人骨骼肌α辅肌动蛋白（α－actinin）的提取及其抗血清的制备和鉴定

本文提取人骨骼肌α辅肌动蛋白（α－ａｃｔｉｎｉｎ）是综合了文献报导有关提取兔肌α－ａｃｔｉｎｉｎ的和提取鸡胗α－ａｃｔｉｎｉｎ的方法,稍加修改而确定的。用Ｈａｓｓｅｌｂａｃｈ－Ｓｃｈｎｅｉｄｅｒ缓冲液提取骨骼肌中的肌球蛋白后,将残余物经硼酸－缓冲液提取、匀浆及高速离心去掉肌动蛋白和肌原纤维的其它成份,上清加硫酸铵至３０％,３５％饱合度所得的沉淀用２２０ｍｍｏｌ／ＬＴｒｉｓ－乙酸溶解、透析、离心后经ＤＥ－５２柱层析可得电泳纯。α－ａｃｔｉｎｉｎ。将人骨骼肌α－ａｃｔｉｎｉｎ纯化制品免疫了三只大耳白纯种家兔,两个多月后,三只兔子都产生免抗人骨骼肌α－ａｃｔｉｎｉｎ的特异抗血清,用双向免疫扩散法和酶联免疫吸附试验（ＥＬＩＳＡ）测定,产生的抗体效价较高,用双扩散法测定效价为１：３２,用ＥＬＩＳＡ测定,用比率法判断结果,效价最高者为１：１００,０００左右,经免疫电镜观察结果证实,上述抗血清可以满足进一步实验要求。     

Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca²⁺ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca²⁺ were monitored by using single channel recordings of the high conductance Ca²⁺-activated K⁺ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na⁺-saline, UTP (>30 nm) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 m. Cells bathed in K⁺ saline (150 m) were less sensitive to UTP (5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K⁺ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca²⁺ entry blockers or by removal of extracellular Ca²⁺. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca²⁺ ionophores (<5 m) to that of UTP indicate that both agents elevate cytosolic Ca²⁺ by mobilization of this ion from intracellular pools. However, the Ca²⁺ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca²⁺ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues. PKC does not appear to be directly involved, even though the UTP response is blocked by low nm levels of PMA. While the latter data implicate PKC in diminishing the UTP response, agents that inhibit either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.     

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K⁺) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the “Independence” hypothesis is correct, tension during the decay of K⁺ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K⁺ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K⁺ contracture tension are not independent.     

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach

Heart tissue contains large amounts of the protein encoded by the Ca²⁺ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms byin situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca²⁺ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca²⁺ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.Deceased August 14, 1992     

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

Acoustic structure of vocalization and stapedius muscle activity during vocal development in chickens (Gallus gallus)

The link between stapedius muscle activity and acoustic structure of vocalization was analysed in cocks of age 20–30 to 90–100 days old. The results show that stapedius muscle activation depends on the acoustic structure of vocalization and changes during vocal development. This dependence was observed in spontaneous calls and in vocalizations elicited by stimulating the mesencephalic calling area. In 30-day-old cocks stapedius muscle EMG response is never associated with vocalizations with an acoustic energy content which is always distributed at frequencies higher than 2000 Hz. The coupling between vocalization and stapedius muscle activity begins later, when birds produce vocalizations with acoustic energy shifted towards lower frequencies. Overall, stapedius muscle activity is related to a bird's production of high amplitude low frequencies. These results support the hypothesis that the primary role of the stapedius muscle during normal vocal development is to dampen the amplitude of low frequency energy that reaches the cochlea during vocalization.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

组织器官通讯对骨骼肌发育的作用及调控机制研究进展

骨骼肌是动物机体最重要的器官之一,研究骨骼肌发育调控机制对于肌肉相关疾病的诊断以及家畜肉质的改善都有着重要意义。骨骼肌发育调控是一个复杂的过程,受到大量肌肉分泌因子和信号通路的调节。此外,为了维持体内代谢稳态并最大限度地利用能量,机体协调多个组织器官形成了复杂而又精密的代谢调控网络,对于调控骨骼肌发育也发挥着重要的作用。随着组学技术的发展,人们对于组织器官通讯的潜在机制进行了深入研究。本文综述了脂肪组织、神经组织、肠道等组织器官通讯对于骨骼肌发育的影响,以期为靶向调控骨骼肌发育提供理论基础。