Myosin Regulatory Light Chain 2 Research Articles

Fustin suppressed melanoma cell growth via cAMP/PKA-dependent mechanism.

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1, which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo.

Validation of protein biological markers of lamb meat quality characteristics based on the different muscle types.

This work investigated the ability of 8 potential biomarkers (phosphoglycerate kinase-1 (PGK1), pyruvate kinase-M2 (PKM2), phosphoglucomutase-1 (PGM1), β-enolase (ENO3, myosin-binding protein-C (MYBPC1), myosin regulatory light chain-2 (MYLPF), troponin C-1 (TNNC1) and troponin I-1 (TNNI1)) to characterize meat quality by analyzing their relative abundance and enzymatic activity. Two different meat quality groups (Quadriceps femoris (QF) and Longissimus thoracis (LT) muscles) were selected at 24h postmortem from 100 lamb carcasses. The relative abundance of PKM2, PGK1, PGM1, ENO3, MYBPC1, MYLPF, and TNNI1 was significantly different between LT and QF muscle groups (P<0.01). Moreover, PKM, PGK, PGM, and ENO activity in LT muscle group was significantly lower than that in QF muscle (P<0.05). Suggesting that PKM2, PGK1, PGM1, ENO3, MYBPC1, MYLPF, and TNNI1 can be used as robust biomarkers of lamb meat quality, providing the reference for understanding the molecular mechanism of postmortem meat quality formation in future.

ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

The bicellular tensile force sorts the localization of LSRs in bicellular and tricellular junctions

Lipolysis-stimulated lipoprotein receptors (LSRs) localize to tricellular tight junctions. Recent studies have shown that changes in the localization and expression profiles of LSRs are associated with malignancy of endometrial carcinomas, although the precise mechanisms by which malignant progression induces changes in the localization of LSRs are still unknown. In this study, we found that changes in cell tension correlated with alterations in the junctional localization of LSRs in endometrial cancer Sawano cells. At high cell densities, myosin phosphatase target subunit 1 (MYPT1) localized to bicellular junctions, whereas activated myosin regulatory light chain 2 (MRLC2) was dislocated from these regions, suggesting that circumferential tensile forces decreased at high cell densities. Under these conditions, LSRs localized to tricellular junctions. In contrast, a phosphorylated form of MRLC2 localized to bicellular regions, while MYPT1 was excluded from these regions, suggesting that tensile forces formed along the circumferential edge at low cell densities. It is noteworthy that, when cells were cultured under these conditions, LSRs localized to bicellular regions. Upon treatment with a myosin inhibitor, LSR localization in bicellular junctions decreased at low cell densities. Overall, our results indicate that the modulation of cellular tension was involved in the translocation of LSRs from bicellular to tricellular tight junctions.

Proteomic Profiling in the Spinal Cord and Sciatic Nerve in a Global Cerebral Ischemia-Induced Mechanical Allodynia Mouse Model.

Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic pain syndrome. At present, there are few studies of pain in regions such as the spinal cord or sciatic nerve in cerebral ischemic animal models. To identify proteomic changes in the spinal cord and sciatic nerve in global cerebral ischemic model mice, in the present study we performed an investigation using proteomic methods. In a comparison between the intensity of protein spots obtained from a sham and that from a bilateral carotid artery occulusion (BCAO) in spinal cord and sciatic nerve, the levels of 10 (spinal cord) and 7 (sciatic nerve) protein spots were altered. The protein levels in the spinal cord were significantly increased in N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), 6-phosphogluconolactonase isoform 1, and precursor apoprotein A-I and decreased in dihydropyrimidinase-related protein 2 (CRMP-2), enolase 1B, rab guanosine 5'-diphosphate (GDP) dissociation inhibitor beta, septin-2 isoform a, isocitrate dehydrogenase subunit alpha, cytosolic malate dehydrogenase, and adenosine triphosphate synthase. The protein levels in the sciatic nerve were significantly increased in a mimecan precursor, myosin light chain 1/3, and myosin regulatory light chain 2 (MLC2), and decreased in dihydropyrimidinase-related protein 3 (CRMP-4), protein disulfide-isomerase A3, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, and B-type creatine kinase. In addition, CRMP-2 and CRMP-4 protein levels were decreased, and DDAH1 and MLC2 protein levels were increased on day 1 after BCAO using Western blotting. These results suggested that changes in these proteins may be involved in the regulation of CPSP.

