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M Anger

Publications and source records attributed to M Anger.

15 recordsLinked to original sources
PubMed1998

Cellular distribution of Ca2+ pumps and Ca2+ release channels in rat cardiac hypertrophy induced by aortic stenosis.

BACKGROUND: The response of ventricular myocytes to pressure overload is heterogeneous and not spatially coordinated. We investigated whether or not the alterations in SERCA and RyR gene expression are homogeneous within the myocardium. METHODS AND RESULTS: The cellular distribution of mRNAs and proteins encoding the 2 sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) isoforms (SERCA 2a and 2b) and 2 Ca2+ release channels (the ryanodine receptor, RyR, and the IP3 receptor, IP3R) were analyzed by in situ hybridization and immunofluorescence, respectively. Analyses were performed during early (1 and 5 days) and late (1 month) stages of cardiac hypertrophy induced in rat by thoracic aortic stenosis (AS). The results indicated that 1 and 5 days after AS, the cellular distribution of SERCA 2a and RyR2 mRNAs in right ventricle and atrium was similar to controls but the mRNA levels appeared to decrease in some areas of the left ventricle (LV). One month after AS, the distribution of SERCA 2a mRNA and protein became heterogeneous throughout the LV, whereas RyR2 mRNA and protein levels were decreased in a homogeneous manner. SERCA 2b, poorly expressed in both cardiomyocytes and vessels of controls, was increased 4-fold 1 month after AS in coronary arteries only. In both sham (Sh) and AS, SERCA 3 and IP3R mRNAs were mainly found in the vessels. CONCLUSIONS: In severe hypertrophy, decreased accumulation of SERCA 2a was heterogeneous and not compensated by an induction of SERCA 2b in the cardiomyocytes. Decrease in RyR2 expression was more homogeneous and not compensated by an increased IP3R expression.

Animals↗
PubMed1998

Automated recording of RNA differential display patterns from pig granulosa cells.

We have developed a protocol for fast, nonradioactive, mRNA differential display reverse transcription PCR (DDRT-PCR) based on a commercial automated sequencer with RNA isolated from pig granulosa cells. We sought to discover conditions that would minimize the problem of using relatively small primers labeled with large infrared dye molecule, IR41, required for the sequencer. Extended IR41-labeled primers IR41-AAGC-T11-A, IE41-AAGC-T11-C and IR41-AAGC-T11-G gave more consistent differential display patterns than shorter anchored primers (IR41-T11A, IR41-T11C and IR41-T11G) without the additional (AAGC) cloning site. The optimal concentration of the extended labeled (downstream) primers was 20 pmol when 13-mer arbitrary (upstream) primers were used at a concentration of 4 pmol. Background smear and the intensity of amplified bands was significantly improved by changing from conventional Taq DNA polymerase to AmpliTaq Gold polymerase, which permits an improved "hot start" for the reaction. Running time (during which a digitized gel image is recorded) for a 26-cm polyacrylamide gel was 4 h, enabling us to analyze 90 reactions in an 8-h day. This protocol offers a rapid and reliable nonradioactive method for comparing gene expression patterns for various research or diagnostic purposes.

Animals↗
PubMed1995

Sarcoplasmic reticulum Ca2+ pumps in heart and diaphragm of cardiomyopathic hamster: effects of perindopril.

The polymyopathy of the Syrian hamster is associated with alterations of cellular calcium regulation and contractile performance of cardiac and skeletal muscles and, in particular, the diaphragm. Angiotensin-converting enzyme (ACE) inhibitors have been shown to preserve contractile performance. Therefore we analyzed the expression of the genes coding for the sarco(endo)plasmic reticulum Ca(2+)-adenosinetriphosphatase (SERCA) in heart and diaphragm of the cardiomyopathic Syrian hamster (CSH) from the dilated strain Bio 53-58, and we tested the influence of ACE inhibition on accumulation of the different SERCA mRNAs. In the diaphragm of healthy hamsters, two SERCA mRNA isoforms were present: SERCA 1 and SERCA 2. At 6 mo of age, the myopathic process resulted in decreased levels of SERCA 1, whereas the level of SERCA 2 was unchanged. The ACE inhibitor perindopril (1 mg.kg-1.day-1), administered by force feeding from 1 to 6 mo of age, had no effect on the SERCA 1 mRNA level. In heart, the myopathy was associated with a depressed level of SERCA 2 mRNA in 9- but not in 6-mo-old animals. Perindopril treatment from 6 to 9 mo reversed cardiac hypertrophy and the relative decrease in SERCA 2 mRNA level. Preventive treatment with perindopril from 1 to 9 mo tended to prevent (not significantly) the development of cardiac hypertrophy and reduction in SERCA gene expression. In conclusion, the myopathic process affects SERCA gene expression in the diaphragm and subsequently in the heart. Perindopril treatment can prevent SERCA mRNA loss in heart but not in diaphragm.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Animals↗
PubMed1994

Measurements of ATP binding on the large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase overexpressed in Escherichia coli.

