyme(s) in different experimental models. In this study we have investigated the effect of monosialoganglioside on oxidation-induced changes in organ-cultured rabbit lenses and in cultured dog lens epithelium and human retinal pigment epithelial cells. Exposure of organ-cultured lenses to 0.5 mM hydrogen peroxide for 1 hr increased the efflux of 86Rb from intact lenses and loss of myoinositol from the capsule epithelium. Pretreatment of the lenses with monosialoganglioside significantly reduced the efflux rate of 86Rb and loss of myoinositol. Monosialoganglioside also prevented morphological changes induced by 0.1 mM hydrogen peroxide in dog lens epithelium and loss of cell viability caused by docosahexaenoic acid in dog lens epithelium and in human retinal pigment epithelial cells. In contrast to the protective effect of monosialoganglioside on permeability and morphological changes in cultured cells, it had no effect against single-strand breaks of DNA in dog lens epithelium resulting from exposure to hydrogen peroxide, X-ray and UV-B radiation. Although the molecular mechanisms by which monosialoganglioside prevents permeability and morphological changes induced by hydrogen peroxide and docosahexaenoic acid are not known, it appears that this ganglioside serves as a membrane stabilizer rather than as a free-radical scavenger.

Animals↗
PubMed1993

Nitric oxide-regulated endogenous ADP-ribosylation of rod outer segment proteins.

Both membrane and cytosolic fractions of retinal rod outer segments contain ADP-ribosylases that modify proteins in the respective fractions. Nitroprusside and endogenously produced NO regulate the activities of these ADP-ribosylases. The ADP-ribosylation of the membrane proteins of molecular weight 116K, 66K and 46K is inhibited by NO and nitroprusside, while that of the 38K cytosolic protein and the 39K membrane-associated protein is activated. The 39K protein is identified as the alpha-subunit of G-protein. The ADP-ribosylation of this protein is activated 6 to 11-fold by NO suggesting that NO may play a significant role in modulating the activity of G-protein in visual transduction.

Adenosine Diphosphate Ribose↗
PubMed1993

Aldose reductase in human retinal pigment epithelial cells.

Sugar alcohols have been reported to accumulate in retinal pigment epithelium (RPE) of diabetic animals. This finding has raised interest in the role of RPE in diabetes-associated retinal changes such as cystoid macular edema. To confirm the presence of aldose reductase in this tissue, the NADPH-dependent enzyme was purified to an apparent homogeneity from cultured human RPE cells, characterized, and its biochemical properties investigated. The induction of aldose reductase by hypertonic stress was also examined. The purification of aldose reductase was performed by a series of chromatographic steps which include gel filtration, affinity chromatography and chromatofocusing. Final purity achieved was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic properties and susceptibility to inhibition of the purified aldose reductase were essentially identical to aldose reductase purified from human placenta and kidney. In addition to aldose reductase, chromatofocusing demonstrated the presence of aldehyde reductase, another NADPH-dependent reductase. However, the amounts of aldehyde reductase present were much smaller than those of aldose reductase and the levels of aldehyde reductase appeared too small to contribute to the polyol production in the RPE cells. Culture of RPE cells in hypertonic medium containing 150 mM sodium chloride (600 mosmol total) increased both reductase activity, monitored with DL-glyceraldehyde as substrate, and immunoblot staining for aldose reductase. Chromatofocusing of RPE cells cultured in hypertonic media resulted in a prominent increase in the peak corresponding to aldose reductase compared to the peak height of cells grown in control medium. No increase in aldehyde reductase from RPE cells cultured in hypertonic medium was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗
PubMed1993

Petri net representations in metabolic pathways.

The present methods for representing metabolic pathways are limited in their ability to handle complex systems, incorporate new information, and to provide for drawing qualitative conclusions from the structure of pathways. The theory of Petri nets is introduced as a tool for computer-implementable representation of pathways. Petri nets have the potential to overcome the present limitations, and through a multitude of properties, enable the preliminary qualitative analysis of pathways.

Computer Simulation↗
PubMed1993

Hypertonic stress increases NaK ATPase, taurine, and myoinositol in human lens and retinal pigment epithelial cultures.

