维甲酸诱导人成骨肉瘤MG-63细胞分化过程中核基质构型及其蛋白质组成变化观察

应用选择性抽提、整装透射电镜观察和双向聚丙烯酰胺凝胶电泳技术，分析维甲酸诱导处理前后人成骨肉瘤MG-63细胞核基质-中间纤维系统的构型变化，以及核基质蛋白表达变化．实验结果显示MG-63细胞核基质和中间纤维数景较少、分布不均匀，核纤层为薄厚不一结构，与两类纤维联系不密切．经1μmol／L维甲酸处理后，细胞核基质纤维和中间纤维数量增多、结构层次丰富、单丝成分多，分布均匀并相互交织成规则网络，两类纤维通过薄层均一的核纤层发生密切联系，形成贯穿整个细胞核质区域的完整体系．此外，核基质蛋白表达也发生显著变化．表明经维甲酸诱导处理后MG-63的核基质-中间纤维系统产生了与正常细胞相似的恢复性改变，并且伴有核基质蛋白表达的差异．这些变化是癌细胞恶性表型逆转的重要特征和功能表现，对于揭示核基质构型及其蛋白组成与细胞癌变和逆转的关系和阐明细胞增殖分化的基因表达调控原理，均具有重要意义．     

维甲酸诱导人成骨肉瘤MG-63细胞分化过程中核基质构型及其蛋白质组成变化观察

应用选择性抽提、整装透射电镜观察和双向聚丙烯酰胺凝胶电泳技术,分析维甲酸诱导处理前后人成骨肉瘤MG-63细胞核基质-中间纤维系统的构型变化,以及核基质蛋白表达变化.实验结果显示MG-63细胞核基质和中间纤维数量较少、分布不均匀,核纤层为薄厚不一结构,与两类纤维联系不密切.经1 μmol/L维甲酸处理后,细胞核基质纤维和中间纤维数量增多、结构层次丰富、单丝成分多,分布均匀并相互交织成规则网络,两类纤维通过薄层均一的核纤层发生密切联系,形成贯穿整个细胞核质区域的完整体系.此外,核基质蛋白表达也发生显著变化.表明经维甲酸诱导处理后MG-63的核基质-中间纤维系统产生了与正常细胞相似的恢复性改变,并且伴有核基质蛋白表达的差异.这些变化是癌细胞恶性表型逆转的重要特征和功能表现,对于揭示核基质构型及其蛋白组成与细胞癌变和逆转的关系和阐明细胞增殖分化的基因表达调控原理,均具有重要意义.     

HMBA对人肝癌SMMC-7721细胞核基质-中间纤维系统构型的影响

应用选择性抽提，整装光镜和电镜样品制备技术，观察人肝癌SMMC-7721细胞经HMBA处理后核基质-中间纤维系统的变化．经HMBA诱导处理后，人肝癌细胞核基质纤维和中间纤维数量增多，结构层次丰富，分布均匀，并通过核纤层使3种纤维之间形成紧密联系．结果表明人肝癌细胞在诱导分化过程中核基质-中间纤维系统构型发生明显变化，对肿瘤细胞恶性表型逆转变化具有重要影响．     

天然抗氧化剂Isoverbascoside对人胃腺癌细胞生长和超微…

ＭＧｃ８０－３细胞经２０μｍｏｌ／Ｌ抗氧化剂Ｉｓｏｖｅｒｂａｓｃｏｓｉｄｅ处理后，细胞生长曲线与分裂指数显下降，倍增时间延长，生长抑制率达５７．８％，电镜观察结果表明，Ｉｓｏｖｅｒｂａｓｃｏｓｉｄｅ处理的细胞，核质比例下降，核形规则，核仁体积缩小，数量减少，核内异染色质减少，线粒体形态结构典型，细胞表面微绒毛减少等显变化，说明天然抗氧化剂Ｉｓｏｖｅｒｂａｓｃｏｓｉｄｅ改变了ＭＧｃ８０－３细胞的     

HMBA对人肝癌SMMC-7721细胞核基质-中间纤维系统构型的影响

应用选择性抽提,整装光镜和电镜样品制备技术,观察人肝癌SMMC-7721细胞经HMBA处理后核基质-中间纤维系统的变化.经HMBA诱导处理后,人肝癌细胞核基质纤维和中间纤维数量增多,结构层次丰富,分布均匀,并通过核纤层使3种纤维之间形成紧密联系.结果表明人肝癌细胞在诱导分化过程中核基质-中间纤维系统构型发生明显变化,对肿瘤细胞恶性表型逆转变化具有重要影响.     

