Biomedical subjects

A Zarain-Herzberg

Publications and source records attributed to A Zarain-Herzberg.

16 recordsLinked to original sources

PubMed2003

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina.

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.

Alternative Splicing↗

PubMed1999

Transcriptional modulators targeted at fuel metabolism of hypertrophied heart.

The transition of nonfailing to failing cardiac hypertrophy cannot be prevented by current drug regimens. This investigation examined whether possible drug targets have remained unexplored because they do not result in acute improvement of heart function. Of major importance, in this respect, is an inadequate performance of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2). In the present approach, binding sequences within the proximal promoter of SERCA2 are described which may be useful in the development of drugs (i.e., transcriptional modulators) that interfere selectively with the transcription of genes of the cardiomyocyte. The proximal promoter region of the SERCA2 genes has a thyroid response element, 9 potential Sp1-binding sites (5'-GGGCGG-3', 5'-CCGCCC-3' and 5'-GGGAGG-3'), and an E-box motif (5'-CACATG-3'), which may function as glucose response elements. This region also has 2 putative fatty-acid response elements (5'-GGGGGA-3'). It is proposed that the beneficial effects of the camitine palmitoyltransferase-1 inhibitor etomoxir arise from a shift in fuel metabolism involving glucose response elements and/or peroxisomal proliferator-activated receptors. Although the relative contribution of these DNA regulatory elements remains to be defined, it appears that they provide the driving force that prevents the decrease in transcriptional activity of the SERCA2 gene in the hypertrophic heart. It is further concluded that etomoxir represents a member of a novel class of transcriptional modulators that improve function of hypertrophied hearts with unimpeded blood flow by modulating gene expression of the cardiomyocyte.

Animals↗

PubMed1999

Captopril treatment improves the sarcoplasmic reticular Ca(2+) transport in heart failure due to myocardial infarction.

Although captopril, an angiotensin-converting enzyme (ACE) inhibitor, has been shown to exert a beneficial effect on cardiac function in heart failure, its effect on the status of sarcoplasmic reticulum (SR) Ca(2+) transport in the failing heart has not been examined previously. In order to determine whether captopril has a protective action on cardiac function, as well as cardiac SR Ca(2+)-pump activity and gene expression, a rat model of heart failure due to myocardial infarction was employed in this study. Sham operated and infarcted rats were given captopril (2 g/l) in drinking water; this treatment was started at either 3 or 21 days and was carried out until 8 weeks after the surgery. The untreated animals with myocardial infarction showed increased heart weight and elevated left ventricular end diastolic pressure, reduced rates of pressure development and pressure fall, as well as depressed SR Ca(2+) uptake and Ca(2+)-stimulated ATPase activities in comparison with the sham control group. These hemodynamic and biochemical changes in the failing hearts were prevented by treatment of the infarcted animals with captopril. Likewise, the observed reductions in the SR Ca(2+) pump and phospholamban protein contents, as well as in the mRNA levels for SR Ca(2+) pump ATPase and phospholamban, in the failing heart were attenuated by captopril treatment. These results suggest that heart failure is associated with a defect in the SR Ca(2+) handling and a depression in the gene expression of SR proteins; the beneficial effect of captopril in heart failure may be due to its ability to prevent remodeling of the cardiac SR membrane.

Animals↗

PubMed1996

Analysis of mRNA expression and cloning of a novel plasma membrane Ca(2+)-ATPase splice variant in human heart.

Four different plasma membrane Ca(2+)-ATPase (PMCA) genes and three sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) genes have been previously cloned and characterized. In this study we have investigated the expression of the mRNA encoding the various PMCA and SERCA proteins in fetal and adult human heart and placenta by the reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cDNA cloning. We have found that PMCA1 and PMCA4 genes were expressed in 8-, 12- and 20-week fetal heart and in adult heart. PMCA2 gene was expressed at low levels in adult heart but was not detected in fetal heart. PMCA3 mRNA was not detected in the heart nor placenta. In contrast, the mRNA encoding SERCA2a, SERCA2b and SERCA3 were expressed in all cardiac developmental stages. Multiple alternatively spliced mRNA transcripts which differ at splice site A and B/C of the PMCA1, PMCA2 and PMCA4 genes were detected in the human heart. Interestingly, a novel tissue specific variant of the PMCA4 gene was detected in both fetal and adult human heart but not in placenta that accounts for about 30% of the total PMCA4 mRNA variant expression. DNA sequence analysis of this novel variant revealed that it corresponds to the equivalent of the PMCA1d variant and accordingly we have named it PMCA4d. We cloned and sequenced eight cDNA inserts encoding for the PMCA1 and PMCA4 variants from a fetal human heart cDNA library confirming that these are the two main PMCA genes expressed in cardiac muscle.

