Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display.

Aiello, Lloyd Paul; Robinson, G. A.; Lin, You-Wei; Nishio, Yoshihiko; King, George L.

doi:10.1073/pnas.91.13.6231

Cited by 87 publications

(45 citation statements)

References 57 publications

(33 reference statements)

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“…In the present study, eight possible candidate genes were suggested by the initial screening of -2,000 mRNA species and two of the eight showed reproducible differences in expression by Northern blot analysis, a rate of confirmed positives similar to that reported in previous studies (17,18). Genes previously identified as altered in the diabetic state such as GLUT-4 (34) and Ca2' ATPase (35) mRNA were not observed in this study, however, this may be due to several potential limitations of the differential display method such as the insensitivity of this method to quantitate small changes in gene expression as compared with Northern blot analysis and differences in primer efficiencies.…”

Section: Discussionsupporting

confidence: 64%

“…Total RNA was extracted using Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). mRNA differential display was performed as previously reported (17,18 SDS, and 60 Mg/ml salmon sperm DNA at 65°C and washed in 0.5 x SSC, 5% SDS at 65°C for over 1 h. mRNA expression was quantified using a phosphorlmager and standardized volume integration with the accompanying ImageQuant Analyzing Software version 3.3 (Molecular Dynamics, Sunnyvale, CA) and loading differences were normalized using 36B4 as standard cDNA probe (17,18).…”

Section: Introductionmentioning

confidence: 99%

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Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Nishio

Warren²,

Buczek‐Thomas³

et al. 1995

J. Clin. Invest.

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IntroductionPrimary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Galj81-3GalNAcacR 81-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of 0-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats. (J. Clin. Invest. 1995. 96:1759-1767

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Section: Discussionsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Nishio

Warren²,

Buczek‐Thomas³

et al. 1995

J. Clin. Invest.

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“…This phenomenon has been reported, but not directly explained, also by others. 31,32 It is presumably related to the exponential nature of the PCR reaction and the probability that cDNA amplification may occasionally fail under the PCR conditions required for the differential display analysis.…”

Section: Differential Display Analysis Of Immunoselected Ma-11 Cell Pmentioning

confidence: 99%

Differential display analysis of breast carcinoma cells enriched by immunomagnetic target cell selection: Gene expression profiles in bone marrow target cells

Ree

Engebraaten

Hovig

et al. 2001

Intl Journal of Cancer

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The red bone marrow (BM) is an important indicator organ of hematogenous micrometastatic spread of carcinomas. Characterization of biological properties specific for BM micrometastatic cells, however, is technically challenging due to the limited number of target cells usually available for the purpose. This report provides referrals to qualitative gene expression profiling of BM micrometastatic cells enriched by immunomagnetic selection. First, an experimental strategy was used to study regulatory mechanisms involved when BM micrometastatic cells colonize distant organs. The MA-11 cells, originating from BM micrometastases in a breast cancer patient clinically devoid of overt metastatic disease, were injected into immunodeficient rats. Metastatic MA-11 cells were subsequently immunoselected from the resulting in vivo lesions. The selected cell populations were compared to the injected cells by differential display analysis, and several genes possibly involved in tumor cell invasion and proliferation were confirmed as differentially expressed among the various MA-11 cell populations. A direct approach to qualitative gene expression profiling of BM micrometastatic cells was also explored. Carcinoma cells were immunoselected from BM and axillary lymph nodes obtained from breast cancer patients, and the isolated cell populations were compared by differential display analysis. Two candidate genes, identified as factors involved in cellular growth control, appeared as differentially expressed by the target cells from BM. Our study provides detailed information on how to combine an immunomagnetic selection procedure and differential display analysis to reveal gene expression profiles that may characterize BM micrometastatic cells. © 2002 Wiley-Liss, Inc. Key words: bone marrow; micrometastasis; breast cancer; immunomagnetic cell preparation; differential cloningClinical and experimental evidence suggests that epithelial tumor cells are able to disseminate to secondary organs at an early stage of primary tumor development. The red bone marrow (BM) compartment represents an important indicator organ of hematogenous micrometastatic spread of carcinomas. According to current concepts, however, disseminated tumor cells detected in the BM are not able to grow as distant metastatic lesions unless they possess certain biological characteristics, among which the ability of invasion into and proliferation within the target organ is considered highly significant. [1][2][3] The classical, experimental works on mechanisms of tumor metastasis demonstrated that only a small subset of cells within the parental population is capable of metastasizing 4 and that the cellular composition of secondary tumors differs from that of the primaries. 5 Subsequent reports introduced the concept of dynamic heterogeneity, which argues that although the metastatic phenotype is a genetically controlled trait, it is inherently unstable. 6 Indeed, cellular properties that are crucial for initiation of distant tumor growth may be obscured by the heterog...

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“…After ultraviolet cross-linking, the membranes were prehybridized and hybridized to a 32 P-labeled bovine preproET-1 cDNA probe prepared by the multiprime DNA labeling system in 0.1 mol/l Pipes, 0.2 mol/l NaPO 4 , 0.1 mol/l NaCl, 1 mmol/l EDTA, 5% SDS, and 30 µg/ml salmon sperm DNA at 65°C. Stringent washing was done in 50% of 1ϫ sodium chloride and sodium citrate, 5% SDS at 65°C for over 1 h. The expression of mRNA was quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and normalized using 36B4 as the standard cDNA probe (34). In situ PKC activity assay.…”

Section: Methodsmentioning

confidence: 99%

Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation.

et al. 2000

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Enhanced actions or levels of endothelin-1 (ET-1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET-1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET-1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET-1 mRNA in parallel levels by 2-fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucoseinduced ET-1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen-activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3-kinase inhibitor. By immunoblot analysis, PKC-␤2 and -␦ isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC-␤1 and -␦ isoforms but not PKC-isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose-induced ET-1 mRNA expression by at least 2-fold. These results showed that enhanced ET-1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC-␤ and -␦ isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.

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Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display.

Cited by 87 publications

References 57 publications

Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Differential display analysis of breast carcinoma cells enriched by immunomagnetic target cell selection: Gene expression profiles in bone marrow target cells

Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation.

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