Biomedical subjects

R Zak

Publications and source records attributed to R Zak.

At least 19 recordsLinked to original sources

PubMed1992

Effects of exercise training and diabetes on cardiac myosin heavy chain composition.

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. alpha-MHC and beta-MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of alpha-MHC. Sedentary diabetics exhibited a shift to beta-MHC. Exercise trained diabetic rats showed a predominance of beta-MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the beta-MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.

Animals↗

PubMed1992

cis-acting elements responsible for muscle-specific expression of the myosin heavy chain beta gene.

The 5' flanking region of the rabbit myosin heavy chain (HC) beta gene extending 295 bp upstream from the cap site provides muscle-specific transcriptional activity. In this study, we have identified and functionally characterized cis-acting elements that regulate the muscle-specific expression within this region. By using linker-scanner (LS) mutants between -295 bp and a putative TATA box, we found five distinct positive cis-acting sequences necessary for transcription: element A, the sequences between -276 and -263, which contains a putative M-CAT motif in an inverted orientation; B, the sequences between -207 and -180; C, the sequences between -136 and -127; D, the sequences between -91 and -80; and E, a TATA consensus sequence at -28. The fragment containing both A and B elements dramatically enhanced the expression of the chloramphenicol acetyltransferase (CAT) gene driven by a heterologous promoter in differentiated muscle cells, whereas fragments containing either A or B elements alone had little or no effect in either muscle or nonmuscle cells. Therefore, these two elements appear to act cooperatively in determining a high level of muscle- and stage-specific expression. Unlike the typical enhancer element, this region functions in an orientation-dependent manner. In contrast, the fragment containing C and D elements activates the heterologous promoter in both muscle and nonmuscle cells in an orientation-independent manner.

Animals↗

PubMed1992

Isolation and characterization of a rat ventricular cDNA expressed specifically in cardiac and skeletal muscles.

We describe the isolation of a novel cDNA named Myomy and show that its transcripts are present in skeletal and cardiac muscles as well as in differentiated Sol 8 skeletal muscle cell line. Sequence analysis revealed that neither nucleotides nor deduced protein product have any significant homology to those previously described. The encoded protein of Myomy cDNA consists of 76 amino acids and has a molecular weight of 8,000 dalton. Based on its muscle specific expression, low abundance and a higher occurrence of SP(T)XX, S(T)S(T)XX motifs, we suggest that Myomy encodes a new muscle specific transcription factor.

Amino Acid Sequence↗

PubMed1992

Expression of alpha-cardiac and alpha-skeletal actin mRNAs in relation to innervation in regenerating and non-regenerating rat skeletal muscles.

The expression of alpha-cardiac and alpha-skeletal actin mRNA in regenerating muscle was examined. Changes in mRNA levels were analyzed in autografted extensor digitorum longus (EDL) muscles in rats using alpha-isoform specific synthetic oligonucleotides and beta-actin cDNA as probes. After autografting, the expression of alpha-cardiac actin mRNA was induced; concomitantly that of alpha-skeletal actin mRNA was reduced. The pattern of alpha-actin mRNA expression appeared to be similar to that seen in embryonic skeletal muscle. In order to evaluate the effects of innervation on alpha-actin mRNA expression in regenerating muscle, nerveless, standard, and nerve-intact autografted muscles were examined. More complete innervation facilitated the recovery of alpha-skeletal actin mRNA to control levels, but had little effect on the amount of alpha-cardiac actin mRNA. We found that regenerating muscle shows that embryonic pattern of alpha-actin mRNAs in the early stage and concluded that the recovery of alpha-skeletal actin mRNA expression to the adult pattern is influenced by innervation, while alpha-cardiac actin mRNA expression is nerve independent.

Actins↗

PubMed1992

Predictors of blinding or serious eye injury in blunt trauma.

Multivariate analysis was used to identify factors predicting injury and visual outcome in 94 blunt trauma patients evaluated for eye injuries among 6700 admissions to a level I trauma center over a 29-month period. Patients with penetrating eye injuries were excluded from this review. Eye injury was detected in 93% or 87 of the patients evaluated. Seven percent of eye injuries resulted in blindness, 22% were serious (visual acuity between 20/40 and 20/200 or eye injury requiring surgery), and 71% were temporary (final visual acuity of 20/40 or better). The presence of an afferent pupillary defect or a nonreactive pupil was the most important factor in predicting the severity of eye injury (p = 0.0023), followed by facial fractures (p = 0.0084), and no eye opening or eye opening to pain within the Glasgow Coma Scale (p = 0.02). Eye injury is an infrequent complication of blunt trauma. Appropriate consultation for evaluation of this problem can be obtained based on findings from the initial history and screening physical examination.

