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1.
Analysis by two-dimensional gel electrophoresis and silver staining of heavy full, light full, and empty bovine papillomavirus particles has shown that the major capsid protein L1 is highly modified. Besides exhibiting at least 13 isoelectric point variants of approximately the same molecular mass (54 kilodaltons), it is suggested that an additional heavier protein chain (69 kilodaltons) is also derived from L1 by glycosylation. These modifications may stabilize the particle structure. Treatment with neuraminidase reduces the number of modification products detectable, with a concomitant increase in the more basic forms of L1. Although it was not possible to detect histones in any of the preparations, proteins of similar molecular mass were detected. Therefore, it is suggested that the basic tails of L1 bind to the DNA in a manner similar to that of histone. Calculation of the theoretical mobilities of the papillomavirus proteins shows good agreement with the actual position of L1 and its isoelectric point variants and suggests that two of the proteins with molecular masses similar to those of the histones may actually be coded by the bovine papillomavirus E7 and E5 open reading frames. 相似文献
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PD Dr. G. F. Jirikowski J. F. Ramalho-Ortigao K. W. Kesse F. E. Bloom 《Histochemistry and cell biology》1990,94(2):187-190
Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide
probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied
this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes
and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present
study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and
an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons
in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining
only, perhaps indicating different stages of synthetic and secretory activity.
The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level.
The method might also have potential for in situ hybridization on the electronmicroscopical level. 相似文献
5.
Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition 总被引:60,自引:26,他引:34
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Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well- preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin- depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold. 相似文献
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Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. 总被引:25,自引:0,他引:25
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Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements. 相似文献
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K. S. Koch X. P. Lu D. A. Brenner G. H. Fey A. Martinez-Conde H. L. Leffert 《In vitro cellular & developmental biology. Plant》1990,26(12):1202-1202
The online version of the original article can be found at 相似文献
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Summary The microproblematicumPycnoporidium ? eomesozoicum
Flügel, 1972, from Upper Triassic reefs of the Alpine-Mediterranean region, Turkey Oman and Iran (originally interpreted as possible
alga) represents the type species of a new strophomenid brachiopod genus (Gosaukammerella n.g.). The genus is characterized by a very small, millimeter-sized plano-convex shell, whose ventral valve is attached to
the substratum (mainly sponges) by symmetrically arranged outgrowths developing from a pseudopunctate, lamellose foliated
shell wall and composed of densely spaced subparallel ‘tubes’ comparable with productide spines secreted by papillose extensions
of the mantle.Gosaukammerella seems to be the only reliable candidate for the existence of post-Paleozoic strophomenid (productid ?) brachiopods.
Gosaukammerella eomesozoica is restricted to possibly cryptic, shaded reef environments inhabited predominantly by sponges serving as substrates for
micromorphic brachiopods. 相似文献