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D Carper

Publications and source records attributed to D Carper.

At least 19 recordsLinked to original sources
PubMed2001

Gene expression analysis of an H(2)O(2)-resistant lens epithelial cell line.

Gene expression patterns were examined in lens epithelial cells conditioned to grow in 125 microM hydrogen peroxide in order to define the protective mechanisms that may be involved in survival during oxidative stress. RNA was extracted from normal and hydrogen peroxide-resistant alphaTN4 mouse lens epithelial cells. Gene expression was evaluated using Differential Display (DD) and RT-PCR. Upregulation of mRNAs for antioxidant and cellular defense enzymes was observed. The highest elevation detected was a 14-fold increase in catalase in the hydrogen peroxide-resistant cells. Glutathione peroxidase, ferritin, and alphaB-crystallin were upregulated 2-fold, and reticulocalbin was upregulated 6-fold in the resistant cells. alphaA-crystallin was downregulated 5-fold, while aldose reductase and mitochondrial gene products were unchanged. Thus, in the alphaTN4 mouse lens cell line, long-term exposure to high levels of hydrogen peroxide elicited an upregulation of transcripts for enzymes involved in hydrogen peroxide degradation, metal binding, and chaperone function. Since mitochondrial gene transcription is sensitive to hydrogen peroxide, the presence of normal levels of mitochondrial transcripts, in this study, demonstrates the effectiveness of the antioxidant defense systems.

Animals↗
PubMed1999

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.

Animals↗
PubMed1999

Osmotic response element is required for the induction of aldose reductase by tumor necrosis factor-alpha.

Induction of aldose reductase (AR) was observed in human cells treated with tumor necrosis factor-alpha (TNF-alpha). AR protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-alpha. An increase in AR transcripts was also observed in human liver cells after 3 h of TNF-alpha treatment, reaching a maximum level of 11-fold at 48 h. Among the three inflammatory cytokines: TNF-alpha, interleukin-1, and interferon-gamma, TNF-alpha (100 units/ml) gave the most induction of AR. Differences in the pattern of AR induction were observed in human liver, lens, and retinal pigment epithelial cells with increasing concentrations of TNF-alpha. A similar pattern of AR promoter response was observed between TNF-alpha and osmotically stressed human liver cells. The deletion of the osmotic response element (ORE) abolished the induction by TNF-alpha and osmotic stress. A point mutation that converts ORE to a nuclear factor-kappaB (NF-kappaB) sequence abolished the osmotic response but maintained the TNF-alpha response. Electrophoretic gel mobility shift assays showed two NF-kappaB proteins, p50 and p52, capable of binding ORE sequence, and gel shift Western assay detected NF-kappaB proteins p50 and p65 in the ORE complex. Inhibitors of NF-kappaB signaling, lactacystin, and MG132 abolished the AR promoter response to TNF-alpha.

Aldehyde Reductase↗
PubMed1998

Oxidative modification of aldose reductase induced by copper ion. Factors and conditions affecting the process.

Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(II)] through an oxygen-independent oxidative modification process. A stoichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inactivation. While metal chelators such as EDTA or o-phenantroline prevent but do not reverse the ALR2 inactivation, DTT allows the enzyme activity to be rescued by inducing the recovery of the native enzyme form. The inactive enzyme form is characterized by the presence of 2 equiv of bound copper, at least one of which present as Cu(I), and by the presence of two lesser equivalents, with respect to the native enzyme, of reduced thiol residues. Data are presented which indicate that the Cu-induced protein modification responsible for the inactivation of ALR2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of ALR2 and induces, through its oxidation to GSSG, the generation of an enzyme form linked to a glutathionyl residue by a disulfide bond.

Aldehyde Reductase↗
PubMed1998

Up-regulation of osteonectin/SPARC in age-related cataractous human lens epithelia.

PURPOSE: To characterize gene expression patterns between epithelia isolated from cataractous and normal human lenses. METHODS: Reverse transcriptase differential display was used to identify differential expression between cataractous and normal epithelia. RT-PCR was used to compare pooled and individual RNA samples. RESULTS: One transcript, up-regulated in cataractous as compared to normal epithelia, was identified as osteonectin which is also known as SPARC (secreted acidic protein rich in cysteines). RT-PCR confirmed over-expression of this RNA. High levels of osteonectin mRNA were also detected in six individual epithelia dissected from cataractous lenses. CONCLUSIONS: The present study provides evidence for up-regulation of osteonectin in human age-related cataract and suggests that osteonectin, a protein involved in cell-cycle control, extracellular matrix and Ca++ binding, plays an important role in human lens homeostasis and may be involved in processes leading to lens opacity.

Adult↗
PubMed1998

Differential display detects altered gene expression between cataractous and normal human lenses.

