Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.     

Force-velocity properties and myosin light chain isoform composition of an identified type of skinned fibres from rat skeletal muscle

Force-velocity relations, myosin heavy chain (MHC) and myosin light chain (MLC) isoform composition of single skinned fibres from rat plantaris muscle were determined. In fibres containing the same (2X) isoform of myosin heavy chain, several parameters derived from the force-velocity relation and isometric force (Po) were tested for relation with the fibre content in alkali myosin light chain (MLC) isoforms. Whereas maximum shortening velocity was found to be proportional to the relative content in the 3f isoform of alkali MLC, velocity of shortening at 5% relative load, maximum power output, and Po were not. These results strengthen the idea that, in mammalian skeletal fibres, alkali MLC isoforms modulate shortening velocity at zero load, but suggest that they do not control the contractile behaviour at loads higher than zero.     

Fibre-type specific expression of fast and slow essential myosin light chain mRNAs in trained human skeletal muscles

The fibre-type specific expression patterns of fast and slow isoforms of essential (alkali) myosin light chains (ELC) was analysed in trained, untrained and pathological human muscles. Biopsies from m. vastus lateralis of moderately trained and untrained persons, as well as highly trained endurance and strength athletes were analysed, by in situ hybridization, for the expression of the `fast' ELC 1f/3f and the `slow' ELC 1sb. We wanted to investigate if changes in the fibre-type specific ELC mRNA pattern could be used as markers for training adaptation, especially, if the mRNA of the slow ELC 1sb isoform would appear in type IIA fibres as a result of endurance training (Baumann et al. 1987). We found the fast/slow ELC expression patterns in the fibre types to be remarkably stable. Physiological stress, even high training loads, did not affect it. No IIA fibres expressing ELC 1sb mRNA were found. They could be detected, however, in pathological muscle samples, where fast/slow ELC patterns not found in normal muscles were frequent. Our data suggest that in healthy muscles, only a subset of the theoretically possible combinations of myosin heavy and light chain isoforms are expressed at the level of their mRNAs.     

Autoantibodies to myosin light chains in the blood as early marker of myocardial injury after aortocoronary bypass surgery

Serum concentration of autoantibodies to myosin light chains was measured after resumption of the bloodflow recovery in patients who underwent hypothermic aortocoronary bypass surgery. The patients were divided into 3 groups according to postoperative hemodynamic parameters and degree of myocardial injury. The studies showed significant differences in the concentrations of autoantibodies to myosin light chains between the groups. High correlation was shown between the duration of aorta clamping and concentration of autoantibodies to myosin light chains. Some factors characterizing initial severity of the disease can modulate blood concentration of autoantibodies to myosin light chains. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 208–212, August, 2007     

先天性心脏病家系心室肌球蛋白轻链1基因的RFLP分析

本研究利用人心室肌球蛋白轻链1cDNA探针对先天性心脏病家系心室肌球蛋白轻链1基固进行了RFLP分析．从患有四种不同先天性心脏病的9个家系40名成员和13名无血缘关系患者的RFLP分析结果发现，有两个家系呈现限制酶切片段多态性，但这种限制性酶切片段多态性似与先天性心脏病的发生没有联系．     

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. Developmental Dynamics 240:1856–1863, 2011. © 2011 Wiley‐Liss, Inc.     

Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle

The relation between the isoform distribution of the myosin 17 kDa essential light chain (LC₁₇) and the mechanical properties of smooth muscle was investigated. The relative content of the basic (LC_17b) and acidic (LC_17a) isoelectric variants of the 17 kDa myosin light chain was determined in different mammalian smooth muscle tissues. The relative content of LC_17b varied between muscles: rabbit rectococcygeus 0%, rabbit trachea 5%, guinea-pig taenia coli 21%, rat uterus 38%, rabbit aorta 56% and rat aorta 60%. The rate of tension development was determined following photolysis of cagedadenosine triphosphate (ATP) in skinned fibres activated with thiophosphorylation of the regulatory light chains. The half-time for force development was 0.67 s in rabbit rectococcygeus, 1.6 s in rabbit trachea, 1.13 s in guineapig taenia coli and 1.38 s in rabbit aorta. The maximal shortening velocity (v _max) was determined with the isotonic quick release technique in skinned fibre preparations activated with thiophosphorylation. v _max was 0.25 muscle lengths per second (ML/s) in rabbit rectococcygeus, 0.24 ML/s in rabbit trachea, 0.17 ML/s in guinea-pig taenia coli, 0.11 ML/s in rat uterus and 0.03 ML/s in rabbit aorta. The range of variation in v _max between muscles was larger than in the half-time for force development. The inverse relationship between v _max and the relative content of LC_17b in the investigated muscles suggests that the type of essential myosin light chain influences the v _max in smooth muscle.     

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca²⁺-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing α-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively α-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, α-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than β-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.     

Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy

 Five disease genes encoding sarcomeric proteins and associated with familial and classical forms of hypertrophic cardiomyopathy have been determined since 1989. In 1996 two other genes encoding ventricular regulatory and essential myosin light chains were shown to be associated with a particular phenotype of the disease characterized by mid left ventricular obstruction. The aim of the present study was to search for mutations in the ventricular regulatory myosin light chain gene (MYL2), located on chromosome 12q23q24.3, in a panel of 42 probands presenting a classical phenotype of familial hypertrophic cardiomyopathy. Single-strand conformation polymorphism analysis was used to search for mutations in the coding segments of the MYL2 gene, and the abnormal products were sequenced. Two novel missense mutations, Phe18Leu in exon 2 and Arg58Gln in exon 4 were identified in three unrelated families. None of the affected patients had hypertrophy localized only at the level of the papillary muscle with mid left ventricular obstruction. By analysis of genetic recombinations, one of these mutations identified in a large family allowed us to refine the localization of the MYL2 gene on the genetic map, in an interval of 6 cM containing six informative microsatellite markers. In conclusion, we show that mutations in the MYL2 gene may be involved in familial and classical forms of hypertrophic cardiomyopathy, and we provide new tools for the genetic analysis of patients with familial hypertrophic cardiomyopathy. Received: 12 August 1997 / Accepted: 20 November 1997     

Use of monoclonal antibodies against myosin light chains 1 to detect the corresponding antigen in blood of patients with myocardial infarction

Research Institute of Human Genetics, All-Union Medical Genetics Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 10, pp. 416–417, October, 1991.     

Effects of low and high frequency patterns of stimulation on contractile properties,enzyme activities and myosin light chain accumulation in slow and fast denervated muscles of the chicken

The effects of denervation and direct stimulation in fast and slow latissimus dorsii muscles were investigated in chicken. In slow ALD muscle, denervation resulted in an incompleteness of the relaxation, a decrease in MDH and CPK activities and an increase in fast myosin light chains (MLC) accumulation. Direct stimulation at either fast or slow rhythm prevented the effects of denervation on relaxation and CPK activity but was ineffective on MDH activity and fast MLC accumulation. Moreover, direct stimulation of denervated ALD caused rhythm-dependent change in tetanic contraction. In fast PLD muscle, the main changes in muscle properties following denervation were a slowing down of the time course of the twitch and an incompleteness of the relaxation, a decrease in LDH and CPK activities and in LC₃F accumulation. Stimulation at a high frequency partly prevented the effects of denervation and resulted in a large accumulation of LC₃F, while a low frequency stimulation did not restore the twitch time to peak, increased MDH activity and induced synthesis of slow MLC. This study emphasizes the role of muscle activity and its pattern in some properties of slow and fast chicken muscles following denervation.This work was supported by the Association des Myopathes de France     

人心肌肌球蛋白轻链基因启动子驱动的GFP和Luc报告基因在人心肌细胞系中的表达

目的:构建人肌球蛋白2v基因启动子(pMLC2v)驱动的绿色荧光蛋白(GFP)和萤光素酶(Luc)报告基因慢病毒载体并观察其在人心肌细胞系(HCM)和人肺癌细胞株A549中的整合表达特征。方法:应用去毒化的人类I型免疫缺陷病毒和pMLC2v-GFP或pMLC2v-Luc报告基因构建慢病毒示踪载体,转染HCM和A549细胞,激光共聚焦显微镜及生物发光检测仪观察2种报告基因在不同细胞生长进程中的表达特征。非特异性启动子驱动的GFP(GFP_C)和红色荧光蛋白(RFP_C)报告基因作为对照。结果:2种细胞转染GFP_C和RFP_C后第3 d,都表达GFP和RFP;而HCM只在转染pMLC2v-GFP和pMLC2v-Luc 21 d后表达GFP和Luc,A549细胞不表达。结论:pMLC2v主要在培养21 d后新增殖的人心肌细胞中驱动GFP和Luc报告基因表达,这为监测干细胞向心肌细胞的分化进程提供了可靠的病理及活体示踪工具。     

Localization of myosin II regulatory light chain in the cerebral vasculature

The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo.     

Marginal zone B cell enrichment and strong follicular B cell reduction correlate with a delayed IgG response in a light chain diversity restricted mouse model

Recently developed B6.kappa(-)lambda(SEG) mice (by crossing kappa(-) and C57BL/6 mice congenic for the wild Mus spretus SEG strain lambda locus lacking genes coding for lambda1 and lambda3) have a very reduced light chain diversity. B6.kappa(-)lambda(SEG) mice produce only lambda2 and lambdax light chains. Regardless of their Igh haplotype, B6.kappa(-)lambda(SEG) mice show a restricted B cell distribution by light chain subtype with lambdax dominance in all peripheral compartments except peritoneal cavity where lambda2 is dominant. This distribution suggests that selection mechanisms act differently in different B cell compartments on lambda2 and lambdax bearing B cells. Sequence analysis before or following immunization did not reveal unusual mechanisms of diversification. B6.kappa(-)lambda(SEG) mice still respond to various challenging antigens using new Ab patterns. In particular, regardless of Igh(a) or Igh(b) haplotypes, the anti-2,4-dinitrophenyl response is characterized by a restricted diversity for both heavy and light chains and a delayed IgG response when compared to B6 and B6.kappa(-) mice. We suggest that the delayed IgG response is due to the expansion of marginal zone B cells whereas follicular B cells are strongly reduced.     

Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion

While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC⁺ B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 – enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.     

Neuronal driven pre-plaque inflammation in a transgenic rat model of Alzheimer's disease

Chronic brain inflammation is associated with Alzheimer's disease (AD) and is classically attributed to amyloid plaque deposition. However, whether the amyloid pathology can trigger early inflammatory processes before plaque deposition remains a matter of debate. To address the possibility that a pre-plaque inflammatory process occurs, we investigated the status of neuronal, astrocytic, and microglial markers in pre- and post-amyloid plaque stages in a novel transgenic rat model of an AD-like amyloid pathology (McGill-R-Thy1-APP). In this model, we found a marked upregulation of several classical inflammatory markers such as COX-2, IL-1β, TNF-α, and fractalkine (CX3CL1) in the cerebral cortex and hippocampus. Interestingly, many of these markers were highly expressed in amyloid beta-burdened neurons. Activated astrocytes and microglia were associated with these Aβ-burdened neurons. These findings confirm the occurrence of a proinflammatory process preceding amyloid plaque deposition and suggest that Aβ-burdened neurons play a crucial role in initiating inflammation in AD.     

慢性房颤患者右心房细胞电压依赖性钾电流变化的研究

目的：研究风湿性心脏病（RHD）慢性房颤（AF）对心房肌细胞膜电压依赖性外向钾电流的影响，探讨和评价心房电重塑（AER）中钾电流变化的作用和意义。 方法： 采用急性分离的人心房肌细胞，应用膜片钳全细胞技术，记录18例风心病非房颤（NAF）和18例慢性AF患者右心房肌细胞两种电压依赖性外向钾电流：瞬间外向钾电流（Ito1）和超快速激活钾电流（IKur），并比较两组的电流-电压曲线。结果： 在钳制电位+10 mV-+60 mV下，AF组的Ito1密度均较NAF组Ito1值明显降低（P<0.05），在钳制电位+60 mV时，AF组Ito1密度较NAF组低57.5%（P<0.05）。在钳制电位0 mV-+40 mV时，AF组IKur值明显低于NAF组（P<0.05）；其中，AF组IKur密度在钳制电位+40 mV时，较NAF组低47.0%（P<0.05）。结论： Ito1和IKur的改变均可能是慢性房颤时AER中多种离子通道重塑作用中的重要表现，可能与AF时心肌细胞的传导性、不应期的改变和频率适应性变化有关，与其它离子通道改变以及AF发生和持续的关系尚需进一步研究证明。     

CD26 (dipeptidyl-peptidase IV)-dependent recruitment of T cells in a rat asthma model

CD26 truncates several chemokines as well as neuropeptides and influences immune responses via modulation of cell adhesion and T cell activation, suggesting an involvement of CD26 in asthmatic and airway inflammation. Therefore, Fischer 344 (F344), Brown Norway (BN) and Lewis (LEW) rat strains, which differ in their CD26-like enzymatic activity, were compared using an asthma model. Additionally, two CD26-deficient mutant F344 rat substrains were included and compared to the wild-type F344 substrain. Immunization was performed twice with ovalbumin (OVA), and 2 weeks later the rats were challenged with OVA intratracheally Flow cytometry (FACS) analysis of different leucocyte subsets as well as enzyme-linked immunosorbent assay (ELISA) for IgE levels in the blood and bronchoalveolar lavage (BAL) were performed 24 h after challenge. LEW rats with the lowest CD26 activity among the rat strains investigated here displayed significantly reduced CD4+ T cell numbers in the BAL compared to wild-type F344 and BN rats. Moreover, in asthma, the ratio of CD26+ to CD26- T cell receptor (TCR)-positive cells increased significantly in F344 and LEW but not BN rats. Most intriguingly, in both CD26-deficient F344 rat substrains the number of CD4+ T lymphocytes was markedly reduced compared to wild-type F344. The decrease in T cell recruitment observed in the CD26-deficient rats was associated with significantly reduced OVA-specific IgE-titres. This is the first report to show a remarkably reduced T cell recruitment in rat strains that either lack or exhibit reduced CD26-like enzymatic activity, suggesting a role for CD26 in the pathogenesis of asthma via T cell-dependent processes such as antibody production.