Myosin Light Chain Gene Research Articles

Widespread Aneurysms Secondary to Myosin Light-Chain Kinase Gene Mutation Leading to Multifocal Right Hemispheric Stroke (P3-5.001)

<h3>Objective:</h3> Familiarize providers with the presence of the Myosin Light-Chain Kinase (MYLK) and the possible vascular manifestations in individuals carrying this autosomal dominant gene mutation. <h3>Background:</h3> Myosin Light-Chain Kinase is a ubiquitously expressed kinase involved in the physiological contraction of smooth muscle cells within hollow organs. Variations in the Myosin Light-Chain Kinase (MYLK) gene lead to decrease in phosphorylation of 20kDa regulatory light-chain leading to a reduction in smooth muscle contractile function. MYLK gene mutations have been associated with autosomal dominant thoracic aortic aneurysms and dissections, however there are limited studies mentioning the involvement of other vascular structures containing these dysfunctional smooth muscle cells. In this report, we present a case of a 43-year-old individual who presented to the hospital for quadrantanopia, hearing loss and headache who was subsequently found to have an acute stroke of multiple foci involving the right ACA and MCA territories. Past medical history was significant for hemochromatosis, early onset coronary artery disease in self and close family members. Etiology workup revealed a right internal carotid artery aneurysm measuring 10cm. Complains of abdominal pain led to imaging revealing focal dissection of the abdominal aorta, a right common iliac artery 2.3cm aneurysm as well as a proximal celiac artery 1.7cm aneurysm. On outpatient genetic testing, the patient was found to be a carrier of the MYLK (p.Val509Met) gene mutation; variance was of unclear significance, however familial thoracic aneurysms/dissections have been reported in the Clinical Variance Database. The patient has since been managed with dual anti-platelet therapy and was instructed to follow closely with neurological and vascular surgery for close monitoring. <h3>Design/Methods:</h3> NA <h3>Results:</h3> NA <h3>Conclusions:</h3> This case highlights the MYLK gene mutation and its possible relationship with widespread malformations in the carotid, celiac, and mesenteric vasculature to familiarize practitioners with manifestations and clinical sequelae of individuals affected with this gene. <b>Disclosure:</b> Dr. Hamzoian has nothing to disclose. Dr. Abdelmasih has nothing to disclose. Dr. Ditamo has nothing to disclose. Dr. Kahila has nothing to disclose.

Identification of Differentially Expressed Genes in the Longissimus Dorsi Muscle of Luchuan and Duroc Pigs by Transcriptome Sequencing.

The Duroc pig originated in the United States and is a typical lean-meat pig. The breed grows fast, and the body size is large, but the meat quality is poor. The Luchuan pig is one of eight excellent local breeds in China; it has tender meat but is small in size. To study the factors that determine growth, we selected the longissimus dorsi muscle of Luchuan and Duroc pigs for transcriptome sequencing. The results of the transcriptome showed that 3682 genes were differentially expressed (DEGs) in the longissimus dorsi muscle of Duroc and Luchuan pigs. We screened out genes related to muscle development and selected the MYL2 (Myosin light chain-2) gene to perform preliminary research. Gene Ontology (GO) enrichment of biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene products were mainly involved in the Akt/FoxO signaling pathway, fatty acid metabolism, arachidonic acid metabolism and glycine, serine and threonine metabolism. Such pathways contributed to skeletal muscle growth, fatty acid metabolism and intramuscular fat deposition. These results provide insight into the mechanisms underlying the formation of skeletal muscle and provide candidate genes to improve growth traits, as well as contribute to improving the growth and development traits of pigs through molecular breeding.

Comparative analysis of the transcriptomes of EDL, psoas, and soleus muscles from mice