Adolescent↗

PubMed1992

Both muscle-specific and ubiquitous nuclear factors are required for muscle-specific expression of the myosin heavy-chain beta gene in cultured cells.

Expression of the myosin heavy-chain beta gene is controlled by multiple cis-acting regulatory elements in the 5' flanking region; two of these, referred to as A (-276 to -263) and B (-207 to -180), are essential for conferring muscle-specific activation on homologous and heterologous promoters. Here we report on the identification of nuclear protein factors that specifically bind to these two elements. By using the A element as a probe, as well as nuclear extracts from muscle cells, we found two protein-DNA complexes that displayed distinct bands in a gel mobility shift assay but had identical methylation interference patterns. One complex was present mainly in nuclear extracts from undifferentiated muscle and nonmuscle cells, whereas the other was observed mainly in nuclear extracts from differentiated muscle cells. Thus, the muscle-specific complex formation with the A element appears to be involved in determining tissue-specific expression. Furthermore, competition analysis demonstrated that the A-element-binding factors also bind to the muscle-CAT motif in the cardiac troponin T gene. By using the B element as a probe, we saw similar patterns of gel-shifted bands and methylation interference in nonmuscle and muscle nuclear extracts. In addition, both elements A and B were found to be necessary for tissue-specific expression, suggesting that the muscle-specific activation of the myosin heavy-chain beta gene may require interaction between a muscle-specific and a ubiquitous protein-DNA complex.

Animals↗

PubMed1992

Reversibility of load-induced changes in myosin heavy chain gene expression.

Changes in myosin expression were studied after the removal of functional overload of skeletal and cardiac muscles. In the first set of experiments the chicken slow tonic anterior latissimus dorsi muscle was overloaded by placing a lead band over the wing. In the overloaded muscles the normal developmental progression toward elimination of slow myosin SM-1 was accelerated. No reexpression of SM-1 took place after weight removal. In the second set of experiments rat heart was overloaded by constriction of abdominal aorta. Isomyosin composition and the abundance of mRNA coding for alpha- and beta-myosin heavy chain (MHC) was determined. The overload resulted in downregulation of the alpha-MHC with corresponding upregulation of beta-MHC expression. Debanding resulted in the regression of hypertrophy and rapid return of alpha-MHC to normal values. In contrast, the recovery in beta-MHC expression was much slower and at 7 wk of debanding still remained substantially elevated.

Animals↗

PubMed1992

Regulation by thyroid hormone of nuclear and mitochondrial genes encoding subunits of cytochrome-c oxidase in rat liver and skeletal muscle.

Biogenesis of mitochondria involves the expression of genes located on nuclear chromosomes as well as on mitochondrial DNA. We studied the coordination of the two genomes by measuring transcript levels for nuclear (IV, Va, and VIc) and mitochondrial (II and III) subunits of cytochrome-c oxidase after altering the mitochondrial content of rat muscle and liver by altering the thyroid state of the animals. Tissue levels of these mRNAs were generally decreased in hypothyroid animals and were up-regulated again after thyroid hormone (T3) treatment. However, significant increases in the levels of all nuclear transcripts were observed in the liver 24 h after T3 treatment, but were delayed or remained unaltered (VIc) in muscle. In contrast, levels of mitochondrial transcripts were elevated early in muscle and late in liver. The abundance of the corresponding polypeptides, which were analyzed by immunoblotting, changed in direction and magnitude according to the changes in their mRNAs, indicating pretranslational control. We conclude that the two genomes are regulated by T3 not through a common coordinating mechanism, but via two separate pathways, which respond to T3 with tissue-specific kinetics. S1-nuclease protection analysis showed that probably only one transcript for subunit VIc is present in both tissues, thus excluding the possibility that the tissue-specific response is due to the expression of two isogenes. The abundance of mitochondrial DNA was unaltered despite the observed changes in mitochondrial transcripts, indicating that mitochondrial gene expression is regulated by transcriptional mechanisms and not by gene dosage as has been postulated by others.

