MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

为探究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌生长发育的作用机制,本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化,使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型,检测设计合成的MSTN siRNA （si-MSTN）干扰效果;采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化,研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示,在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调;转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）;在转染si-MSTN的GM期牛骨骼肌卫星细胞中,肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05）,PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05;P<0.01）;在转染si-MSTN的DM3牛骨骼肌卫星细胞中,ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05）,RhoA基因mRNA水平极显著升高（P<0.01）,PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明,干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达,探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制,为进一步研究MSTN对牛成肌分化的调控机制提供参考。     

Cutaneous leiomyosarcoma with visceral metastases in a White Carneau pigeon and literature review

Cutaneous leiomyosarcomas are malignant mesenchymal tumors of smooth muscle origin and are reported occasionally in avian species. A 14-y-old male laboratory White Carneau pigeon (Columba livia) was presented for surgical excision of a cervical soft tissue mass. Ultrasonography with color flow Doppler imaging revealed multiple cavitations of mixed echogenicity within the mass and vascularization. Histologically, the dermis and subcutis were expanded by a densely cellular multinodular mass comprised of fusiform cells forming haphazardly arranged broad streams and short interwoven bundles, often surrounding blood vessels and variably sized cavitations. Neoplastic cells were strongly immunopositive for desmin and α–smooth muscle actin, and negative for pancytokeratin, S100, and von Willebrand factor. Based on histopathology and IHC findings, the cutaneous mass was diagnosed as leiomyosarcoma (LMS). The pigeon died 312 d post-operatively. Postmortem examination revealed masses infiltrating the left and right pulmonary airways and one hepatic nodule, but no regrowth at the surgical site. Histologic and IHC evaluation of the pulmonary and hepatic masses were consistent with LMS, representing metastatic foci from the primary cutaneous LMS. Our case highlights the malignant behavior and histomorphologic features of cutaneous LMS in an avian species.     

西伯利亚白刺肌动蛋白基因的分离与特性分析

利用兼并PCR及RACE技术克隆了西伯利亚白刺的肌动蛋白基因(Nitraria sibirica Actin, NsAct),并对其基因结构和表达特性进行了分析。所克隆的NsAct cDNA全长序列为1 831 bp,包含1 134 bp的开放阅读框,编码377个氨基酸;基因组DNA全长序列为2 913 bp,包含5个外显子和4个内含子。系统进化分析结果显示,NsAct与其他植物的肌动蛋白基因具有较高的同源性,与芒果(无患子目)亲缘关系最为接近。基因表达特性分析的结果显示,该基因在根、茎、叶中的表达量基本一致;在干旱、盐、低温及外源脱落酸胁迫下,表达量无明显变化,这些结果验证了该基因作为分子内标的可靠性。     

Effects of synthetic kainic acid on microfilament expression and gap junction in astrocytes

AIM：To study the effects of synthetic kainic acid (SKA) and 1-heptanol (1-Hep), a gap junction blocker, on the cytoskeletal filament expression in the astrocytes. METHODS：The neonatal rat brain was obtained from the Wistar rats (1 day old) and primary purified astrocytes were obtained by differential attachment for removing filamentoblasts and orbital shaker for removing the oligodendrocytes. The effects of SKA and other interventions on the morphologic changes and expression levels of skeleton protein filamentous actin (F-actin) were observed in the astrocytes after 24 h of the exposures by laser scanning confocal microscopy. The effect of 1-Hep on the expression of F-actin was also explored. RESULTS：Compared with control group, the fluorescence intensities in KCl group and KCl+SKA group were increased, and highly increased in KCl+SKA group. The F-actin filaments in the above 2 groups were more intensive, thickened and concentrated than those in control group, and more obvious in KCl+SKA group. l-Hep significantly decreased the expression of F-actin in KCl group and KCl+SKA group as compared with control group, and parts of the filamentous fracture were seen in the astrocytes in all 1-Hep-treated groups, in which some of the filamentous lines were crosscut. CONCLUSION：Increase in the expression of F-actin in the astrocytes affects the structure and function of the intercellular gap junctions, which may be involved in the mechanism of SKA-induced epilepsy.     

Dynamics of Actin Polymerization

The process of actin polymerization and depolymerization have some relation with its function,the activation of such process is compatible to its functions. The polymerization progress is divided into four steps: The first step is the activation of the monomer actin. The nucleation of actin nucleus forms in the second step which limits the polymerization speed. The elongation of the F-actin filaments happens in the following step. In the last step, a steady-state is attained, the length of the filament does not change because the polymerizing rate in the barbed end is the same with the depolymerizing rate in the pointed end. Some factors which affect the polymerizing process, such as nucleotide, actin related proteins are discussed. The aim is to know more about how to assemble the complicated system with biomacromolecule and what to function.     