天然抗氧化剂Isoverbascoside对人胃腺癌细胞生长和超微结构的影响

ＭＧｃ８０－３细胞经２０μｍｏｌ／Ｌ抗氧化剂ｌｓｏｖｅｒｂａｓｃｏｓｉｄｅ处理后，细胞生长曲线与分裂指数显著下降，倍增时间延长，生长抑制率达５７．８％．电镜观察结果表明：ｌｓｏｖｅｒｂａｓｃｏｓｉｄｅ处理的细胞，核质比例下降，核形规则，核仁体积缩小，数量减少，核内异染色质减少；线粒体形态结构典型；细胞表面微绒毛减少等显著变化．说明天然抗氧化剂Ｉｓｏｖｅｒｂａｓｃｏｓｉｄｅ改变了ＭＧｃ８Ｏ－３细胞的恶性表型特征，具有明显的诱导分化作用．     

熊去氧胆酸对人胃腺癌细胞系MGc80－3生长和形态结构的影响

研究了中药熊胆的主要成份之一熊去氧胆酸对ＭＧｃ８０－３细胞生长和形态结构的影响．结果表明，熊去氧胆酸对ＭＧｃ－８０－３细胞的增殖活动有明显的抑制作用，使其群体倍增时间延长及分裂指数下降，并使其细胞形态与超微结构发生一系列的变化：细胞体积增大并呈充分铺展状态，核质比值下降；细胞表面微绒毛减少或消失并出现小泡状突起；细胞器如线粒体等数厨增多，结构趋向正常化，核内异染色质团块减少，胞质游离多聚核糖体减少等等。这些结果说明熊去氧胆酸能在定程度上逆转ＭＧｃ８０－３细胞的恶性表型．     

亚硒酸钠和硫杂脯氨酸组合对人胃癌细胞的诱导分化作用

以亚硒酸钠和硫杂脯氨酸为诱导剂组合，处理人胃腺癌ＭＧｃ８０－３细胞．通过细胞生长曲线和分裂指数的测定、光镜细胞化学和酶标法检测碱性磷酸酶活性，以及免疫胶体金技术标记ｃ－ｅｒｂＢ－２癌基因蛋白等方法，证实诱导剂组合能有效地抑制ＭＧｃ８０－３细胞恶性生长，改变碱性磷酸酶活性和分布，引起ｃ－ｅｒｂＢ－２癌基因蛋白表达的变化，具有一定的诱导分化作用．     

包囊游仆虫细胞的类中间纤维细胞骨架体系

应用生化分级抽提,并结合DGD包埋 去包埋透射电镜样品制备方法显示,包囊游仆虫营养细胞和休眠细胞中,均存在由直径10 nm左右的单根纤维及单根纤维聚集成的纤维束为结构单元形成的类中间纤维细胞骨架体系.其中,营养细胞的类中间纤维构成的细胞质三维网架,在细胞膜内缘以较密集的纤维网占有了整个表质层,在表质层内缘的细胞质深部纤维形成较松散的网络,网内常见附着有细胞器及一些电子密度颗粒;核纤层位于大核核膜内缘,纤维紧密聚集成网;核骨架纤维网分布比较致密,未见有电子密度颗粒附着.休眠细胞中含有与营养细胞相似的纤维网架结构,但位于细胞内不同层次的纤维网比营养细胞中的同种结构要致密得多,这可能与纤毛虫形成包囊时细胞大范围的收缩有关.并且值得注意的是,在休眠细胞包囊壁的内层壁中也观察到相似于中间纤维的纤维网络,其纤维网均匀和致密地分布在整个包囊壁层中.电泳图谱显示,纤毛虫形成包囊后,保留了营养细胞中的部分蛋白条带,失去了部分条带,新产生了一些特异的条带.结果表明,包囊游仆虫的类中间纤维 核骨架体系,是细胞在营养条件下和休眠状态下都稳定存在的结构,它可能起到比微管类骨架更重要的作用.并且休眠细胞中该体系产生的一些特异蛋白条带,可能是纤毛虫休眠生命活动中的重要蛋白.     

熊去氧胆酸对人胃腺癌细胞系MGc80—3生长和形态结构的…

研究了中药熊胆的主要成份之一熊去氧胆酸对ＭＧｃ８０－３细胞生长和形态结构的影响。结果表明，熊去所氧胆酸对ＭＧｃ－８０－３细胞的增殖活动有明显的抑制作用，使其群体倍增时间延长及分裂分指数下降，并使其细胞形态与超微结构发生一系列的变化。     

天然家蚕抗菌肽对人白细胞及巨噬细胞淋巴瘤细胞骨架系统作用的比较

报道了天然家蚕抗菌肽ＣＭ４对离体Ｕ９３７癌细胞骨架及核骨架损伤作用的扫描电镜观察。随着时间的延长，经天然家蚕抗菌肽ＣＭ４作用后的癌细胞骨架断裂，固缩成团状；癌细胞核骨架断裂，部分凝聚成团，结构不完整。相同剂量的天然家蚕抗菌肽ＣＭ４与正常人白细胞作用后细胞骨架及核骨架未见损伤现象。说明天然抗菌肽与重组抗菌肽的抗癌作用相同。     

Redistribution of intermediate filaments during capping of lymphocyte surface molecules

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenstr?m's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.     