The large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase (LCL), situated between Lys329 and Phe740, is believed to contain both its phosphorylation and ATP binding domains. A cDNA fragment coding for this amino acid sequence was generated in vitro and cloned in vector pQE8 which allowed the overexpression in Escherichia coli of this Ca(2+)-ATPase domain fused with a cluster of 6 histidines at its NH2 terminus. The fusion protein produced in an insoluble form within bacteria was solubilized in 4 M urea, purified on immobilized Ni2+, and then renatured by elimination of urea. More than 4 mg of purified renatured fusion protein was obtained from 500 ml of culture. ATP binding on the refolded protein was demonstrated by two methods: 1) detection of ATP-induced intrinsic fluorescence change and 2) binding of the fluorescent ATP analogue 2',3'-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP) and its chase by ATP. It is shown that the LCL protein has one single TNP-ATP binding site having a dissociation constant (Kd) of 1.6-1.9 microM. Both methods yielded a Kd for ATP around 200 microM. Binding of other nucleotides was detected with a sequence of Kd identical to that found for native Ca(2+)-ATPase: ATP < ADP < GTP < AMP < ITP. A Mg2+ binding site was also found on the LCL protein (Kd = 100 microM at pH 7.2). The fluorescence of bound TNP-ATP was found to be highly dependent on Mg2+ binding on this site.

Adenosine Triphosphate↗
PubMed1994

In situ mRNA distribution of sarco(endo)plasmic reticulum Ca(2+)-ATPase isoforms during ontogeny in the rat.

The Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) plays a crucial role in maintaining the Ca2+ homeostasis, which itself, controls various essential cellular function. The existence of several SERCA isoforms, encoded by three different genes and produced by alternative splicing of pre-mRNA transcripts, has been established by cDNA cloning. However, the temporo-spatial evolution of their expression during ontogeny was unknown. We have used in situ hybridization to determine the cellular distribution of three of these mRNA isoforms, SERCA 2a, SERCA 2b and SERCA 3 during rat ontogeny and focused our study on the cardiovascular system. We demonstrate that early in embryogenesis, SERCA 3 mRNA is highly expressed in the heart tube and is also present in the yolk sac. In 14-16 days embryos, SERCA 3 mRNA has disappeared from the heart but is expressed in the aorta and in discrete foci of the liver. Later on, its expression in the cardiovascular system is restricted to the arterial endothelium. SERCA 2a mRNA is coexpressed with SERCA 3 mRNA in the heart tube and remains expressed in the cardiomyocytes throughout life. It is transiently expressed in skeletal muscle at the onset of differentiation. In early foetal life, SERCA 2b is expressed in the mesenteric area and thereafter in all cell types at various levels. Our data indicate that (i) expression of SERCA 2b is neither tissue-specific nor developmentally regulated (ii) expression of SERCA 2a and SERCA 3 isoforms is regulated in a cell specific manner during development and suggest that the SERCA 3 gene plays a role in controlling the function of endothelial cells during vasculogenesis.

Aging↗
PubMed1993

The sarco(endo)plasmic reticulum Ca(2+)-ATPase mRNA isoform, SERCA 3, is expressed in endothelial and epithelial cells in various organs.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase mRNA isoform, SERCA 3, was previously shown to be expressed in a great variety of muscle and non-muscle tissues [(1989) J. Biol. Chem. 264, 18568] but its cellular localization within these organs was unknown. We have used in situ hybridization and RNase protection techniques to demonstrate that SERCA 3 mRNA is expressed in specific cell types, namely the endothelial and epithelial cells.

Animals↗
PubMed1993

Rabbit masseter expresses the cardiac alpha myosin heavy chain gene. Evidence from mRNA sequence analysis.

The presence of myosin alpha heavy chain in the rabbit masseter has been previously suggested at the protein level [(1991) Basic App. Myol. 1, 23-34; (1991) Histochem. J. 23, 160-170]. To confirm this finding, we cloned most of the mRNA corresponding to the myosin heavy chain S2 subfragment. PCR analysis and subsequent nucleotide sequence determination of the amplified cDNA demonstrates the presence of a myosin alpha heavy chain mRNA in rabbit masticatory muscles.

Amino Acid Sequence↗
PubMed1993

Age-related changes in sarcoplasmic reticulum Ca(2+)-ATPase and alpha-smooth muscle actin gene expression in aortas of normotensive and spontaneously hypertensive rats.

The expression of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase gene and the SR Ca2+ pump function were investigated in thoracic aortas of 5- and 17-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The relative level of the two isoforms of SR Ca(2+)-ATPase mRNA expressed in the aorta (i.e., SERCA 2a and SERCA 2b) was determined by quantitative S1 nuclease protection analysis and normalized to the level of alpha-smooth muscle (alpha-Sm) actin mRNA. The level of alpha-Sm actin mRNA itself was normalized to the level of 18S ribosomal RNA using slot-blot hybridization assays. Total SR Ca2+ pump activity was estimated by measuring the rate of oxalate-supported Ca2+ uptake in homogenates. At 5 weeks, the amount of SERCA 2a and SERCA 2b mRNA, normalized to 18S ribosomal RNA, and the ratio of alpha-Sm actin mRNA to 18S RNA were identical in SHR and WKY rats. The Ca2+ pump activity was similar in the two strains of rats at 5 weeks. From 5 to 17 weeks, the amount of SERCA 2a mRNA increased in both strains while the level of SERCA 2b mRNA remained constant. The Ca2+ pump activity was unchanged in SHRs and tended to decrease in WKY rats. Accordingly, the change in the ratio of the SR Ca(2+)-ATPase mRNA isoforms does not appear to influence SR function. The level of alpha-Sm actin mRNA and SERCA 2a mRNA increased in parallel from 5 to 17 weeks in both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMedDate not supplied

[Lip sinus].

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Child, Preschool↗