Adult↗

PubMed1996

Modification of sarcoplasmic reticulum gene expression in pressure overload cardiac hypertrophy by etomoxir.

Pressure overload on the heart is known to produce hypertrophy of cardiomyocytes and distinct changes in protein phenotype, including reduced expression of the gene for the sarcoplasmic reticulum (SR) Ca2+ATPase (SERCA2). In this study we have shown that the decrease in SERCA2 gene expression (normalized by poly(A)+ mRNA or 18 S rRNA) in rats with 8 wk of aortic constriction was prevented by treatment with etomoxir, an inhibitor of carnitine palmitoyltransferase 1. The reduction in steady-state mRNA levels for SR phospholamban (PLP) and Ca2+ release channel (CRC) in the pressure-overloaded animals was also prevented without any reduction in the extent of cardiac hypertrophy by treatment with etomoxir. Although no changes in mRNA levels for GAPDH were evident in rats with pressure overload, the expression of the alpha-skeletal actin was increased; this change was prevented by etomoxir. Similar beneficial effects of etomoxir treatment were also evident when the gene expression for SR SERCA2, PLP, and CRC in the hypertrophied heart was normalized with respect to mRNA for GAPDH. These results support the view that drugs such as etomoxir may increase the abundance of the mRNA for SR proteins in the hypertrophied heart and thus may prevent the transition of cardiac hypertrophy into heart failure.

Animals↗

PubMed1994

Cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in streptozotocin-induced diabetic rat heart.

Chronic diabetes due to streptozotocin administration has been shown to induce heart dysfunction characterized by prolonged relaxation time as well as decreased Ca2+ transport and Ca(2+)-ATPase (SERCA2) activities of the cardiac sarcoplasmic reticulum (SR). Rats made diabetic with 65 mg/kg of streptozotocin for 3 and 5 weeks exhibited decreased SR Ca(2+)-pump activities; these were normalized upon treatment with insulin. Northern blot and slot blot analyses did not show statistically significant reduction in the relative level of SERCA2 mRNA expression in diabetic or insulin treated rats. Quantitation of SERCA2 protein by Western blot did not reveal any change in diabetic and insulin treated animals. These results suggest that the defect in SR Ca(2+)-pump may not be due to changes at the transcriptional or translational levels in the diabetic heart.

Animals↗

PubMed1994

Sarcoplasmic reticulum calsequestrins: structural and functional properties.

Calsequestrin is the major Ca(2+)-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its alpha-helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsequestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.

Amino Acid Sequence↗

PubMed1994

Thyroid hormone receptor modulates the expression of the rabbit cardiac sarco (endo) plasmic reticulum Ca(2+)-ATPase gene.

We have analyzed the effect of thyroid hormone (T3) on SERCA2 gene expression using fetal chicken primary cardiac myocytes and C2C12 skeletal muscle cells in culture. Northern blot analysis of both cell types demonstrated that T3 induced a 3-fold accumulation of SERCA2 mRNA compared with cells grown in medium lacking T3. We have engineered deletion constructs containing various lengths of the 5'-flanking region from the rabbit SERCA2 gene which we have cloned previously (Zarain-Herzberg, A., MacLennan, D. H., and Periasamy, M. (1990) J. Biol. Chem. 265, 4670-4677). A stable transfectant in C2C12 containing a chimeric SERCA2/CAT gene construct including -254 base pairs (bp) of SERCA2 5'-flanking region showed increased transcription activity upon the addition of 50 nM T3. We have analyzed the expression of several deletion constructs spanning 1,102 bp of the 5'-up-stream sequence of the SERCA2 gene by functional expression assays. Transient coexpression of the T3 receptor alpha 1 with various SERCA2/CAT deletion constructs showed trans-activation of chimeric constructs containing more than -267 bp, indicating that a thyroid hormone-responsive element was localized, at least in part, to the region -267 to -72 bp. T3 receptor-DNA binding assays demonstrated binding of the rat T3 receptor alpha 1 to a fragment containing a proposed T3 response element located between position -254 and -72 in the 5'-flanking region of the SERCA2 gene.

