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Biomedical subjects

A Barrieux

Publications and source records attributed to A Barrieux.

At least 19 recordsLinked to original sources
PubMed1995

A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-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 transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

Animals↗
PubMed1995

Fibronectin expression during physiological and pathological cardiac growth.

Fibronectin (FN) is a dimeric glycoprotein found in the extracellular matrix of most tissues that serves as a bridge between cells and the interstitial collagen meshwork and influences diverse processes including cell growth, adhesion, migration, and wound repair. Multiple FN forms arise by the alternative splicing of a primary transcript originating from a single gene. The spatial and temporal alterations in FN expression in the myocardium has been studied in models of cardiac growth in vivo such as fetal development, and hypertrophy secondary to pressure overload. This review focuses on the differential expression of FN isoforms that are observed in different models of cardiac growth. Using a combination of qualitative and quantitative analyses it is shown that in the rat myocardium: (1) the FN phenotype is developmentally regulated, (2) the re-expression of the fetal FN isoforms is observed in different models of cardiac hypertrophy secondary to a sudden or progressive hypertension and (3) the changes in cardiac FN expression affect mostly the coronary artery smooth muscle cells.

Alternative Splicing↗
PubMed1994

Differential splicing of fibronectin pre-messenger ribonucleic acid during cardiac ontogeny and development of hypertrophy in the rat.

BACKGROUND: Fibronectin, an extracellular matrix protein, exists as multiple isoforms expressed in a time- and cell-dependent manner. Since the developmental pattern of fibronectin expression has not been determined in the heart, the first issue of this study was to investigate the expression of total fibronectin mRNA as well as its isoforms during cardiac ontogeny. In adults, pressure overload induces a shift towards the fetal form of proteins expressed by either muscle or nonmuscle cardiac cells. Fetal forms of fibronectin mRNA being found in smooth and nonmuscle cardiac cells soon after imposition of pressure overload, the pattern of fibronectin expression during the development of pathological growth was analyzed to determine whether the two conditions of cardiac growth resulted in an identical pattern of fibronectin expression. EXPERIMENTAL DESIGN: Total RNA were isolated from rat heart (a) during in utero and postnatal life and (b) at varying periods of time after imposition of a pressure overload induced by coarctation of the thoracic aorta in 25-day-old rats. Fibronectin-EIIIA+ or -EIIIB+ and total fibronectin mRNAs were quantitated by reverse transcription-polymerase chain reactions and dot-blot analysis, respectively. RESULTS: Fibronectin mRNA, abundant in the 14-day-old fetal heart, rapidly decreased during cardiac physiologic growth (> 5-fold); no changes in the fibronectin mRNA level was observed during the development of pressure-induced cardiac hypertrophy. The percentages of fibronectin transcripts containing EIIIA or EIIIB exons, very high in the early fetal heart (> 45%), harmoniously decreased during cardiac maturation (< 12%). Aortic coarctation resulted in an early, transient (12 to 48 hours) and preferential expression of fibronectin-EIIIA+ mRNA (approximately 40%). CONCLUSIONS: In rat heart, neither physiologic nor pressure-induced growth requires increased amounts of fibronectin mRNA but the growth conditions specifically modulated the fibronectin pre-mRNA splicing.

Animals↗
PubMed1993

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.

Actins↗
PubMed1992

Pyrimidine nucleotide synthesis is preferentially supplied by exogenous cytidine in adult rat cultured cardiomyocytes.

The metabolism of pyrimidine nucleotides was studied in non-contracting myocytes isolated from adult rat hearts and compared to that observed in freshly prepared myocardium. The myocytes were cultured for up to 96 hrs in a commercial medium containing 50 microM cytidine, uridine, adenosine and adenine; 20 microM guanosine, thymidine and D-ribose; and 5 microM hypoxanthine, xanthine, guanine, thymine and uracil. Nucleotide pool sizes were measured by HPLC. Nucleotide and RNA labelling were followed by incorporation of [U-14C]-cytidine or [U-14C]-uridine added in trace amounts to the medium. The adenine nucleotide pool was 2.4-fold larger than in situ after 7 hrs of incubation and then returned to values 30% higher than that found in the myocardium after 25 hrs. Cytosine and uracil nucleotide pools after 25 hrs of culture were respectively 2 and 4-fold larger than in situ and remained at these levels thereafter. Intracellular cytidylate and uridylate equilibrated very rapidly with exogenous [U-14C]-cytidine but not with [U-14C]-uridine. We conclude that, under the experimental conditions used here, the synthesis of pyrimidine nucleotides in isolated myocytes is mainly supplied by exogenous nucleosides. Furthermore, extracellular cytidine is rapidly converted to both uracil and cytosine nucleotides while uridine serves only as the precursor for uracil nucleotide synthesis.

Adenine Nucleotides↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Ischemia of the dog heart induces the appearance of a cardiac mRNA coding for a protein with migration characteristics similar to heat-shock/stress protein 71.

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.

Animals↗
PubMed1986

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.

Cell Count↗
PubMed1985

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)

Animals↗
PubMed1984

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.

Animals↗
PubMed1983

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart.

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.

Animals↗