BackgroundIndividual skeletal muscles have evolved to perform specific tasks based on their molecular composition. In general, muscle fibers are characterized as either fast-twitch or slow-twitch based on their myosin heavy chain isoform profiles. This approach made sense in the early days of muscle studies when SDS-PAGE was the primary tool for mapping fiber type. However, Next Generation Sequencing tools permit analysis of the entire muscle transcriptome in a single sample, which allows for more precise characterization of differences among fiber types, including distinguishing between different isoforms of specific proteins. We demonstrate the power of this approach by comparing the differential gene expression patterns of extensor digitorum longus (EDL), psoas, and soleus from mice using high throughput RNA sequencing.ResultsEDL and psoas are typically classified as fast-twitch muscles based on their myosin expression pattern, while soleus is considered a slow-twitch muscle. The majority of the transcriptomic variability aligns with the fast-twitch and slow-twitch characterization. However, psoas and EDL exhibit unique expression patterns associated with the genes coding for extracellular matrix, myofibril, transcription, translation, striated muscle adaptation, mitochondrion distribution, and metabolism. Furthermore, significant expression differences between psoas and EDL were observed in genes coding for myosin light chain, troponin, tropomyosin isoforms, and several genes encoding the constituents of the Z-disk.ConclusionsThe observations highlight the intricate molecular nature of skeletal muscles and demonstrate the importance of utilizing transcriptomic information as a tool for skeletal muscle characterization.

Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation.

Aims Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants.Methods and results We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants.Conclusion Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.

The Molecular Mechanisms of Mutations in Actin and Myosin that Cause Inherited Myopathy.

The discovery that mutations in myosin and actin genes, together with mutations in the other components of the muscle sarcomere, are responsible for a range of inherited muscle diseases (myopathies) has revolutionized the study of muscle, converting it from a subject of basic science to a relevant subject for clinical study and has been responsible for a great increase of interest in muscle studies. Myopathies are linked to mutations in five of the myosin heavy chain genes, three of the myosin light chain genes, and three of the actin genes. This review aims to determine to what extent we can explain disease phenotype from the mutant genotype. To optimise our chances of finding the right mechanism we must study a myopathy where there are a large number of different mutations that cause a common phenotype and so are likely to have a common mechanism: a corollary to this criterion is that if any mutation causes the disease phenotype but does not correspond to the proposed mechanism, then the whole mechanism is suspect. Using these criteria, we consider two cases where plausible genotype-phenotype mechanisms have been proposed: the actin “A-triad” and the myosin “mesa/IHD” models.

A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Cardiac Myocyte De Novo DNA Methyltransferases 3a/3b Are Dispensable for Cardiac Function and Remodeling after Chronic Pressure Overload in Mice.

BackgroundRecent studies reported altered DNA methylation in failing human hearts. This may suggest a role for de novo DNA methylation in the development of heart failure. Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.MethodsMice with specific ablation of Dnmt3a and Dnmt3b expression in cardiomyocytes were generated by crossing floxed Dnmt3afl and Dnmt3bfl alleles with mice expressing Cre recombinase under control of the atrial myosin light chain gene promoter. The efficacy of combined Dnmt3a/3b ablation (DKO) was characterized on cardiomyocyte-specific genomic DNA and mRNA levels. Cardiac phenotyping was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (TAC). Under similar conditions, cardiac genome-wide transcriptional profiling was performed and DNA methylation levels of promoters of differentially regulated genes were assessed by pyrosequencing.ResultsDKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts. Cardiac phenotyping revealed no significant differences between DKO and control mice under sham and TAC conditions. Transcriptome analyses identified upregulation of 44 and downregulation of 9 genes in DKO as compared with control sham mice. TAC mice showed similar changes with substantial overlap of regulated genes compared to sham. Promoters of upregulated genes were largely unmethylated in DKO compared to control mice.ConclusionThe absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

Cloning and molecular characterization of a myosin light chain gene from Puccinia striiformis f. sp. tritici

The fungus Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Many studies have found that myosins play important roles during fungal growth and propagation. However, there are few reports on the myosins of Pst. In this study, we cloned and obtained the myosin light chain gene PsMLC1 from Pst and characterized its expression. Furthermore, the function of PsMLC1 was identified by mutant complementation. As a result, we found that expression of PsMLC1 in Schizosaccharomyces pombe mostly complemented the defects of the cdc4 mutant, indicating that PsMLC1 belongs to the myosin light chain family member. Expression studies showed that the transcript levels of PsMLC1 little changed before 24 h post inoculation then was suddenly down-regulated during Pst infection of wheat. By using ML-7, we observed that inactivity of PsMLC1 greatly reduced the germination rate of urediniospores. These results suggest that PsMLC1 is essential for the early stages of Pst infection of wheat but unnecessary for the later stages of infection. This work elucidates the function of the myosins in Pst and may provide some theoretical basis for controlling strip rust.