Animals↗

PubMed1991

Structure of the rabbit phospholamban gene, cloning of the human cDNA, and assignment of the gene to human chromosome 6.

We have isolated and characterized genomic DNA clones encoding rabbit phospholamban. Only a single gene for phospholamban was detected in the rabbit genome. The phospholamban gene of 13.2 kilobases contains only one 10.5-kilobase intron, which separates exonic sequences located in the 5'-untranslated region. Two potential transcription initiation sites were mapped to 335 and 185 nucleotides upstream from the translation initiation site in the mRNA or 239 and 89 nucleotides upstream from the exon/intron boundary. Phospholamban gene expression in various smooth muscle tissues, as well as in cardiac and slow twitch skeletal muscle, was detected by Northern blotting. The phospholamban gene was mapped to human chromosome 6, using a human phospholamban cDNA.

Adenosine Triphosphatases↗

PubMed1990

Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene.

We have isolated three overlapping genomic clones extending over 39 kilobases (kb), which encodes the rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene (SERCA2). S1 nuclease mapping and primer extension analysis of the 5' end of the cardiac/slow-twitch (SERCA2a) and smooth/non-muscle (SERCA2b) mRNAs showed that both transcripts are initiated from the same transcription initiation site, located 528 base pairs (bp) upstream of the translation initiation codon AUG. The putative promoter revealed a "TATA box" like element at -24 bp and a "CAAT box" at -78 bp relative to the cap site. A number of DNA sequence elements that could bind trans-acting factors were also found within the 1.8 kb of DNA sequence upstream from the transcription initiation site. To determine the DNA sequences governing transcriptional regulation, we have stably transfected the myogenic cell line C2C12 with a plasmid containing the putative promoter and 946 bp upstream sequence of the SERCA2 gene, coupled to the chloramphenicol acetyltransferase gene. Our results show that this chimeric plasmid construct exhibits appropriate activation and coordinate expression with the endogenous SERCA2 gene during the terminal differentiation of myoblasts into myotubes, suggesting that it contains the promoter and upstream sequence elements required for the regulated expression of the SERCA2 gene.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase.

We have isolated and sequenced full-length cDNA clones from a rabbit uterine library which encode the smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. These cDNAs resulted from an alternative splice of the cardiac/slow-twitch Ca2+-ATPase gene transcript, and encoded a protein identical to rabbit cardiac/slow-twitch Ca2+-ATPase except for the replacement of the carboxyl-terminal four amino acids with an extended and relatively hydrophobic sequence of 49 amino acids. This cDNA was virtually identical to the alternatively spliced product of the cardiac/slow-twitch Ca2+-ATPase gene recently identified in human kidney (Lytton, J., and MacLennan, D. H. (1988) J. Biol. Chem. 263, 15024-15031) and rat non-muscle tissues (Gunteski-Hamblin, A.-M., Greeb, J., and Shull, G. (1988) J. Biol. Chem. 263, 15032-15040). S1 nuclease mapping of total cellular RNA from a variety of tissues demonstrated that cardiac muscle expressed the cardiac/slow-twitch isoform almost exclusively, most smooth muscle and non-muscle tissues expressed the alternatively spliced smooth/non-muscle isoform almost exclusively, and a few tissues expressed both isoforms in varying amounts. Thus, regulation of alternative splicing of the cardiac/slow-twitch Ca2+-ATPase gene transcript is tissue-specific. The expression of the smooth/non-muscle isoform in every tissue tested supports the hypothesis that this molecule represents the "housekeeping" endoplasmic reticulum Ca2+-ATPase.

Amino Acid Sequence↗

PubMed1989

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.

Animals↗

PubMed1988

Structure of the rabbit fast-twitch skeletal muscle calsequestrin gene.

We have isolated two overlapping 17-kilobase (kb) genomic clones which, together, encode the rabbit fast-twitch skeletal muscle calsequestrin. The calsequestrin gene is 8.6 kb in length and consists of 11 exons. S1 nuclease protection and primer extension analyses position the transcription initiation site 191 nucleotides upstream of the translation initiation codon. The 5'-flanking sequence contains a CCAAT sequence and two CG-rich sequences but does not have an identifiable TATA box sequence in the anticipated position just upstream of the transcription initiation site. The sequence of the calsequestrin gene confirmed the sequence deduced from cDNA clones derived from neonatal muscle mRNA (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171). We could not detect any potential alternative splicing of the transcript in an analysis of this region of the gene that would account for the different sequence found at the carboxyl terminus of adult rabbit fast-twitch skeletal muscle calsequestrin.

Amino Acid Sequence↗

PubMed1988

Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene.

We have isolated two genomic clones which together encode the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. One of the two 16.5 kilobase (kb) genomic inserts in the lambda phage vector Charon 4A contains 23 exons extending from the polyadenylation site at the 3' end of the ATPase gene to within 38 nucleotides of the translation initiation codon in the 5' exon. An overlapping genomic insert of 16.5 kb contains the remainder of the 5' exon and a further 8 kb of upstream sequence. S1 nuclease mapping and primer extension analysis of the 5' end of the Ca2+-ATPase mRNA indicate that the transcription initiation site is located 185 base pairs (bp) upstream of the translation initiation codon. A "TATA" box (CA-TAAA) was found at position -30 and the sequence CCAAT was found at position -78 relative to the transcription initiation site. In a previous study (Brandl, C. J., de Leon, S., Martin, D. R., and MacLennan, D. H. (1987) J. Biol. Chem. 262, 3768-3774) cDNAs for neonatal and adult forms of the fast-twitch Ca2+-ATPase were shown to encode different carboxyl-terminal sequences, presumably as a result of alternative splicing. We have now found that these different DNA sequences encoding different carboxyl-terminal sequences are located in different exons. Exon boundaries of the Ca2+-ATPase gene did not correlate well with proposed domain boundaries for the Ca2+-ATPase protein. The locations of exon/intron boundaries were only partially conserved between the Ca2+-ATPase gene and a Na+/K+-ATPase gene (Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., Allikmets, R. L., Ushkaryov, Y. A., Melkov, A. M., Smirnov, Y. V., Malyshev, I. V., Dulubova, I. E., Petrukhin, K. E., Gryshin, A. V., Sverdlov, V. E., Kiyatkin, N. I., Kostina, M. B., Modyanov, N. N., and Sverdlov, E. D. (1987) FEBS Lett. 213, 73-80) and they did not follow closely the boundaries of amino acid sequences that are highly conserved among a group of ion transport ATPases.

Amino Acid Sequence↗

PubMed1985

Characterization of a calcium-dependent ATPase in Entamoeba invadens.

A high-affinity calcium-dependent ATPase (Ca2+-ATPase) was identified in a crude plasma membrane fraction from Entamoeba invadens (IP-1 strain). The Ca2+-ATPase activity was solubilized from the membrane by utilizing the non-ionic detergent octylglucoside. The activity had an apparent half maximal saturation constant of 0.4 +/- 0.05 microM for free calcium. The calcium activation of ATPase activity followed a cooperative mechanism (Hill number of 2.3 +/- 0.13) which suggests that two interacting sites were involved. The high-affinity Ca2+-ATPase appeared to be magnesium-independent, since by lowering contaminant free magnesium with trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid did not modify the activity observed with Ca2+. The apparent Km of the enzyme for ATP was 31 microM. The observed activity had an optimum pH of 8.8. The enzyme was insensitive to various agents such as Na+, K+, ouabain, dicyclohexylcarbodiimide, KCN, NaN3, mersalyl, quercetin, ruthenium red and vanadate. Only lanthanum (0.5 mM) inhibited 100% the enzymatic activity. Calmodulin and trifluoperazine at the concentrations tested did not modify the Ca2+-ATPase activity.

Adenosine Triphosphate↗

PubMedDate not supplied

Decreased expression of cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase in congestive heart failure due to myocardial infarction.

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca(2+)-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca(2+)-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca(2+)-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.

Animals↗