Contractile units in stress fibers of fetal human astroglia in tissue culture

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca2⁺-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed ‘contractile units’, suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.     

An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin, in food

Shellfish are a common cause of food reactions in hypersensitive individuals and are among the eight foods that account for over 90% of food allergies. At present, the only way to prevent these serious consequences of food allergies is to avoid the foods that trigger the reactions. A sandwich-ELISA kit has been developed for the detection of crustacean meat in food, based on the major heat-stable shellfish allergen, tropomyosin. Tropomyosin was purified from whole prawn (Penaeus latisulcatus) and used to immunize rabbits after confirming its identity by MALDI-TOF MS. A sandwich-ELISA based on the rabbit antibodies takes less than 2 h to perform, including the food extraction, and has a detection limit of 1 ppm crustacean (prawn, lobster), without detectable cross-reactivity with fish or mammalian meat.     

Correlación genotipo-fenotipo en miocardiopatía hipertrófica: un estudio multicéntrico en Portugal y España sobre la variante p.Arg21Leu de TPM1

Introduction and objectivesTPM1 is one of the main hypertrophic cardiomyopathy (HCM) genes. Clinical information on carriers is relatively scarce, limiting the interpretation of genetic findings in individual patients. Our aim was to establish genotype-phenotype correlations of the TPM1 p.Arg21Leu variant in a serie of pedigrees.MethodsTPM1 was evaluated by next-generation sequencing in 10 561 unrelated probands with inherited heart diseases. Familial genetic screening was performed by the Sanger method. We analyzed TPM1 p.Arg21Leu pedigrees for cosegregation, clinical characteristics, and outcomes. We also estimated the geographical distribution of the carrier families in Portugal and Spain.ResultsThe TPM1 p.Arg21Leu variant was identified in 25/4099 (0.61%) HCM-cases, and was absent in 6462 control individuals with other inherited cardiac phenotypes (P < .0001). In total, 83 carriers (31 probands) were identified. The combined LOD score for familial cosegregation was 3.95. The cumulative probability of diagnosis in carriers was 50% at the age of 50 years for males, and was 25% in female carriers. At the age of 70 years, 17% of males and 46% of female carriers were unaffected. Mean maximal left ventricular wall thickness was 21.4 ± 7.65 mm. Calculated HCM sudden death risk was low in 34 carriers (77.5%), intermediated in 8 (18%), and high in only 2 (4.5%). Survival free of cardiovascular death or heart transplant was 87.5% at 50 years. Six percent of carriers were homozygous and 18% had an additional variant. Family origin was concentrated in Galicia, Extremadura, and northern Portugal, suggesting a founder effect.ConclusionsTPM1 p.Arg21Leu is a pathogenic HCM variant associated with late-onset/incomplete penetrance and a generally favorable prognosis.     

Cephalopod tropomyosins: Identification as major allergens and molecular cloning

Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod tropomyosins are cross-reactive with one another and also with crustacean tropomyosins. Molecular cloning experiments first elucidated the primary structures of tropomyosins from five species of cephalopods. The cephalopod tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63–64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod tropomyosins, supporting the cross-reactivity between cephalopod and crustacean tropomyosins.     

A Spatially Explicit Nanomechanical Model of the Half-Sarcomere: Myofilament Compliance Affects Ca2+-Activation

The force exerted by skeletal muscle is modulated by compliance of tissues to which it is connected. Force of the muscle sarcomere is modulated by compliance of the myofilaments. We tested the hypothesis that myofilament compliance influences Ca2+ regulation of muscle by constructing a computational model of the muscle half sarcomere that includes compliance of the filaments as a variable. The biomechanical model consists of three half-filaments of myosin and 13 thin filaments. Initial spacing of motor domains of myosin on thick filaments and myosin-binding sites on thin filaments was taken to be that measured experimentally in unstrained filaments. Monte-Carlo simulations were used to determine transitions around a three-state cycle for each cross-bridge and between two-states for each thin filament regulatory unit. This multifilament model exhibited less "tuning" of maximum force than an earlier two-filament model. Significantly, both the apparent Ca(2+)-sensitivity and cooperativity of activation of steady-state isometric force were modulated by myofilament compliance. Activation-dependence of the kinetics of tension development was also modulated by filament compliance. Tuning in the full myofilament lattice appears to be more significant at submaximal levels of thin filament activation.     