Genomic Organization, Intronic Duplications, and Promoter Characteristics of the Fast Skeletal Myosin Light Chain-2 Gene (mlc2f) from Javanese Ricefish Oryzias javanicus

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

Characterization of stable fluorescent transgenic marine medaka (Oryzias dancena) lines carrying red fluorescent protein gene driven by myosin light chain 2 promoter

Stable transgenic germlines carrying the red fluorescence protein (RFP) gene (rfp) driven by fast skeletal myosin light chain-2 gene (mlc2f) promoter were established in a truly euryhaline fish species, the marine medaka (Oryzias dancena; Beloniformes). Transgenic lines contained transgene copy numbers varying from a single copy to more than 230 copies per genome. Although the transgenic founders displayed mosaic and/or ectopic expression of the RFP signal, the resultant F1 transgenics and their progeny showed consistently stable transmission of the transgenic locus and uniform RFP signal through several subsequent generations. In adult transgenics, an authentic brilliant red fluorescence was achieved over the skeletal muscles of the transgenic individuals, which might be sufficient for ornamental display. Expression analysis of the transgenic mRNAs indicated that rfp transcripts were predominantly expressed in the skeletal muscles. Different transgenic lines displayed different levels of transgene expression at the mRNA, protein, and phenotypic levels. However, the efficiency of transgene expression was independent of the transgene copy number. The RFP protein levels were consistently stable in the transgenic fish muscles through several generations, up to F5. The results of this study suggest that transgenic marine medaka that acquire strong fluorescent signals in their skeletal muscles can be developed as a promising, novel ornamental fish for display in both freshwater and seawater aquaria.

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771bp, including a 5′-terminal untranslated region (UTR) of 36bp, 3′-terminal UTR of 279bp and an open reading frame (ORF) of 456bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24h) as much as that in the control (P<0.05) challenged by poly I:C and 5.0 times (at 24h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.

Analysis of ventricular myosin light chain genes in cardiomyopathy

Recently, numerous mutations in sarcomeric genes are reported as factor of cardiomyopathy. Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are general disease in cardiomyopathy and causes sudden death in young people. In this study, ventricular myosin regulatory light chain gene (MYL2) and ventricular myosin essential light chain gene (MYL3) were analyzed in order to elucidate the cause of cardiomyopathy. In MYL3, a significant missense mutation (Ala57Gly) was detected in HCM case. This mutation was located in the EF-hand domain calcium-binding site that regulates the cardiac contraction. No genetic mutation was found in our previous report except this gene, it indicated that the mutation of the MYL3 was the main factor for the HCM case. It is necessary to survey the other disease causing genes to clarify the relationship with etiology.

Dynamic Regulation of Vascular Myosin Light Chain (MYL9) with Injury and Aging

BackgroundAging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels.Methodology/Principal FindingsWe studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI's GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers.Conclusions/SignificanceThe consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.

Alkali‐like myosin light chain‐1 (myl1) is an early marker for differentiating fast muscle cells in zebrafish

During myogenesis, muscle precursors become divided into either fast- or slow-twitch fibres, which in the zebrafish occupy distinct domains in the embryo. Genes encoding sarcomeric proteins specific for fast or slow fibres are frequently used as lineage markers. In an attempt to identify and evaluate early definitive markers for cells in the fast-twitch pathway, we analysed genes encoding proteins contributing to the fast sarcomeric structures. The previously uncharacterized zebrafish alkali-like myosin light chain gene (myl1) was found to be expressed exclusively in cells in the fast-twitch pathway initiated at an early stage of fast fibre differentiation. Myl1 was expressed earlier, and in a more fibre type restricted manner, than any of the previously described and frequently used fast myosin light and heavy chain and troponin muscle markers mylz2, mylz3, tnni2, tnnt3a, fMyHC1.3. In summary, this study introduces a novel marker for early differentiating fast muscle cells.