Animals↗

PubMed1991

Correlations between a nuclear and a mitochondrial mRNA of cytochrome c oxidase subunits, enzymatic activity and total mRNA content, in rat tissues.

Cytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for III; p less than 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.

Animals↗

PubMed1991

Egr-1, a serum-inducible zinc finger protein, regulates transcription of the rat cardiac alpha-myosin heavy chain gene.

Egr-1 is an early growth response gene that encodes a protein with three zinc fingers and is involved in transcriptional regulation. In adult heart myocytes, in contrast to c-fos and c-myc, high levels of Egr-1 mRNA expression have been shown. Here we report that Egr-1 transactivates rat cardiac alpha-MHC gene expression. In serum-starved primary cultures of 18-day-old fetal rat heart myocytes, addition of serum evoked expression of both Egr-1 and alpha-MHC gene transcripts. Inclusion of 10 microM cycloheximide in these cultures for 48 h caused a greater increase in Egr-1 mRNA, whereas the expression of alpha-MHC transcripts was ablated. To examine the involvement of Egr-1 in alpha-MHC induction, we transfected primary cultures of cardiac myocytes with plasmids pCMVEgr-1 (Egr-1 expression vector) and pMP3.3CAT containing -2.9- to +0.42-kilobase sequences of the alpha-MHC gene fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of pCMVEgr-1 stimulated expression of pMP3.3CAT 10-15-fold. Furthermore, pCMVEgr-1 also stimulated expression of the endogenous alpha-MHC gene in primary cultures of cardiac myocytes. Transactivation of pMP3.3CAT expression by pCMVEgr-1 was also observed by transfecting the myogenic cell line Sol 8, but not in L6E9 cells or in NIH3T3 fibroblasts. By creating progressive 5' deletions of the alpha-MHC gene, we found that the region extending between -1698 and -1283 base pairs is necessary for Egr-1-induced expression of the alpha-MHC/CAT construct. These results define a physiological target for the Egr-1 transcription factor and delineate a novel mechanism for regulation of the alpha-MHC gene.

Animals↗

PubMed1991

Characterization of cDNA and genomic sequences encoding a chicken phospholamban.

We report on the isolation and characterization of cDNA and genomic sequences encoding a chicken cardiac phospholamban. Three mRNAs, 0.6, 1.1, and 3.3 kilobase pairs in size, were detected in cardiac and slow-tonic muscle RNAs on Northern blots. We determined that these mRNAs differ in the length of their 3'-untranslated regions, perhaps because they utilize alternative polyadenylation signals. We studied the developmental and tissue-specific expression of phospholamban mRNA using nuclease mapping analysis. There appeared to be considerable homology between coding and 3'-untranslated regions of phospholamban mRNAs from cardiac and slow-tonic muscles during development, strongly suggesting that these mRNAs are encoded by the same gene. The phospholamban gene, which is present as a single copy in the chicken genome, is about 10 kilobase pairs long, with approximately 6.5 kilobase pairs representing the intron sequences. Primer extension and nuclease mapping analyses of the 5' end of the phospholamban mRNA showed that the three transcripts are initiated from the same initiation site. Analysis of the putative promoter region revealed "TATA box" and "CAAT box" sequences, putative muscle-specific elements, and a cyclic AMP-responsive element.

Adenosine Triphosphatases↗

PubMed1991

Purification of mitochondrial DNA from total cellular DNA of small tissue samples.

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.

Animals↗

PubMed1991

Activation of alpha-myosin heavy chain gene expression by cAMP in cultured fetal rat heart myocytes.

The effect of cAMP on cardiac myosin heavy chain (MHC) gene expression in primary cultures of 18-day-old fetal rat heart myocytes was investigated. When myocytes were treated with either 10 microM forskolin or 1 mM 8-bromo-cAMP for 48 h, the relative amount of the V1 to -V3 myosin isoform ratio increased 3-fold. The abundance of alpha-MHC mRNA was also increased 3-to-4-fold in forskolin treated vs control cells. However, no appreciable change was observed in the level of beta-MHC mRNA. In addition, a 70% increase in the transcription rate of the cardiac MHC gene was observed by nuclear run-on assay following treatment of cells with 10 microM forskolin for 12 h. These results demonstrate the preferential induction of alpha-MHC mRNA by cAMP which is, in part, mediated by transcriptional activation of the gene.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.

