Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5′-regions of the three known genes encoding light meromyosin isoforms (10 °C, intermediate and 30 °C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 °C and intermediate types, between the 10 °C and 30 °C types, and between the intermediate and 30 °C types, respectively. Isoform-specific differences were clearly observed between the 10 °C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 °C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 °C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.     

Temperature-dependent expression patterns of grass carp fast skeletal myosin heavy chain genes

Three types of myosin heavy chain cDNA clone named 10 °C, intermediate and 30 °C types were isolated from fast skeletal muscles of thermally acclimated grass carp Ctenopharyngodon idellus. Three clones encompassing parts of 3′-translated and entire 3′-untranslated regions showed high heterogeneities in their nucleotide sequences in the 3′-untranslated region. The comparison in the deduced amino acid sequence of the 10 °C-type clone with those of the intermediate- and 30 °C-type clones showed 88% and 89% identities, respectively. By contrast, the deduced amino acid sequence of the intermediate-type clone shared much higher identity of 97% with its 30 °C-type counterpart. Northern blot analysis demonstrated that the 10 °C- and 30 °C-type clones were predominantly expressed in grass carp acclimated to 10 and 30 °C, respectively. The intermediate type was expressed both in grass carp acclimated to 20 and 30 °C. Furthermore, expression patterns of the three myosin heavy chain genes were altered in accompaniment with seasonal temperature fluctuation. In autumn and winter grass carp expressed the 10 °C-type gene almost exclusively, whereas it was completely replaced by the intermediate- and 30 °C-type genes in spring and summer.These results suggest that tetraploid grass carp also undergo an adaptation to fluctuating environmental temperatures by selectively expressing fast skeletal myosin heavy chain isoforms as do diploid common carp previously reported.     

Histopathology of Ichthyophthirim multifiliis infections in fishes

Histopathological changes involved in Ichthyophthirius multifiliis infections are described from a wide range of host fishes—carp, crucian carp hydrids, Carassius carassius × C. auralus , goldfish, eels, African catfish, Glorias lazera , several species of cichlid fishes and rainbow trout. Fish were obtained from diverse geographical regions: Israel, South Africa, Portugal and Venezuela, as well as from laboratory experimental infections. Observed histopathological changes could not demonstrate any interspecific differences, but individual fish or different groups of fish of the same species demonstrated wide variability in susceptibility to infection and consequently in the nature and severity of the histopathological response. Low to moderate primary infection and secondary reinfestations did not induce significant histopathological changes in the integument. The proliferative response of the integumentary epithelium appeared only following subsequent infections. Repeated heavy reinfections induced massive cellular necrosis and in very heavy infections extensive lysis of the epithelial tissue. Host-parasite interactions in the light of the histological findings are discussed.     

Characterization of the carp myosin heavy chain multigene family

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.     

Reptilian class I major histocompatibility complex genes reveal conserved elements in class I structure

The polymerase chain reaction was used to isolate clones with class I major histocompatibility complex sequences from fish (carp), amphibian (axolotl), and two species of reptile (lizard and snake). The lizard and snake clones were used to isolate class I cDNA clones. All the sequence showed the expected evolutionary relatedness. The carp and axolotl clones and one lizard cDNA clone lacked the first systeine in the ₃ domain which in other class I heavy chains forms an intradomain disulfide bond. A small number of amino acid residues are conserved in the class I heavy chain sequences from all five classes of vertebrates. In the first two domains they are symmetrically clustered and contribute to intra-and interdomain contacts. None of these invariant residues are at peptide-binding, T-cell receptor-interacting, or CD8-binding positions.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: A2, M81089; C4,M8109 M81092; S1, M81093; LC1, M81094; LC5, M81095; LC13, M81096; LC25, M81097; LC27, M81098; SCI, M81099; SC2, M81100.     

鲢鱼轻酶解肌球蛋白的cDNA克隆及结构解析

与栖息温度相关的鲢鱼两种轻酶解肌球蛋白重链(light meromyosin,LMM)同工型(低温型,sc-w;高温型,sc-s)的氨基酸序列解析结果表明:sc-w与sc-s在LMM的氨基酸序列上显示91.8%的同源性,但与已经报道的草鱼(Ctenopharyngodon idella)低温型(gc10)有96.9%的...     

Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies

Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.     

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Coordinate changes in Myosin heavy chain isoform gene expression are selectively associated with alterations in dilated cardiomyopathy phenotype

BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.     

吴城鄱阳湖自然保护区鱼体重金属的富集及安全性评价

为研究吴城鄱阳湖自然保护区鱼类重金属的污染情况, 以鄱阳湖吴城6种常见淡水鱼(鲤、鳙、鲢、草鱼、青鱼、鳊)为研究对象, 分别研究鱼心脏、肝脏、鳃丝和肌肉等部位中铜、锌、铅、镉4种重金属的富集情况, 并采用目标危险系数(THQ)评价其健康风险。结果显示, 重金属元素在鱼体不同器官中的富集程度不同, Cd在肝脏中的含量最高, Cu在肝脏和心脏中的含量最高, Pb在肝脏中的富集含量最低, Zn在各器官的总体含量要高于其他3种元素。4种重金属在鱼肌肉中的含量均符合国内外标准。目标危险系数(THQ)分析结果表明, Pb导致的健康风险最高, Cd最低, 单一重金属THQ值和复合重金属TTHQ值均小于1, 说明吴城鄱阳湖自然保护区鱼类重金属的污染程度较低, 不会对消费者健康产生潜在危害。     

Full-length rat alpha and beta cardiac myosin heavy chain sequences. Comparisons suggest a molecular basis for functional differences

The two cardiac myosin heavy chain isoforms, alpha and beta, differ functionally, alpha Myosin exhibits higher actin-activated ATPase than does beta myosin, and hearts expressing alpha myosin exhibit increased contractility relative to hearts expressing beta myosin. To understand the molecular basis for this functional difference, we determined the complete nucleotide sequence of full-length rat alpha and beta myosin heavy chain cDNAs. This study represents the first opportunity to compare full-length fast ATPase and slow ATPase muscle myosin sequences. The alpha and beta myosin heavy chain amino acid sequences are more related to each other than to other sarcomeric myosin heavy chain sequences. Of the 1938 amino acid residues in alpha and beta myosin heavy chain, 131 are non-identical with 37 non-conservative changes. Two-thirds of these non-identical residues are clustered, and several of these clusters map to regions that have been implicated as functionally important. Some of the regions identified by the clusters of non-identical amino acid residues may affect actin binding, ATP hydrolysis and force production.     

环境毒物对金黄色葡萄球菌毒性影响的研究

采用毒物在营养琼脂中垂直扩散方法,通过测定毒物在营养球脂中抑制金黄色葡萄球菌生长产生蓝色抑菌带的长度,研究Hg、Cr^6 、Pb、CN^—、As、NO2^—、F^—及苯酚对金黄色葡萄球菌的毒性影响。结果表明：受试毒物的浓度与抑菌带有相关性,相关系数具显著意义;对毒物的敏感性为Cr^6 ＞Hg＞As＞CN^—＞Pb＞NO2^—＞苯酚＞F—;多种毒物共同作用其毒性影响增加。     

Carp transferrin can protect spermatozoa against toxic effects of cadmium ions

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.