Inhibition of the AMP-Activated Protein Kinase-α2 Accentuates Agonist-Induced Vascular Smooth Muscle Contraction and High Blood Pressure in Mice

The aim of the present study was to determine the effects and molecular mechanisms by which AMP-activated protein kinase (AMPK) regulates smooth muscle contraction and blood pressure in mice. In cultured human vascular smooth muscle cells, we observed that activation of AMPK by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited agonist-induced phosphorylation of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1). Conversely, AMPK inhibition with pharmacological or genetic means potentiated agonist-induced the phosphorylation of MLC and MYPT1, whereas it inhibited both Ras homolog gene family member A and Rho-associated kinase activity. In addition, AMPK activation or Rho-associated kinase inhibition with Y27632 abolished agonist-induced phosphorylation of MLC and MYPT1. Gene silencing of p190-guanosine triphosphatase-activating protein abolished the effects of AMPK activation on MLC, MYPT1, and Ras homolog gene family member A in human smooth muscle cells. Ex vivo analyses revealed that agonist-induced contractions of the mesenteric artery and aortas were stronger in both AMPKα1(-/-) and AMPKα2(-/-) knockout mice than in wild-type mice. Inhibition of Rho-associated kinase with Y27632 normalized agonist-induced contractions of AMPKα1(-/-) and AMPKα2(-/-) vessels. AMPKα2(-/-) mice had higher blood pressure along with decreased serine phosphorylation of p190-guanosine triphosphatase-activating protein. Finally, inhibition of the Ras homolog gene family member A/Rho-associated kinase pathway with Y27632, which suppressed MYPT1 and MLC phosphorylation, lowered blood pressure in AMPKα2(-/-) mice. In conclusion, AMPK decreases vascular smooth muscle cell contractility by inhibiting p190-GTP-activating protein-dependent Ras homolog gene family member A activation, indicating that AMPK may be a new therapeutic target in lowering high blood pressure.

Deletion of Cardiomyocyte Mineralocorticoid Receptor Ameliorates Adverse Remodeling After Myocardial Infarction

Mineralocorticoid receptor (MR) blockade improves morbidity and mortality among patients with heart failure; however, the underlying mechanisms are still under investigation. We studied left ventricular remodeling after myocardial infarction in mice with cardiomyocyte-specific inactivation of the MR gene (MR(MLCCre)) that were generated with a conditional MR allele (MR(flox)) in combination with a transgene expressing Cre recombinase under control of the myosin light-chain (MLC2a) gene promoter. Control (MR(flox/flox), MR(flox/wt)) and MR(MLCCre) mice underwent coronary artery ligation. MR ablation had no detectable baseline effect on cardiac morphology and function. The progressive left ventricular chamber enlargement and functional deterioration in infarcted control mice, detected by echocardiography and conductance catheter analysis during the 8-week observation period, were substantially attenuated in MR(MLCCre) mice. Chronically infarcted MR(MLCCre) mice displayed attenuated pulmonary edema, reduced cardiac hypertrophy, increased capillary density, and reduced accumulation of extracellular matrix proteins in the surviving left ventricular myocardium. Moreover, cardiomyocyte-specific MR ablation prevented the increases in myocardial and mitochondrial O(2)(·-) production and upregulation of the NADPH oxidase subunits Nox2 and Nox4. At 7 days, MR(MLCCre) mice exhibited enhanced infarct neovessel formation and collagen structural organization associated with reduced infarct expansion. Mechanistically, cardiomyocytes lacking MR displayed accelerated stress-induced activation and subsequent suppression of nuclear factor-κB and reduced apoptosis early after myocardial infarction. Cardiomyocyte-specific MR deficiency improved infarct healing and prevented progressive adverse cardiac remodeling, contractile dysfunction, and molecular alterations in ischemic heart failure, highlighting the importance of cardiomyocyte MR for heart failure development and progression.

Molecular cloning, expression pattern and phylogenetic analysis of myosin light chain 2 gene from Antheraea pernyi: A potential marker for phylogenetic inference

Myosin light chain 2 (MLC-2) gene was isolated and characterized from Antheraea pernyi, a well-known wild silkmoth. The isolated cDNA sequence is 905 bp in length with an open reading frame of 612 bp encoding a polypeptide of 203 amino acids. Semi-quantitative RT-PCR analysis showed that the MLC-2 gene was transcribed during four developmental stages (egg, larva, pupa, and moth), and present in all tissues tested. Alignment analysis revealed that the deduced protein sequence has over 95% identity to myosin light chain 2 of lepidopteran species, and 57–88% identity to other insect species, suggesting that insect MLC-2 proteins are highly conserved throughout evolution. The protein sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate MLC-2 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of MLC-2 gene in phylogenetic study.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Characterization of myosin light chain in shrimp hemocytic phagocytosis

Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp.