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Biomedical subjects

M Periasamy

Publications and source records attributed to M Periasamy.

At least 19 recordsLinked to original sources
PubMed2006

Multiplex RT-PCR, a novel technique for the simultaneous detection of the DNA and RNA viruses causing rice tungro disease.

Rice tungro, economically the most important viral disease of rice, is a complex disease caused by two morphologically and genomically dissimilar viruses, Rice tungro bacilliform virus (RTBV), a double stranded DNA virus replicating through RNA intermediate and Rice tungro spherical virus (RTSV), a single stranded RNA virus with 3' poly (A) tail. A novel multiplex RT-PCR technique for the simultaneous detection of RTBV and RTSV from the total RNA extracted from tungro-infected plants has been developed. It involves a one-step reaction initiating first strand cDNA synthesis by oligo (dT) primer with poly (A) tailed RTBV transcript and RTSV genomic RNA as template for the PCR amplification. The results indicate that adaptation of this technique will strengthen the screening for tungro resistance among rice varieties and hybrids.

Badnavirus↗
PubMed2006

Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration.

The AV promoter expressing AV1 and AV2 genes and AC1 promoter expressing AC1 gene are present in opposite orientation in the intergenic region of Mungbean yellow mosaic India virus (MYMIV). Transient Agrobacterium-mediated delivery of putative promoter constructs into Nicotiana benthamiana and different legumes, followed by reporter gene (beta-d-glucuronidase, GUS) assay, identified the promoter region of both AC1 and AV genes that is necessary for transcriptional initiation. Transcription activator protein-independent activity of AV promoter and differential regulation of AC1 promoter are unique to MYMIV. The AV promoter is a composite core promoter having both TATA box and Initiator elements (TATA(+)Inr(+)). Many transcription factor binding sites were identified in the upstream promoter sequences of both virion and complementary sense genes, which might be used in the transcription regulation studies of the host plant as well as the virus.

Artificial Gene Fusion↗
PubMed2004

Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line.

The kinetics of smooth muscle are thought to be partially determined by the level of the expression of the 7 amino acid insert, SMB, in the myosin heavy chain, as SMB is generally expressed at higher levels in faster smooth muscle. In this study, we determined the role of this insert on shortening velocity and force regeneration following rapid reduction in muscle length (k(step)) in bladder tissue from a transgenic mouse line expressing the insert at three different levels: wild type (WT, +/+, SMB/SMB), an SMA homozygous type (SMB KO, -/-), and a heterozygous type (+/-, SMB/SMA). Smooth muscle from +/+ bladder shorten faster than both the +/- and -/- bladder smooth muscle when activated with Ca2+, consistent with SMB determining the shortening velocity of smooth muscle. The addition of Pi to the fully activated skinned bladder strips did not affect the rate of shortening for either the +/+ or -/- bladder types but did significantly decrease the rate of shortening for the +/- type. In contrast, the addition of ADP to fully Ca2+ activated bladder strips increased the rate of shortening for all three bladder types. However after thiophosphorylation, ADP slowed the shortening velocity. These data are consistent with shortening velocity being determined by the level of activation (or crossbridge attachment) in smooth muscle. The rates of force regeneration according to the k(step) protocol showed no differences between bladder types and also proved insensitive to either Pi or ADP. These data suggest that the rates of force regeneration were determined not only by the kinetics of the crossbridge cycle, but also by factors outside the contractile apparatus.

Adenosine Diphosphate↗
PubMed2001

Replacement of the muscle-specific sarcoplasmic reticulum Ca(2+)-ATPase isoform SERCA2a by the nonmuscle SERCA2b homologue causes mild concentric hypertrophy and impairs contraction-relaxation of the heart.

The cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a(-/-) mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a(-/-) mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a(-/-) mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a approximately 20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a(-/-) animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved beta-adrenergic response. Ca(2+) uptake levels in SERCA2a(-/-) cardiac homogenates were reduced by approximately 50%. In isolated cells, relaxation and Ca(2+) removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Alternative Splicing↗
PubMed2001

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Animals↗
PubMed2001

New methods of resolution and purification of racemic and diastereomeric amino alcohol derivatives using boric acid and chiral 1,1'-bi-2-naphthol.

Resolution of the racemic amino alcohol derivatives 1-6 is readily achieved to obtain enantiomerically enriched compounds using chiral 1,1'-bi-2-naphthol and boric acid in solvents such as CH(3)CN, THF, and MeOH. Purification of the diastereomeric mixture 7 has also been carried out following this method. The corresponding intermediate ammonium borate complexes were also characterized by X-ray diffraction methods.

Journal Article↗
PubMed2001

Squamous cell tumors in mice heterozygous for a null allele of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump.

