High glucose induces DNA damage in cultured human endothelial cells.

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 less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) 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.

Accumulation of fetal fibronectin mRNAs during the development of rat cardiac hypertrophy induced by pressure overload.

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. 1-3 d after aortic stenosis, the fetal forms of c-FN mRNAs were found in the wall of coronary arteries and in focal areas of the myocardium. Thus nonmuscle cells and smooth muscle cells, like myocytes, do respond to pressure overload by reexpressing fetal gene transcripts.

Nonidentity of the 48,000-dalton protein of mRNA-protein particles and the beta subunit of eukaryotic initiation factor 2.

The beta subunits of the GTP-dependent initiator methionyl-tRNA-binding protein and the 48,000-dalton protein, which is one of the two major proteins present in isolated mRNA . protein particles, have been shown to share a number of similar structural and mRNA-binding properties. The nonidentity of these two proteins is now established by documentation of differing molecular weights, isoelectric focusing points, and products of protease or cyanogen bromide cleavage.

mRNA-induced dissociation of initiation factor 2.

The GTP-dependent initiator methionyl-tRNA (Met-tRNAi) binding protein, eukaryotic initiation factor 2 (eIF2), binds mRNA, as well as Met-tRNAi with high affinity and both RNA species appear to bind to the 48,000-dalton subunit of eIF2. Binding of mRNA by eIF2 produces an alteration in its molecular configuration resulting in dissociation of component subunits as assayed by isoelectric focusing. In contrast, GTP, GDP, or Met-tRNAi produce no subunit dissociation. Phosphorylation of the 37,000-dalton eIF2 subunit by the heme-controlled translational repressor does not alter the ability of mRNA to produce subunit dissociation of eIF2. A role for mRNA binding by eIF2 in initiation of protein synthesis or in recycling of eIF2, or both, is postulated.

Messenger RNA binding protein purified from reticulocyte polyribosomes.

One of the proteins in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes which bind poly(A)-rich mRNA has been purified to apparent homogeneity using ammonium sulfate fractionation and phosphocellulose, hydroxylapatite, and diethylaminoethylcellulose column chromatography. The protein appears to contain two subunits of 66 700 and 56 400 apparent molecular weights with a 1:1 stoichiometry, since an apparent molecular weight of 110 000 was determined using Sephadex G-200 chromatography and an s020,w of 5.6 was obtained with rate-zonal sedimentation. The mRNA binding activity banded at pH 5.2-5.5 on isoelectric-focusing polyacrylamide gel electrophoresis. Protein-dependent binding appeared to be specific, since other natural or synthetic RNAs, including tRNA, ribosomal RNA, and poly(riboadenylic acid), were 90- to 250-fold less effective than mRNA at competing for binding of [3H]poly(adenylic acid)-rich mRNA. Poly(riboguanylic acid), however, was even more efficiently bound by this protein than mRNA.

Comparison of mRNA binding by Met-tRNAf binding protein and mRNA-associated proteins.

One of the heterogeneous mRNA binding activities in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes co-purified to apparent homogeneity through phosphocellulose and DEAE-cellulose chromatography and isoelectric focusing with the GTP-dependent Met-tRNAf binding protein. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following iodination revealed putative subunits of 51,000 and 39,000 apparent molecular weights. Specificity of mRNA binding by this protein was suggested since the ability of poly(A)-rich mRNA to compete for binding of [3H]poly(A)-rich mRNA exceeded by 10- to 100-fold that of most natural or synthetic RNAs tested, except for the hybrid poly(G) - poly(C) which was almost as effective, and poly(G), which was more effective, at competing for protein-dependent binding. The mRNA binding activity exhibited complete GTP independence and no apparent divalent cation requirement. GDP inhibited Met-tRNAf binding but neither GDP, GMP, nor 7-methylguanosine 5'-monophosphate inhibited mRNA binding by this protein. Similar data were obtained with respect to the ability of natural or synthetic RNAs to compete for binding of [3H]poly(A)-rich mRNA by proteins associated with purified rabbit reticulocyte polyribosomal mRNA-protein particles; while poly(A) was an ineffective competitor, poly(G) was more effective than even mRNA at competing for protein-dependent binding. No significant binding of Met-tRNAf by mRNA-protein particles was detected. Polyacrylamide gel electrophoresis following reduction of mRNA-protein particles revealed apparent co-migration of a major protein with one subunit of the GTP-dependent Met-tRNAf binding protein, but no protein comparable to the 39,000 dalton subunit protein.

