Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells-but not from fibroblasts-exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P < 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333±153 cpm/105 cells vs. 88±42 in control cells, mean±SD, P = 0.04), which is indicative of-increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage-a process that is under genetic control-may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication.

Recent evidence indicates that different forms of stress, including hypoxia, can induce specific proteins called heat-shock or stress proteins in various types of mammalian cells. These studies examined whether myocardial ischemia can result in increased levels of proteins with molecular weight and isoelectric point characteristics similar to those described for heat-shock or stress proteins. The left anterior descending coronary artery of the dog heart was completely occluded; normal and ischemic myocardial samples were obtained 6 hours after occlusion; and total cardiac proteins and RNA were prepared. Ribonucleic acid was translated in vitro in a modified rabbit reticulocyte lysate system, and [35S]-methionine-labelled translational products as well as unlabelled cardiac proteins were separated by two-dimensional gel electrophoresis. Total proteins were visualized by silver staining and in vitro translation products quantified by fluorometry. A translatable mRNA coding for a 71,000 dalton peptide with an isoelectric point of 5.8 was markedly increased in the ischemic myocardium after 6 hours of ischemia. A protein with similar migration characteristics was detected in ischemic myocardium but not in normal myocardium. These results indicate that an mRNA coding for a translational product with similar migration characteristics of heat-shock protein 71 is induced by ischemia in the dog heart.

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Cardiac pressure overload induces a shift towards the fetal form of major proteins expressed by the myocytes, and an accumulation of extracellular matrix proteins. One of them, fibronectin (FN), accumulates soon after the imposition of pressure overload. Because FN exists both as cellular FN (c-FN) locally synthesized by nonmuscle cells and as "plasma-FN" (p-FN) synthesized by the hepatocytes, the first issue of this study was to determine whether FN accumulation within the myocardium in response to pressure overload is paralleled by a local increase in mRNA. The expression of c-FN isoforms being developmentally regulated in a tissue-specific manner, the types of FN exons expressed by cardiac cells were analyzed. Pressure overload was induced in 25-d-old rats by stenosis of the thoracic aorta. Using in situ hybridization, we show that the mRNAs encoding the fetal forms of c-FN are accumulated in the interstitial tissue of fetal rat hearts but are absent in adult.

The mKAT-2 gene encodes a Na+-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA (3). Interest in cationic AA transport regulation was stimulated by the discovery that arginine is the exclusive precursor of NO, and "de novo" arginine transport is required for NO production in some tissues (e.g., refs. 4-6).The mCAT genes were, to our knowledge, the first mammalian AA transporters cloned (reviewed in ref. 7). Their isolation permits a molecular and genetic analysis of cationic AA transporter expression and regulation (8-11). The mCAT proteins share substantial sequence (9), structural (11), and functional (12, 13) similarity when expressed and assessed in Xenopus oocytes (8,10,12,13). Based on their transport characteristics, the mCAT genes are considered to encode the y+ transport system (8,10,(12)(13)(14). Although the two genes share many similarities, they differ in their chromosomal location (9, 15) and expression patterns (7,12). Their apparent functional redundancy raises questions about mCAT gene regulation. Although little molecular information is available regarding the mechanism of AA transporter gene regulation, it is well established that AA transport systems are responsive to metabolic demands (3). We have shown that mCAT-2 mRNA accumulates during T-cell activation (9, 12) and in other tissues in response to surgical trauma (16). In contrast, mCAT-1 mRNA levels are more constant in parallel test conditions (7,12,16), although the mRNA levels are altered in response to glycogen and during liver regeneration (17).Here we report evidence that mCAT-2 gene transcription is initiated from multiple promoters. Sequence analysis of several cDNA clones revealed distinct 5' untranslated regions (UTRs) that could result from multiple promoters, alternative splicing, or trans-splicing. To analyze this further, the entire coding region of the structural gene was isolated. Moreover, the regions surrounding three of the 5' UTRs were sequenced and found to contain classical promoter and enhancer elements. We present evidence that these promoters are utilized and result in regulated gene expression in response to surgical stress. Finally, we document the differential utilization of these putative promoters in different cell types. Our data provide strong evidence that mCAT-2 transcription initiates from several distinct, widely spaced promoters.t MATERIALS AND METHODS Animals and Cell Culture. Six-to 8-week-old female AKR/J mice were purchased from The Jackson Laboratories. Tissues were harvested for RNA preparation in accordance with University of California and National Institutes of Health guidelines. The SL12.3 a...