巴西橡胶树ADF6基因的克隆与表达分析

肌动蛋白解聚因子(Actin Depolymerizing Factor,ADF)是一种重要的肌动蛋白结合蛋白。ADF通过参与调控肌动蛋白的装配和解聚在多种生理过程中发挥重要作用。本研究通过RT-PCR技术从橡胶树胶乳中获得HbADF6基因的cDNA和基因组序列,序列分析显示HbADF6基因的开放阅读框为441 bp,共编码146个氨基酸,基因组序列由2个内含子和3个外显子组成。实时荧光定量PCR结果表明,HbADF6基因在所有检测组织中均表达,其中树皮中表达量最高,叶片中表达量最低。HbADF6基因的表达受乙烯利(ET)和碘化钾(KI)调控,并与排胶时间和死皮有关。此结果表明HbADF6基因可能在橡胶树乙烯响应、死皮和排胶过程中发挥重要作用。      

Muscle protein expression of pikeperches (Stizostedion lucioperca and S. volgense)

The purpose of the present study was to investigate the muscle protein expression in two pikeperches (Stizostedion lucioperca and S. volgense) through intra‐ and intermyomeric composition of white muscles. Using denaturing 10% sodium dodecylsulfate‐polyacrylamide gel electrophoresis, muscle protein expression was studied in relation to within‐ and between‐species morphological development, sex, maturity and age of pikeperches. Myosin, actin and troponin have a distinct role in the contraction and length tension of muscle fibers of these species. No obvious intramyomeric differences were found in the myosin heavy chain of both species. Myosin light chains (15–38 kDa) have different expression in different age groups. The muscle protein of the fingerling and adult S. lucioperca had high molecular weight (50 kDa) myosin in contrast to the other Percid species. The molecular weight of actins increased comparatively in low‐age‐group fish. ATP is stored in myosin and released to cause contraction when myosin comes in contact with actin of the experimental fish. Troponin regulates increasing concentration of light‐chain myosin in mature fish. Because troponin T has been implicated in the regulation of skeletal muscle kinetics, muscle contraction kinetics was predicted in different age groups. The muscle proteins of both sexes of these species have polymorphism in various age groups but have no difference in similar aged fish. No muscle protein dimorphism was found in these Percid species. The white muscle protein composition and contractile properties affect power production during fast, unsteady movement and swimming.     

Laryngeal rhabdomyoma in a dog

A 3‐year‐old, female spayed Australian Shepherd dog was presented to Veterinary Healthcare Associates in Winter Haven, FL with a history of respiratory stridor, difficulty swallowing, and a change in bark for approximately 3 months. Radiographs revealed a soft tissue mass caudal to the epiglottis. Oral and computerized tomographic (CT) examinations were performed under general anesthesia and revealed a small firm mass in the caudal larynx on the right side. Cytologic evaluation of a fine‐needle aspirate of the mass revealed cells consistent with a rhabdomyoma, oncocytoma, or granular cell tumor. Histopathologic interpretation was rhabdomyoma or oncocytoma. Histochemical staining with periodic acid‐Schiff was mostly negative with a low number of weakly positive cells and with phosphotungstic acid‐hematoxylin was strongly positive with visible cross striations. Tumor cells did not express pancytokeratin AE1/AE3, were strongly immunoreactive for myoglobin and muscle‐specific actin, and did not express vimentin except for a low number of weakly immunoreactive cells. These findings confirmed a diagnosis of rhabdomyoma. Primary neoplasia of the larynx is rare, and widely varying types of neoplasms have been documented in this location. Due to the cytologic and histologic similarities between rhabdomyoma, oncocytoma, and granular cell tumor, these neoplasms should be differentiated using histochemical staining and immunohistochemical analysis.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

蛋白质磷酸化对肌动球蛋白解离及其乙酰化水平的影响

[目的]研究肌球蛋白重链和肌动蛋白磷酸化对其乙酰化水平、肌动球蛋白解离及ATP酶活性的影响,为通过调控磷酸化水平改善肉品嫩度提供理论依据.[方法]以羊背最长肌为材料制备肌肉匀浆液,采用碱性磷酸酶抑制剂(抑制去磷酸化)和蛋白激酶抑制剂(抑制磷酸化)调控其磷酸化水平,在4℃分别孵育0、0.5、4、12、24、48和72 h...