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1.
Expression of new proteins of the intermediate filament protein family in differentiating F9 embryonal carcinoma cell cytoskeleton 总被引:3,自引:0,他引:3
Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B. 相似文献
2.
Poehland R Tomek W Becker F Kurth J Kanitz W Bhojwani S 《Molecular reproduction and development》2008,75(7):1109-1119
The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos. 相似文献
3.
Changes in macromolecule syntheses, especially RNA synthesis, and the energy providing system were investigated in seeds ofAgrostemma githago aged for different periods. In embryos of aged seeds all macromolecule syntheses start later and reach a lower level than
young ones. It was found that the synthesis of rRNA in embryos of aged seeds is reduced whereas the synthesis of poly (A+) RNA in relation to the total RNA synthesis is highly increased as well as the amount of this RNA species with long poly
(A) chains. The results are discussed in connection with the decreased protein synthesis and the reduced ATP content and ATP
formation ability in embryos during the long time storage of seeds. 相似文献
4.
Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study 总被引:7,自引:6,他引:1
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The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments. 相似文献
5.
ERIKA SCHMID STEPHEN TAPSCOTT GUDRUN S. BENNETT JAMES CROOP STEVEN A. FELLIN HOWARD HOLTZER WERNER W. FRANKE 《Differentiation; research in biological diversity》1979,15(1-3):27-40
The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation. 相似文献
6.
Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo. 总被引:9,自引:0,他引:9
E Schmid S Tapscott G S Bennett J Croop S A Fellini H Holtzer W W Franke 《Differentiation; research in biological diversity》1979,15(1):27-40
The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation. 相似文献
7.
Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins 总被引:2,自引:2,他引:0
RYOICHI MATSUDA TAKASHI KITAJIMA HIROSHI OHINATA YUKO KATOH TORU HIGASHINAKAGAWA 《Development, growth & differentiation》1988,30(1):25-33
A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [35 S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca2+ . Cell-free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification. 相似文献
8.
The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos. 相似文献
9.
We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins. 相似文献
10.
Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [3H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation. 相似文献
11.
Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated. 相似文献
12.
The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins. 总被引:3,自引:0,他引:3
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The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed. 相似文献
13.
Isolation of endo A cDNA from mouse 8-cell stage embryos 总被引:1,自引:0,他引:1
M Nozaki K Murata T Morita A Matsushiro 《Biochemical and biophysical research communications》1988,154(3):890-894
To analyse the species of genes expressed in a cleavage stage mouse embryo, we have constructed a cDNA library containing 3.0 x 10(5) independent clones from about 2 x 10(3) embryos at the 8-cell stage of development. Endo A cDNA prepared from parietal yolk sac endoderm like PYS-2 cells was used to screen the library. Southern blot analyses using the endo A sequence as a probe and restriction mapping analyses revealed that four independent recombinants had been inserted endo A sequence. Sequencing data of these clones showed that endo A mRNA present in the 8-cell stage embryo is identical to that of parietal yolk sac endoderm cells. 相似文献
14.
Seu-Mei Wang Miao-Chia Lo Ching Shang Shih-Chu Kao Yung-Zu Tseng 《Journal of cellular biochemistry》1998,71(1):82-95
A rat polyclonal anti-M-line protein antiserum and three mouse monoclonal anti-titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M-line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M-line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage (day 3 cultures), M-line proteins were incorporated into dot-like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M-line proteins were registered in periodic pattern in the mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morphological data indicate that the early registration of M-line proteins in primitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin. In order to investigate the role of M-line proteins in the assembly of titin filaments, anti-M-line protein or anti-titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins, and the profile of myofibril assembly was examined. Cardiomyocytes from day 2–3 cultures with incorporated anti-M-line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti-titin antibody E2, which recognizes the M-line end domain of titin, resulted in the failure of M-line proteins organized into the M-line structure, as shown by random, sporadic staining with anti-M-line protein antibody. These studies confirm the essential role of M-line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure. J. Cell. Biochem. 71:82–95, 1998. © 1998 Wiley-Liss, Inc. 相似文献
15.
Vitale AM Calvert ME Mallavarapu M Yurttas P Perlin J Herr J Coonrod S 《Molecular reproduction and development》2007,74(5):608-616
In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage. 相似文献
16.
Intermediate filament protein partnership in astrocytes. 总被引:20,自引:0,他引:20
C Eliasson C Sahlgren C H Berthold J Stakeberg J E Celis C Betsholtz J E Eriksson M Pekny 《The Journal of biological chemistry》1999,274(34):23996-24006
Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes. 相似文献
17.
5 protein fractions were identified and their relative mobility was determined in the rat oocytes and cleaving embryos by means of vertical capillary microdisc-electrophoresis in 7.5% polyacrilamide gel (PAA-gel). The same fractions were identified in the cleaving embryos devoid of zona pellucida. A conclusion was drawn that these proteins were present in the oocyte cytoplasm and kept in the cleaving embryos until the stage of implantation. 4 groups of proteins with different anodic mobility were identified in the isolated zona pellucida by means of microdisc-electrophoresis in 7.5% PAA-gel added with 1% sodium dodecylsulfate (SDS). The molecular weight of low molecular weight proteins of oocytes and preimplantation embryos was determined by means of disc-electrophoresis in 14% PAA-gel with 1% SDS. The zona pellucida of one embryo contained, according to the data of capillary spectrophotometry, 5 ng of protein. 相似文献
18.
Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system 总被引:5,自引:4,他引:1
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《The Journal of cell biology》1988,107(2):587-596
The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool. 相似文献
19.
Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis 总被引:2,自引:0,他引:2
Carol A. Erickson Richard P. Tucker Benjamin F. Edwards 《Differentiation; research in biological diversity》1987,34(2):88-97
We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes. 相似文献
20.
L F Lemanski 《The Journal of cell biology》1979,82(1):227-238
Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin. 相似文献