Protofilament and Hexagon: A Three-Dimensional Mechanical Model for the Junctional Complex in the Erythrocyte Membrane Skeleton

It is a long-standing mystery why erythrocyte actin filaments in the junctional complex (JC) are uniformly 37 nm and the membrane skeleton consists of hexagons. We have previously proposed that a molecular ruler formed by E-tropomodulin and tropomyosin 5 or 5b functions to generate protofilaments of 12 G actin under mechanical stress. Here, we illustrate that intrinsic properties of actin filaments, e.g., turns, chemical bonds, and dimensions of the helix, also favor fragmentation into protofilaments under mechanical stress. We further construct a mechanical model in that a pair of G actin is wrapped around by a split and spectrin, which may spin to two potential positions, and stabilize to one when the tail end is restricted. A reinforced protofilament may function as a mechanical axis to anchor three (top, middle, and bottom) pairs of Sp. Each Sp pair may wrap around the protofilament with a wide dihedral angle (166.2°) and a minimal axial distance (2.75 nm). Such three Sp pairs may spiral down (right handed) the protofilament from the pointed end with a dihedral angle of 55.4° in between the Sp pairs. This first three-dimensional model of JC may explain the hexagonal geometry of the erythrocyte membrane skeleton. © 2003 Biomedical Engineering Society. PAC2003: 8716Ka, 8716Dg, 8714Ee, 8716Ac     

Neonatal-onset nemaline myopathy mimicking congenital diaphragmatic hernia

In a newborn with severe respiratory failure and abnormal elevation of the right diaphragm, congenital diaphragmatic hernia with sac was diagnosed during surgery. However, microscopic examination of the sac showed atrophic striated muscle cells, indicating eventration instead of hernia. After several extubation failures, the final diagnosis of nemaline myopathy was made by skeletal muscle biopsy. In diaphragmatic defects with sac, diaphragm microscopic analysis should be recommended in order to discriminate between hernia and eventration. Congenital myopathies may underlie such diaphragmatic defects and should be promptly recognized, given their prognostic implications.     

周期型马来丝虫副肌球蛋白基因克隆、序列分析及编码产物B细胞表位预测

目的：克隆周期型马来丝虫副肌球蛋白（BmPmy）基因，进行序列测定、分析及编码产物的B细胞表位预测。方法：从周期型马来丝虫虫体中抽提总RNA，以mRNA为模板，RT-PCR法体外扩增BmPmy基因，扩增产物经初步鉴定后将其克隆人pGEM—T载体，转化E．coliDH5a，筛选阳性克隆，进行双酶切及PCR扩增鉴定，获得阳性重组质粒pGEM—TBmPmy，经测序验证，并进行同源性比较。应用5种参数和方法对其编码产物进行B细胞表位预测。结果：RT-PCR扩增出一条约2640bp的特异性条带，重组质粒双酶切的PCR结果与预期相符，DNA序列分析与基因库已知的基因序列同源性为99％。经表位预测分析，BmPmy的B细胞表位可能在54～66位、144～152位、770～780位和834～845位氨基酸区域。结论：成功构建了BmPmy重组质粒pGEM—T克隆载体，为进一步研究该基因的功能提供了条件。     

Transformation-related expression of a low-molecular-mass tropomyosin isoform TM5/TM30nm in transformed rat fibroblastic cell lines

We cloned a full-length rat TM5/TM30nm cDNA. Using this cDNA as a probe, we demonstrated that expression of TMT/TM30nm mRNA was higher in the tumorigenic rat fibroblastic cell lines SR-3Y1-2 and fos-SR-3Y1-202 than in the normal cell line 3Y1. High expression of TM5/TM30nm protein in SR-3Y1-2 and fos-SR-3Y1-202 cells was also detected by Western blot analysis using anti-TM5/TM30nm antiserum. In addition, the cellular localization of this protein differed between 3Y1 cells and tumorigenic ones. These findings suggest that TM5/TM30nm is involved in malignant transformation of rat fibroblastic cells.     

Tenascin-C induced signaling in cancer

Tenascin-C is an adhesion modulatory extracellular matrix molecule that is highly expressed in the microenvironment of most solid tumors. High tenascin-C expression reduces the prognosis of disease-free survival in patients with some cancers. The possible role of tenascin-C in tumor initiation and progression is addressed with emphasis on underlying signaling mechanisms. How tenascin-C affects malignant transformation, uncontrolled proliferation, angiogenesis, metastasis and escape from tumor immunosurveillance is summarized. Finally, we discuss how the phenotypes of tenascin-C knock-out mice may help define the roles of tenascin-C in tumorigenesis and how this knowledge could be applied to cancer therapy.