Effects of Myosin Light Chain Phosphorylation on Length-Dependent Myosin Kinetics in Rat Cardiac Ventricles

Myosin's capacity to generate force is Ca2+-regulated by thin-filament proteins and sarcomere length, which dictates the number of potential actin-myosin cross-bridge interactions throughout a heartbeat. However, the ventricular form of myosin regulatory light chain-2 (RLC) binds to the neck of myosin and is thought to modulate contraction via its phosphorylation state. Previous studies have shown that RLC phosphorylation can vary throughout the left ventricle wall, prompting the hypothesis that regional effects of RLC phosphorylation could alter myosin behavior throughout the heart. Thus, we performed biochemical analyses of flash-frozen myocardial samples to assess the relative percentage of RLC phosphorylation in the left and right ventricles. We found that RLC phosphorylation varied across the left ventricle, being significantly greater near the outside vs. the inside of the left ventricle free wall, and average RLC phosphorylation was greater in the right vs. left ventricle free wall. To assess the functional consequences of RLC phosphorylation on Ca2+-regulated contractility as sarcomere length varied, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure myosin cross-bridge kinetics as RLC phosphorylation varied with myosin light chain kinase incubation. Increases in RLC phosphorylation and sarcomere length both led to increased Ca2+-sensitivity of the force-pCa relationship, but RLC phosphorylation influenced this relationship more greatly at 2.2 vs. 1.9 µm sarcomere length. We found that RLC-phosphorylation slowed myosin rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC-phosphorylation only slowed myosin MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium

Myosin force production is Ca2+-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca2+-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca2+-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

Proteomic and peptidomic insights on myofibrillar protein hydrolysis in a sausage model during fermentation with autochthonous starter cultures

The hydrolysis of myofibrillar proteins during fermentation of sausage models by an autochthonous starter culture was investigated. In order to provide a whole map of the generated products, proteomic and peptidomic were used and complemented with the amino acid profile. Beaker sausages (BS) were used as models which were inoculated or not with Lactobacillus curvatus CRL705 and Staphylococcus vitulinus GV318 as starter cultures. The hydrolysis of actin, myosin light chain 1/3 (MLC 1/3), myosin regulatory light chain-2 (MRLC-2) and myosin heavy chain (MHC) was evidenced by two-dimensional gel electrophoresis (2-DE). In addition, a total of 33 peptides arisen from troponin T, MRLC-2 and particularly from actin were identified by LC–MS/MS. These results showed that the starter culture significantly enhanced the proteolysis of the proteins named above, even when the endogenous enzymes induced a clear breakdown. L. curvatus CRL705 highly enriched both peptide pattern and amino acid concentrations. When the autochthonous starter culture was inoculated, although proteolysis was remarkably reinforced, a reduction in peptide and amino acid composition was observed. Regarding actin primary structure, three regions of this protein were highly susceptible to degradation by the starter culture. Additionally, the essential role of exopeptidases – from meat and bacteria – in diversity of actin peptides during fermentation was shown. This study improved the knowledge of the proteolysis of myofibrillar proteins and the involved enzymes, as well as, completed the previously reported degradation of sarcoplasmic proteins by the same autochthonous starter culture. The singular peptides and amino acids pattern generated might contribute to the uniqueness of produced fermented sausages while they may be used as quality markers.

Abstract 268: Myofilament Tyrosine Phosphorylation by Src Kinase is upregulated in ErbB2 Transgenic Mice

Objectives: Tyrosine (Tyr) phosphorylation of the myofilament is an emerging, and potentially important, post-translational modification in cardiomyopathy. ErbB2, a Tyr receptor kinase, was overexpressed in transgenic mice (ErbB2-Tg) resulting in significant cardiac hypertrophy. We hypothesize that the development of cardiac hypertrophy in ErbB2-Tg is associated with increased myofilament Tyr phosphorylation and may implicate myofilament Tyr phosphorylation in cardiac hypertrophy. Methods: Proteins were isolated from ErbB2-Tg and Ntg heart homogenates ( n =4 per group). Reduction/alkylation was followed by trypsinization. Resulting peptides were desalted in C 18 columns and lyophilized. Phosphorylated Tyr (p-Tyr) enrichment was performed on 20 mg of peptides using a p-Tyr Mouse mAb kit (Cell Signaling). Immuno-precipitated and desalted peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo). Raw data were searched with Mascot 2.3. Label-free quantification with MS1 extracted ion chromatograms was performed using Skyline. Western blot analysis for total and phosphorylated Src kinase was performed per manufacturer’s protocol (Cell Signaling). Results: We found a total of 286 p-Tyr modified peptides in ErbB2-Tg compared to 226 in control NTg mice. Over 70 p-Tyr sites on myofilament protein were up-regulated in ErbB2-Tg, including troponin I, myosin heavy chain, titin, α-tropomyosin, myosin-binding protein-C3, myosin regulatory light chain-2 and myosin light chain-1. We used PhosphoMotif Finder to search the potential responsible kinase. Most of the p-Tyr sites were consistent with Src kinase motifs. Furthermore, Western blot analysis showed that total, and phospho-Src (Y416) expression was increased in ErbB2-Tg mice. Conclusion: We concluded that these novel p-Tyr sites on myofilament proteins are increased in ErbB2-Tg mice and correlate with up-regulated Src kinase activity. Thus increased tyrosine myofilament phosphorylation may be involved in the development of cardiac hypertrophy. Since ErbB2 is a therapeutic target of trastuzumab therapy this may also have translational implications to ameliorate off target effects of cancer treatment.

Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes

Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also down-regulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway.

A Two-Stage Association Study Suggests BRAP as a Susceptibility Gene for Schizophrenia

Schizophrenia (SZ) is a neurodevelopmental disorder in which altered immune function typically plays an important role in mediating the effect of environmental insults and regulation of inflammation. The breast cancer suppressor protein associated protein (BRAP) is suggested to exert vital effects in neurodevelopment by modulating the mitogen-activated protein kinase cascade and inflammation signaling. To explore the possible role of BRAP in SZ, we conducted a two-stage study to examine the association of BRAP polymorphisms with SZ in the Han Chinese population. In stage one, we screened SNPs in BRAP from our GWAS data, which detected three associated SNPs, with rs3782886 being the most significant one (P = 2.31E-6, OR = 0.67). In stage two, we validated these three SNPs in an independently collected population including 1957 patients and 1509 controls, supporting the association of rs3782886 with SZ (P = 1.43E-6, OR = 0.73). Furthermore, cis-eQTL analysis indicates that rs3782886 genotypes are associated with mRNA levels of aldehyde dehydrogenase 2 family (ALDH2) (P = 0.0039) and myosin regulatory light chain 2 (MYL2) (P < 1.0E-4). Our data suggest that the BRAP gene may confer vulnerability for SZ in Han Chinese population, adding further evidence for the involvement of developmental and/or neuroinflammatory cascades in the illness.

Molecular cloning and functional analysis of MRLC2 in Tianfu, Boer, and Chengdu Ma goats

To determine the molecular basis of heterosis in goats, fluorescence quantitative polymerase chain reaction (PCR) was performed to investigate myosin-regulatory light chain 2 (MRLC2) gene expression in the longissimus dorsi muscle tissues of the Tianfu goat and its parents, the Boer and Chengdu Ma goats. The goat MRLC2 gene was differentially expressed in the crossbreed, and the purebred mRNA were isolated and identified using fluorescence quantitative reverse transcription-PCR (RT-PCR). The complete coding sequence of MRLC2 was obtained using the cDNA method, and the full-length coding sequence consisted of 513 bp encoding 172 amino acids. The EF-hand superfamily domain of the MRLC2 protein is well conserved in caprine and other animals. The deduced amino acid sequence of MRLC2 shared significant identity with MRLC2 from other mammals. Phylogenetic tree analysis revealed that the MRLC2 protein was closely related to MRLC2 in other mammals. Several predicted miRNA target sites were found in the coding sequence of caprine MRLC2 mRNA. Analysis by RT-PCR showed that MRLC2 mRNA was present in the heart, stomach, liver, spleen, lung, small intestine, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscles. In particular, the high expression of MRLC2 mRNA was detected in the longissimus dorsi, leg muscle, abdominal muscle, stomach, and heart, but low levels of expression were also observed in the liver, spleen, lung, small intestine, and kidney. The expression of the MRLC2 gene was upregulated in the longissimus dorsi muscle of Boer and Tianfu goats, and it was moderately upregulated in Chengdu Ma goats.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

Slow Cardiac Myosin Regulatory Light Chain 2 (MYL2) was Down‐Expressed in Chronic Heart Failure Patients

Genetic studies have shown that many slow cardiac myosin regulatory light chain 2 (MYL2) gene mutations can cause hypertrophic cardiomyopathy, which is one of the most common causes of heart failure (HF). But until now there has been no pathological or histological evidence that MYL2 may be associated with HF development. Recent microarray studies indicated that myosin heavy chain expression changed in the pathological process of HF. Because MYL2 is a regulatory component of myosin heavy polypeptide, the role of MYL2 protein in HF needs to be studied. The level of expression of MYL2 may change in the heart tissue of patients with chronic HF. We collected 28 human right auricle samples, 16 from chronic HF patients and 12 from healthy control subjects. Immunohistochemistry was carried out to observe the tissue-expression pattern of the MYL2 protein and Western blot methods were performed to quantify the relative MYL2 expression level. In chronic HF patients, the MYL2 protein level decreased significantly compared with normal controls (t test P < 0.05). Among the 16 HF patients, MYL2 expression in the severe HF group (New York Heart Association [NYHA] class III or IV) was even lower than that of the moderate HF group (NYHA class II) (t test P < 0.05). MYL2 was down-expressed in HF tissues, and our findings suggested that MYL2 may play a role in the development and progression of chronic HF.