人胃腺癌细胞系恶性表型逆转过程中细胞骨架变化的初步研究

应用显示细胞骨架的光镜和电镜方法观察表明,MGc80-3细胞质内微管很少,中间纤维数量稀少、构型改变,质膜内缘有较丰富的微丝层,具有恶性细胞典型的、不发达的细胞骨架特征.但经dBcAMP诱导后,细胞质内微管和中间纤维数量增多,分布排列有规则,质膜内缘微丝大量减少,细胞骨架组成、构型、数量与分布均产生与正常细胞大体相似的恢复性改变.这种变化是由于dBeAMP诱导胃癌细胞内cAMP水平的提高而实现的.细胞骨架正常构型和功能的恢复,对于逆转细胞形态结构的改变、细胞增殖的调控和细胞表面特性的改变均具有重要影响,是癌变细胞恶性表型逆转的一种重要的形态和功能表现.     

双丁酰环腺苷酸对人胃腺癌体外培养细胞DNA合成的影响

应用~3H-TdR光镜和电镜放射自显影观察表明,MGc80-3 细胞标记指数达31.51％,标记银粒集中于分裂间期的S期细胞核中,细胞核呈重标记状态,绝大多数标记银粒分布于细胞核常染色质区域,少数见于核孔附近的常染色质或核孔附近的细胞质中,核仁未见标记,显示细胞内 DNA合成十分活跃。但经 dBcAMP诱导后,标记指数则降至 4.35％,细胞核呈弱标记状态,电镜放射自显影则未在细胞核内见到标记银粒,表明其DNA合成受到明显抑制。这种变化是由于dBcAMP诱导胃癌细胞内cAMP水平提高而实现的,认为DNA合成抑制是导致MGc80-3细胞走向分化的一个重要原因,对于癌变细胞恶性表型逆转具有重要作用。     

The nuclear lamina is a meshwork of intermediate-type filaments

The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane, is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromatin. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three immunologically related polypeptides of relative molecular mass (Mr) 60,000-75,000 (60-70K) termed lamins. Three lamins (A, B, and C) are typically present in mammalian somatic cells. Previous studies on nuclear envelopes of rat liver and Xenopus oocytes suggested that the lamina has a fibrillar or filamentous substructure. Interestingly, protein sequences recently deduced for human lamins A and C from complementary DNA clones indicate that both of these polypeptides contain a region of approximately 350 amino acids very similar in sequence to the coiled-coil alpha-helical rod domain that characterizes all intermediate-type filament (IF) proteins. Here we analyse the supramolecular organization of the native nuclear lamina and the structure and assembly properties of purified lamins, and show that the lamins constitute a previously unrecognized class of IF polypeptides.     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.     

Formation of reverse rigor chevrons by myosin heads

The uniform angle and conformation of myosin subfragment 1 (S1) bound to actin filaments (F-actin) attest to the precise alignment and stereospecificity of the binding of these two contractile proteins. Because actin filaments are polar, myosin heads must swing or rotate about the head-tail junction in order to bind. Electron microscopy of isolated thick filaments and of myosin molecules suggests that the molecules are flexible, but myosin fragments and crossbridges have been reported not to interact with inappropriately oriented actin filaments. Here we describe myofibrillar defects engendered by a site-directed mutation within the flight-muscle-specific actin gene of the fruitfly Drosophila. The mutation apparently retards sarcomere assembly: peripheral thick and thin filaments are misregistered and not incorporated into the Z-line. Therefore, a myosin filament encounters thin filaments with the 'wrong' polarity. We show that myosin heads tethered in a single thick filament can bind with opposite rigor crossbridge angles to flanking thin filaments, which are apparently of opposite polarities. Preservation of identical actomyosin interfaces requires that sets of heads originating from opposite sides of the thick filament swivel 180 degrees relative to each other, implying that myosin crossbridges are as flexible as isolated molecules.     

Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle

Muscle contraction results from a sliding movement of actin filaments induced by myosin crossbridges on hydrolysis of ATP, and many non-muscle cells are thought to move using a similar mechanism. The molecular mechanism of muscle contraction, however, is not completely understood. One of the major problems is the mechanochemical coupling at high velocity under near-zero load. Here, we report measurements of the sliding distance of an actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle in an unloaded condition. We used single sarcomeres from which the Z-lines, structures which anchor the thin filaments in the sarcomere, had been completely removed by calcium-activated neutral protease (CANP) and trypsin, and measured both the sliding velocity of single actin filaments along myosin filaments and the ATPase activity during sliding. Our results show that the average sliding distance of the actin filament is less than or equal to 600 A during one ATP cycle, much longer than the length of power stroke of myosin crossbridges deduced from mechanical studies of muscle, which is of the order of 80 A (for example, ref. 15).