Isolation of messenger ribonucleic acid (mRNA)-protein particles from cytosol or dissociated polyribosomes yielded complexes in which several proteins were consistently associated with mRNA. Some of the mRNA-associated proteins appeared to have a high affinity for mRNA since they remained complexed to mRNA during centrifugation in CsCl gradients. Quantitation of RNA and protein in polyribosomal mRNPs suggested that each molecule of mRNA bound a molecule of each of the two major proteins of 78 000 and 52 000 apparent molecular weights and/or one or more of several minor proteins found in mRNPs. Of the several mRNP proteins, only the protein of 78 000 apparent molecular weight appeared to form a stable complex with the polyriboadenylic acid [poly(A)]-tract of mRNA, suggesting that the remaining mRNA-associated proteins bind to other regions which may be common to many or all mRNAs. Binding of [3H]poly(A)-rich RNA to mRNP proteins was effectively inhibited by unlabeled poly(A)-rich RNA or the homopolymer polyriboguanylic acid [poly(G)], but not by poly(A) or other natural or synthetic mRNAs. The properties of non-poly(A)-dependent binding of mRNA by mRNP protein were similar to those of mRNA binding by the guanosine triphosphate dependent Met-tRNAfMet-binding protein.

Subribosomal and polyribosomal messenger ribonucleoproteins (mRNPs) were isolated from Ehrlich ascites tumor cells by a method involving sedimentation of polyribosomal and subribosomal particles, dissociation with EDTA, and rate-zonal sedimentation. The fractions containing mRNA protein particles were applied to glass fiber filters and extensively washed with buffer containing 0.5 M KCl. The eluted material was demonstrated to be an RNA-protein complex containing poly(A)-rich RNA, heterogeneous in size, and free of 18S or 28S rRNA. mRNA function for the RNA was suggested by its ability to direct protein synthesis in a cell-free protein-synthesizing system derived from wheat germ embryos. Analysis of the proteins associated with subribosomal and polyribosomal mRNPs by iodination and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed at least seven similar proteins. The apparent molecular weights of the three most prominent proteins were 78,000, 52,000, and 34,000. Analysis of reticulocyte polyribosomal mRNPs revealed an increased prominence of the 78,000 and 52,000 molecular weight proteins relative to the other protein bands.

SUMMARY. During diabetes mellitus, total proteins and ribonucleic acids are significantly decreased in the rat heart, and these parameters can be increased by insulin administration. To determine whether all ribonucleic acids are equally sensitive to insulin, we examined the influence of this hormone on individual translatable ribonucleic acids. Cardiac ribonucleic acid prepared from control, untreated, and insulin-treated diabetic animals was translated in vitro in the presence of [ 3! S]methionine. The radiolabeled peptides were separated by two-dimensional gel electrophoresis and were analyzed by fluorometry. We found that diabetes induces both qualitative and quantitative changes in the predominance of a few specific translatable messenger ribonucleic acid species. The translation of 11 messenger ribonucleic acid species was significantly decreased and that of eight messenger ribonucleic acid species was significantly increased in diabetic preparations. Twelve of the 19 translation products were quantified by digital matrix photometry: three labeled peptides were observed only when cardiac ribonucleic acid from diabetic animals was added to the cell-free translation system, four new peptides appeared when cardiac ribonucleic acid from control animals was added, and although the remaining five peptides were translated in vitro after either control or diabetic ribonucleic acid was added, their relative predominance was altered 2-to 200-fold. When translation products coded for by messenger ribonucleic acids prepared from either diabetic or hypothyroid hearts were compared, we found that most of the alterations induced by diabetes were also induced by hypothyroidism. However, two of the 19 products were exclusively regulated by insulin, whereas two products unchanged in the diabetic heart were regulated exclusively by thyroid hormones. Thus, although each hormone specifically influenced the abundance of a few translatable messenger ribonucleic acids, most hormonesensitive translatable messenger ribonucleic acids are under the dual regulation of insulin and thyroid hormone. (Circ Res 57: 296-303, 1985)