Neonatal Asphyxia Induces the Nitration of Cardiac Myosin Light Chain 2 That is Associated with Cardiac Systolic Dysfunction

Hypoxia followed by reoxygenation (H-R) observed during perinatal asphyxia is a serious complication with high mortality and morbidity rates that may cause adverse cardiovascular effects in neonates. Our aim was to determine if oxidative stress related to H-R induces peroxynitrite-dependent modifications of the cardiac contractile protein, myosin regulatory light chain 2 (MLC2), and whether this is associated with development of cardiac systolic dysfunction. Twelve newborn piglets were acutely instrumented for hemodynamic monitoring and randomized to a control group ventilated with only atmospheric air or to the H-R study group exposed to alveolar normocapnic hypoxia followed by reoxygenation. Afterward, animals were euthanized, and the hearts were harvested for biochemical analyses. Systolic function as well as cardiac MLC2 levels decreased in H-R animals, whereas nitrates and nitrotyrosine levels increased. Negative correlations between nitrates, nitrotyrosine, and MLC2 levels were observed. Moreover, H-R induced nitration of two tyrosine residues within the MLC2 protein. Similarly, in vitro exposure of MLC2 to peroxynitrite resulted in the nitration of tyrosine, which increased the susceptibility of MLC2 to subsequent degradation by matrix metalloproteinase 2. Substitution of this tyrosine with phenylalanine prevented the matrix metalloproteinase 2-dependent degradation of MLC2. In addition, a large decrease in MLC2 phosphorylation caused by H-R was observed. Oxidative stress related to asphyxia induces nitration of cardiac MLC2 protein and thus increases its degradation. This and a large decrease in MLC2 phosphorylation contribute to the development of systolic dysfunction. Inhibition of MLC2 nitration and/or direct inhibition of its degradation by MMP-2 could be potential therapeutic targets aiming at reduction of myocardial damage during resuscitation of asphyxiated newborns.

Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells

Digitalis has been used to treat congestive heart failure for more than 200 years, although the dual effects (proliferation and death) induced by digitalis on cell growth have been known for many years, the mechanisms by which digitalis causes the actions were not completely known. The aim of this work was to characterize the proliferative effect of ouabain on cell growth in endothelial cells, and, to do the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to ouabain and examine changes in protein expression. HUVEC were exposed to different concentrations (0.1-100 nM) of ouabain at 12-48 h intervals. Cell growth and morphological changes of HUVEC treated with ouabain were compared with cells under nontreated conditions. Ouabain stimulated HUVEC cell proliferation at low concentrations and induced cell death at higher concentrations. Using proteomics study, we identified 32 proteins of HUVEC with various important cellular functions and revealed 8 proteins such as Annexin A1, Annexin A2, Malate dehydrogenase, Myosin regulatory light chain 2 (MRLC2), Profilin-1, S100 calcium-binding protein A13, Triosephosphate isomerase and Translationally controlled tumor protein, regulated by low-dose ouabain treatment and MRLC2 was subsequently confirmed by Western blot. Our results give new insights into the cellular and molecular mechanisms of the proliferation action of low-dose ouabain on HUVEC and provide new avenues for the treatment of cardiovascular diseases.

Molecular Cloning and Functional Analysis of &amp;lt;italic&amp;gt;MRLC2&amp;lt;/italic&amp;gt; Differential Expressed in Meishan&amp;times;Yorkshire F1 Crossbreeds and their Parents, Meishan Pigs

In order to detect the molecular basis of heterosis in pigs, suppression subtractive hybridization was carried out to investigate the difference in gene expression in the Longissimus dorsi muscle tissues between MeishanxYorkshire F1 crossbreeds and their parents, Meishan pigs. The swine myosin regulatory light chain 2 (MRLC2) gene differentially expressed between the crossbreeds and the purebreds was isolated and identified using semi-quantitative reverse transcriptase polymerase chain reaction and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis reveals that the open reading frame of this gene encodes a protein of 172 amino acids containing the putative conserved domain of the EF-hand superfamily. This predicted amino acid sequence of porcine MRLC2 protein exhibits 99%, 98%, 98%, 98% and 97% identity with that of cattle, human, dog, rat and mouse, respectively. The homology analysis revealed that the MRLC2 protein was very much conserved in evolution. The tissue expression analysis indicated that the swine MRLC2 gene is highly expressed in muscle, fat, heart, liver, spleen, lung, kidney, stomach, small intestine, ovary and testis, but not expressed in pancreas.