PURPOSE: Recent evidence suggests that taurine and myoinositol may serve as organic osmolytes in a number of cells, including lens and retinal pigment epithelia, but the mechanism for their increased accumulation in response to hypertonic stress is not known. To assess whether NaK ATPase contributed to the elevated levels of taurine and myoinositol in cells exposed to hypertonic media, we measured the activity of NaK ATPase, which is known to be implicated in the transport of these substances, in human lens and retinal pigment epithelia cultured in isotonic and hypertonic media. METHODS: Primary cultures of human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells were maintained in isotonic and hypertonic media for varying periods of time, and the activity of NaK ATPase and the levels of taurine and myoinositol were measured in cells cultured under two different conditions. The possible involvement of the transport enzyme in the accumulation of the two osmolytes was also investigated by inhibiting the enzyme with ouabain. RESULTS: When primary cultures of HLE and HRPE were exposed to hypertonic medium containing NaCl (600 mOsm) or cellobiose (500m Osm) for 72 hours, the concentration of taurine and myoinositol in HLE cells increased by 218% and 558% of control, respectively, in NaCl medium, whereas the corresponding increases in cellobiose medium were 147% and 439%. In HRPE cells, the increase in myoinositol levels in the two hypertonic media was more dramatic than that in taurine. Concomitant with the increase in the concentration of the osmolytes, there was an increase in NaK ATPase activity in both cell types. Although the accumulation of taurine in HLE cells in hypertonic media in a 6-hour culture was essentially prevented by 10(-8) mmol/l ouabain, myoinositol levels were affected to a lesser, but still significant, extent. In HRPE cells, which were cultured for 24 hrs in the presence of 10(-6) mmol/l ouabain, there was a more direct correlation between the inhibition of NaK ATPase and the decreased accumulation of taurine and myoinositol in the hypertonic media. CONCLUSION: Although the exact mechanism by which NaK ATPase activity increases in response to hypertonic stress remains to be established, the increased activity of the enzyme is related to the enhanced accumulation of the organic osmolytes, taurine, and myoinositol, in HLE and HRPE cells cultured in hypertonic medium.

Cellobiose↗
PubMed1993

Synthesis of lens capsule in long-term culture of human lens epithelial cells.

PURPOSE: This study examined the extent to which human lens epithelial (HLE) cells in tissue culture retain the potential for differentiation, expression of lens-specific marker proteins, and the synthesis of lens capsule, the major characteristics of lens epithelium in vivo. METHODS: Primary cultures of HLE cells were maintained for up to 450 days. Transmission and immunoelectron microscopy were used to study the thickness of the synthesized capsule and the formation of type IV collagen and laminin, two major protein components of the basement membrane of lens capsule in vivo. RESULTS: In a long-term HLE culture system, without subcloning, lens fiber differentiation and capsular synthesis were maintained over a period of 450 days. In these cultures, the cell sheet showed three distinct zones: (1) a central zone with tight monolayer; (2) a mild peripheral zone with irregularly aggregated multilayer; and (3) a peripheral zone with loose monolayer. The basement membrane-like material was synthesized in the central zone and lentoids, which serve as a model for fiber differentiation, developed primarily in the mid peripheral zone. No capsular material or lentoids were observed in the peripheral zone. The capsule-like material was 2 to 2.5 microns thick and showed the presence of type IV collagen and laminin, as detected by antibody reaction. CONCLUSION: This study demonstrates for the first time that HLE cells in long-term cultures synthesize a continuous sheet of capsule-like material. The findings also suggest that reformation of a tight cell-to-cell relationship or generation of high cell density similar to that found in vivo may be an important factor for the synthesis of lens capsule.

Basement Membrane↗
PubMed1993

The effect of hypertonicity on aldose reductase, alpha B-crystallin, and organic osmolytes in the retinal pigment epithelium.

PURPOSE: Aldose reductase (AR), an enzyme implicated in diabetic complications of ocular tissues, has been suggested to play a physiologic role in kidney and, possibly, other tissues by elevating the organic osmolytes in conditions of heightened extracellular tonicity. Hypertonicity has been shown to induce AR and alpha B-crystallin in some cells. To examine if similar mechanisms are operating in the retinal pigment epithelium (RPE), another target tissue of diabetic complications, we studied the effect of hypertonic media on the induction of AR, alpha B-crystallin, myoinositol, taurine, and other free amino acids. METHODS: Human RPE cells were cultured in normal and hypertonic media containing 150 mmol/l NaCl or 200 mmol/l cellobiose in combination with 30 mmol/l galactose from 0-8 days. Western blot analysis with antibodies were used to measure the expression of AR and alpha B-crystallin. Hybridization of northern blots using AR and alpha B-crystallin complementary DNA probes were employed for the measurement of the respective messenger RNA for these proteins. Changes in the levels of myoinositol, galactitol, taurine, and other free amino acids were determined biochemically. RESULTS: AR and alpha B-crystallin messenger RNA levels rose 16-fold and 4-fold, respectively, when human RPE cells were cultured for 3 days in media supplemented with either 150 mmol/l NaCl or 200 mmol/l cellobiose. AR and alpha B-crystallin protein levels also increased significantly, as seen by western blots. Consistent with the increased AR, galactitol accumulated to a greater extent when human RPE cells were grown in media containing 30 mmol/l galactose plus 150 mmol/l NaCl compared with cells grown in 30 mmol/l galactose alone. An 11-fold increase in cellular myoinositol and a 1.4-fold increase in taurine was observed in cells exposed to hypertonic media. CONCLUSIONS: These findings suggest that human RPE cells are responsive to hypertonic stress by elevating AR activity and use intracellular organic solutes in an interactive manner to help regulate intracellular tonicity.

Adult↗