PURPOSE: To identify and analyze differentially genes expressed between lens epithelia dissected from age-related cataractous and noncataractous human lenses. METHODS: RNAs from 50 pooled cataractous and 25 pooled noncataractous epithelia were compared by reverse transcription-polymerase chain reaction differential display (RT-PCR-DD). Two differentially displayed bands were chosen for further study. These were reamplified, cloned, and sequenced. Expression of these genes was further evaluated in pooled and individual epithelia by RT-PCR with gene-specific primers. RESULTS: Significant differences in gene expression were detected between the cataractous and the noncataractous epithelia. Three mRNAs displayed higher levels and 12 mRNAs displayed lower levels of expression in the cataractous samples compared with that in the noncataractous samples. Of the mRNAs expressed at higher levels, one was identified as metallothionein IIa (METII). Of the mRNAs with decreased expression, one was identified as protein phosphatase 2A regulatory subunit (P2A-RS). Overexpression of METII and underexpression of P2A-RS were confirmed in pooled and individual epithelia. CONCLUSIONS: These results provide evidence that age-related cataract is associated with alterations in the expression of multiple epithelial genes including METII and P2A-RS. METII is a detoxification protein induced by oxidative stress, and P2A-RS is a mitotic suppressor involved in cell-cycle control. These results implicate these proteins and their associated functions in the maintenance of lens transparency.

Adult↗
PubMed1997

Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

A new and essential cis-element AEE (aldose reductase enhancer element), necessary for the constitutive activity and the osmotic stress response of rat aldose reductase transcription in a rat liver cell line, has been identified. In transient transfection assays, an increase in promoter activity, up to 3.8-fold, was observed with osmotic stress (600 mosm/kg H2O) using a luciferase reporter gene construct containing aldose reductase promoter sequence from -1,094 base pair (bp) to +23 bp. A deletion between -1,071 and -895 bp reduced the constitutive activity and abolished the osmotic response of the promoter. Exonuclease III mediated in vivo DNA footprinting and dimethyl sulfate in vivo footprinting revealed DNA protection of a 32-bp region and two guanosines (G) within this region protected from methylation, respectively. Electrophoretic gel mobility shift assays using whole liver cell extracts showed protein binding, under both normal and stressed conditions. Deletion of the sequence between the two guanosines protected by in vivo dimethyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct (-1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum constitutive activity of 7.7-fold and a maximum osmotic response activity of 6. 7-fold.

Aldehyde Reductase↗
PubMed1995

Structural organization of the human sorbitol dehydrogenase gene (SORD).

The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximately 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA)5 were observed in the 5' noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and zeta-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis.

Amino Acid Sequence↗
PubMed1993

Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13.

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, we mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q42, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other active genes, non-aldose reductase homologous sequences, or pseudogenes.

Aldehyde Reductase↗
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↗
PubMed1992

Hypertonic stress induces alpha B-crystallin expression.

Alpha B-crystallin, a major lens protein, was induced in primary cultures of dog lens epithelial cells and glomerular endothelial cells when they were grown under conditions of hypertonic stress. With Western blot analysis using a specific alpha B-crystallin antibody, we observed a significant increase in the concentration of alpha B-crystallin protein in cells grown for 4-6 days in media supplemented with 150 mM NaCl or 250 mM cellobiose. These supplements increased the osmolarity of the medium from 300 to 550-600 mosmol kg-1. Alpha B-crystallin mRNA was also increased reaching a maximum four-fold increase in lens and 16-fold increase in kidney cells within 1-2 days. These studies demonstrate a type of regulation of alpha B-crystallin expression in cells from lenticular and non-lenticular tissues.

Animals↗
PubMed1991

Characterization of the aldose reductase-encoding gene family in rat.

Although the enzyme aldose reductase (AR) is implicated in the development of tissue pathology in diabetes, the exact mechanism of this involvement remains unclear. To better understand the role that expression of the aldose reductase-encoding gene (ALR) may play in diabetic complications, we have begun to analyze the gene and its regulatory regions, and we present here the sequence of four ALR genes in the rat. The putative functional gene is 14.1 kb long, has ten exons which show perfect sequence identity to the rat lens AR RNA sequence, and nine introns with classical splice-site consensus sequences. Potential regulatory elements in the 5'-flanking region of this gene include a TATA box and two CCAAT boxes. Probing rat genomic Southern blots with a fragment from the first intron indicates that there is probably only one copy of this gene in the rat genome. The other three genes are processed pseudogenes which show approx. 90% identity to the rat lens AR RNA sequence, contain no introns, and have poly(A) regions at their 3' ends. Chromosomal localization studies show the presence of ALR genes on chromosomes 3, 4 and 6 in the rat with the putative functional gene mapped on chromosome 4.

Aldehyde Reductase↗