Characterization of GTP-dependent Met-tRNAf binding protein.

Purified GTP-dependent Met-tRNAf binding protein (EIF2) prepared from the 0.5 M KCl eluate of reticulocyte polyribosomes was successfully resolved into its three-component subunits by isoelectric focusing in the presence of urea. The 37,000-dalton subunit focused at a pH of 5.8 and was resolved into two spots; the 48,000-dalton subunit focused at a pH of 6.6 and was resolved into three spots; and the 52,000-dalton subunit exhibited an isoelectric point of 8.9 and migrated as a single spot. When isolated 37,000- and 48,000-dalton subunit was found to possess Met-tRNAf and mRNA binding activities while the 37,000-dalton subunit was found to possess GDP binding activity. Phosphorylation of EIF2 by protein kinases present in reticulocyte lysates was demonstrated using [gamma-32P]GTP or [gamma-32P]ATP as the phosphate donor. The 37,000-dalton subunit was preferentially phosphorylated when [gamma-32P]ATP was used as substrate; the 48,000-dalton subunit was preferentially phosphorylated when the [gamma-52P]GTP was used as phosphate donor, although some phosphorylation of the 37,000-dalton subunit was also observed. The 37,000-dalton subunit of ribosome-associated EIF2 was present predominantly in a dephosphorylated form following purification.

A novel detection system for submicroscopic human metastases in athymic mice.

We developed a sensitive assay system to accurately detect the amount of human tumor DNA, if present, in athymic mouse organs. Genomic DNA was prepared from a human lung carcinoma cell line and from athymic mouse lungs (tumor-bearing and non-tumor bearing). Mixtures and dilutions of extracted DNA were slot-blotted onto nylon filters and probed with labeled, human-specific Alu sequences. The equivalent of one human cell per 2000 mouse cells could be detected using this assay. This sensitive assay may now be used to confirm the presence or absence of human neoplasm metastases in the athymic mouse model system.

Diabetes-induced alterations in the translational activity of specific messenger ribonucleic acids isolated from rat hearts.

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 [35S]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 preparation. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes-induced changes of proteins synthesized by adult cardiac myocytes are partially reversed by insulin.

In the rat heart diabetes mellitus leads to a change in myosin heavy chain (MHC) mRNAs and corresponding alterations in myosin isoenzymes as well as a decrease in total cardiac protein synthesis. However, it is still unknown whether cardiac proteins other than MHC are altered by diabetes and if so whether these abnormalities are mediated by insulin deficiency. To answer these questions we analyzed proteins synthesized by isolated cardiac myocytes in the presence or absence of insulin. Enzymatically dispersed adult cardiac myocytes from control and streptozotocin-induced diabetic rats were incubated in medium containing [35S]-methionine for 4 h; diabetic cells were incubated with or without the addition of 5 x 10(-7)M insulin. The labelled peptides were then separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by fluorometry. The abundance of six individual polypeptides was consistently affected by diabetes: one protein was significantly decreased while four others were increased in diabetic myocytes. The remaining protein showed a shift in isoelectric point without a change in molecular weight possibly representing isoforms of a single polypeptide. The addition of insulin reverted the predominance of three proteins back to normal while it did not affect the other three at all. In conclusion diabetes induces changes in the abundance of a few proteins synthesized in vitro by cardiac myocytes and only half of them show an acute response to insulin.

Thyroid hormone induced changes in cardiac proteins and mRNAs.