Mutations in the human ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease, an autosomal dominant skin disease characterized by multiple keratotic papules in the seborrheic regions of the body. Mice with a single functional Atp2a2 allele (the mouse homolog of ATP2A2) were shown previously to have reduced levels of SERCA2 in heart and mildly impaired cardiac contractility and relaxation. Here we show that aged heterozygous mutant (Atp2a2(+/-)) mice develop squamous cell tumors of the forestomach, esophagus, oral mucosa, tongue, and skin. Squamous cell tumors occurred in 13/14 Atp2a2(+/-) mice but were not observed in age- and sex-matched wild-type controls. Hyperkeratinized squamous cell papillomas and carcinomas of the upper digestive tract were the most frequent finding among Atp2a2(+/-) mice, and many animals had multiple tumors. Western blot analyses showed that SERCA2 protein levels were reduced in skin and other affected tissues of heterozygous mice. The development of squamous cell tumors in aged Atp2a2(+/-) mice indicates that SERCA2 haploinsufficiency predisposes murine keratinocytes to neoplasia. These findings provide the first direct demonstration that a perturbation of Ca(2+) homeostasis or signaling can serve as a primary initiating event in cancer.

Alleles↗
PubMed2001

Nitric oxide regulates smooth-muscle-specific myosin heavy chain gene expression at the transcriptional level-possible role of SRF and YY1 through CArG element.

Nitric oxide (NO) plays an important role in vascular regulation through its vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits platelet adhesion and aggregation and modulates smooth muscle cell (SMC) proliferation and migration. In animals with experimentally induced vascular injury, ec-NOS gene transfection not only restored NO production to normal levels but also increased vascular reactivity of the injured vessels. However, it is unclear whether NO regulates smooth-muscle-specific gene expression. We report here that addition of PDGF-BB to vascular smooth muscle cells suppressed SM-MHC expression but treatment with the NO donors FK409 and SNAP restored SM-MHC mRNA/protein expression. In vitro transfection and subsequent CAT assays demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC promoter activity. Promoter deletion analysis revealed that a CArG-3 box located at -1276 bp in the SM-MHC promoter was important for NO-dependent promoter regulation and as well as high level promoter activity. Gel mobility shift assays showed that CArG-3 contained the SRF binding site and a binding site for YY1, a nuclear factor which acts as a negative regulator on muscle-specific promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3 element but increased SRF binding, suggesting that these two factors bind competitively to the overlapping sites. We also found that mutation to the YY1 binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced suppression of the SM-MHC promoter activity. These findings indicate that NO regulates SM-MHC gene expression at the transcriptional level at least partially through the regulation of transcription factor binding activities on the CArG element. Thus we propose that NO plays a positive role in maintaining the differentiated state of VSMCs.

Animals↗
PubMed2001

SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility.

The control of intracellular calcium is central to regulation of cardiac contractility. A defect in SR Ca(2+)transport and SR Ca(2+)ATPase pump activity and expression level has been implicated as a major player in cardiac dysfunction. However, a precise cause-effect relationship between alterations in SERCA pump level and cardiac contractility could not be established from these studies. Progress in transgenic mouse technology and adenoviral gene transfer has provided new tools to investigate the role of SERCA pump level in the heart. This review focuses on how alterations in SERCA level affect Ca(2+)homeostasis and cardiac contractility. It discusses the consequences of altered SERCA pump levels for the expression and activity of other Ca(2+)handling proteins. Furthermore, the use of SERCA pump as a therapeutic target for gene therapy of heart failure is evaluated.

Animals↗
PubMed2001

Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

Animals↗
PubMed2000

Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function.

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.

Animals↗
PubMed2000

Smooth muscle myosin heavy chain isoforms and their role in muscle physiology.

Unlike vertebrate skeletal muscle, smooth muscle myosin heavy chain isoforms are encoded by a single gene. Alternative splicing of the primary transcript from a single gene generates four smooth muscle myosin heavy chain isoforms. These isoforms differ both at the carboxyl terminus (SM1 and SM2 isoforms) and at the amino terminus (SM-A and SM-B isoforms). The smooth muscle myosin heavy chain isoforms are differentially expressed during smooth muscle development and in different smooth muscle cell types. The mechanical properties of smooth muscle may be correlated with the myosin heavy chain content/isoform expression. However, the precise function of each smooth muscle myosin heavy chain isoform to muscle contraction remains to be determined. This review mainly focuses on the molecular basis of smooth muscle myosin heavy chain isoform diversity, its expression during development and disease, and its role in muscle physiology.

Amino Acid Sequence↗
PubMed2000

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility.

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.

Animals↗
PubMed2000

The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism.

The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca(2+)transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca(2+)ATPase and other Ca(2+)transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na(+)/Ca(2+)-exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na(+)/Ca(2+)-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels.

Animals↗
PubMed2000

Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy.

Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.

Adrenergic alpha-Agonists↗