Animals↗

PubMed1991

The role of adrenergic system in regulation of cardiac myosin heavy chain gene expression.

The cardiac phenotype exhibits considerable plasticity, being under the regulation of numerous factors, such as developmental stage, functional load, as well as nutritional and hormonal states of the animal. Several lines of evidence indicate that the adrenergic nervous system plays an important role in the redistribution of myosin isoforms in the heart. For example, chemical sympathectomy favors the expression of V3 isomyosin at the expense of V1. In this study, we have examined the effect of adrenergic pathways on the expression of cardiac myosin heavy chain (MHC) genes. The level of cAMP was modulated by either adding forskolin or 8-bromo-cAMP to primary cultures of embryonic (18 d) cardiac myocytes. We have found that the level of mRNA coding for MHC-alpha was increased two- to three-fold. The effect was dose- and time-dependent and was potentiated further when the 8-Br-cAMP was given together with a phosphodiesterase inhibitor. The same changes were found in KCl arrested cells, indicating independence of contractile activity. Treatment of cells known to activate the protein kinase C (TPA) and inositol triphosphate pathways has increased the level of beta-MHC mRNA while that of alpha-MHC remained unchanged. These data lend strong support to direct effect of the adrenergic system on activity of cardiac genes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Structural and phylogenetic analysis of the chicken ventricular myosin heavy chain rod.

We have isolated and characterized five overlapping clones that encompass 3.2 kb and encode a part of the short subfragment 2, the hinge, and the light meromyosin regions of the myosin heavy chain rod as well as 143 bp of the 3' untranslated portion of the mRNA. Northern blot analysis showed expression of this mRNA mainly in ventricular muscle of the adult chicken heart, with trace levels detected in the atrium. Transient expression was seen in skeletal muscle during development and in regenerating skeletal muscle following freeze injury. To our knowledge, this is the first report of an avian ventricular myosin heavy chain sequence. Phylogenetic analysis indicated that this isoform is a distant homolog of other ventricular and skeletal muscle myosin heavy chains and represents a distinct member of the multigene family of sarcomeric myosin heavy chains. The ventricular myosin heavy chain of the chicken is either paralogous to its counterpart in other vertebrates or has diverged at a significantly higher rate.

Amino Acid Sequence↗

PubMed1991

Quantitative approaches for studying gene expression.

The methods currently available for measuring mRNAs and proteins are reviewed, with a special emphasis on their application to physiological questions. The article focuses on the quantitative determination of cellular contents, but also on assessment of rates of synthesis and degradation, and turnover.

Animals↗

PubMed1991

Myosin heavy chain turnover during cardiac mass changes by glucocorticoids.

One aim of this investigation was to determine whether the cardiac enlargement observed with glucocorticoid treatment is temporary or remains a permanent adaptation if steroid treatment is prolonged. A second aim was to study whether myosin heavy chain (MHC) synthesis rates are coordinated with the cardiac mass responses. Female rats received either a vehicle (1% aqueous carboxymethyl cellulose in saline) or hydrocortisone 21-acetate for 1, 3, 7, 11, and 15 days. Peak cardiac enlargement (10-15%) was observed after 7 days of hormone treatment in two separate series of experiments. The enlargement was maintained through 11 days of steroid injections but by 15 days had declined toward control levels. MHC synthesis measurements were performed by constant infusion of [3H]leucine. Leucine specific activities were similar among precursor pools (intracellular, extracellular, and leucyl-tRNA) and did not vary with steroid treatments. Fractional synthesis rates of ventricular MHC (%/day) did not change during the period of increase in ventricular mass but were reduced to 56-59% of controls (-11/19.5) at 7 and 11 days of treatment, when ventricular mass increases were highest. MHC breakdown (%/day) was reduced to approximately 60% (-11.5/18.7) of controls at 7 and 11 days. Changes in total protein synthesis, which was measured in isolated perfused hearts, were similar to the MHC responses and indicated that the alterations in MHC synthesis are synchronized with the hormonal effects on total protein metabolism. These results demonstrate that peak cardiac enlargement is not maintained with long-term glucocorticoid treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