Contraction of the hypothyroid heart is characterized by delayed diastolic relaxation and decreased velocity of systolic contraction. In order to determine if these alterations could be mediated by the changes in the mRNA coding for the Ca++ ATPase of the sarcoplasmic reticulum and alterations of the mRNAs coding for myosin heavy chain (MHC) alpha and beta, the levels of these specific mRNAs were quantitated using a Northern blotting technique. We find that the Ca++ ATPase mRNA was 3-fold lower in hypothyroid hearts. After T3 administration to hypothyroid rats, Ca++ ATPase mRNA increased to 66% of control levels within 2 hrs and to 100% of control levels 5 hrs after T3 administration. In the hypothyroid heart, MHC beta mRNA was the predominant message with MHC alpha mRNA barely detectable. Administration of 2 mg of T3 led to a significant increase in MHC alpha mRNA levels first detectable 2 hrs after T3 administration. Twenty-four hrs after T3 administration, MHC alpha mRNA levels had normalized. The results of these studies indicate that thyroid hormone mediates significant alterations in the level of the mRNA coding for the Ca++ ATPase of the sarcoplasmic reticulum and of the mRNAs coding for MHC alpha and beta. Changes in the level of these specific mRNAs resulting in lower levels of the corresponding proteins may explain the delayed diastolic relaxation and the decreased velocity of contraction of the hypothyroid heart.

Influence of thyroid hormone on myosin heavy chain mRNA and other messenger RNAs in the rat heart.

The level of myosin heavy chain (MHC) alpha mRNA and of MHC-beta mRNA was quantitated in the rat heart using a specific cDNA probe. In hypothyroid and diabetic hearts MHC-beta mRNA predominates, whereas in normal hearts MHC-alpha mRNA represents 80% of all MHC mRNA. Administration of 0.2 mg T3/100 g body wt. to hypothyroid rats led to an increase in MHC-alpha mRNA beginning at 3 h after injection and continued to rise until at 24 h control level of MHC-alpha mRNA were reached. In contrast, after administration of 2 units regular insulin to diabetic rats, MHC-alpha mRNA levels showed a small but significant increase already 30 min after insulin administration reaching a peak at 3 h and returning to diabetic values 5 h after insulin. The T3 response of other cardiac mRNAs was quantitated using in vitro translation, separation of 35S methionine labeled translational products and their quantitation by digital matrix photometry. An mRNA (spot 72b) coding for a translational product with a Mr 81,000 and pI of 5.4 showed a 3-fold increase in its level 1 h after T3 administration. In view of the rapid response of spot 72b and the early response of MHC-alpha mRNA to insulin, it is currently unclear if the T3 response of MHC-alpha mRNA represents a primary effect of T3.

Effect of diabetes and hypothyroidism on the predominance of cardiac myosin heavy chains synthesized in vivo or in a cell-free system.

Rat cardiac ventricular myosins and RNA were prepared from normal, diabetic, insulin-treated diabetic, hypothyroid, and 3,3',5-triiodo-L-thyronine-treated hypothyroid rats. Myosin heavy chains isolated from purified myosin or synthesized in vitro from cardiac RNAs were subjected to partial protease digestion during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that peptide maps obtained from cardiac myosin heavy chains of hypothyroid and diabetic rats were identical but differed from the maps of myosin heavy chain from control and hormone-treated animals. The same results were obtained, whether the heavy chains were isolated from purified myosin synthesized in the intact heart or from translation products coded for by cardiac RNAs added to the modified reticulocyte lysate. These results indicate that the myosin heavy chain RNA species present in the hypothyroid heart is also expressed during insulin deficiency but differs from the species expressed in normal animals. The expression of the two myosin heavy chain RNA species found in the rat cardiac ventricle appears to be independently regulated by these two hormones.

Contractile protein gene expression in serum-free cultured adult rat cardiac myocytes.

The effects of two adhesion substrates (serum and laminin) and time in culture on the expression of genes encoding myosin heavy chain (MHC) isoforms and alpha-skeletal actin were analysed in myocytes isolated from adult rat heart and maintained in serum-free culture. Relative messenger ribonucleic acid (mRNA) abundances were quantitated by dot-blot analysis. Gene expression was not influenced by the substrate used. Time in culture induced a decrease in total mRNA abundance and an up-regulation of beta-MHC and alpha-skeletal actin genes. It is proposed that atrophy of adult myocytes is associated with a pattern of gene expression similar to the fetal program.