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1.
An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest. 相似文献
2.
Antibodies to sperm surface antigens and the c-myc proto-oncogene product inhibit early embryonic development in mice. 总被引:1,自引:0,他引:1
The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab' antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab' identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab' did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development. 相似文献
3.
Butyrate, a 4-carbon short chain fatty acid, is responsible for the protective effects of fiber in colorectal cancer prevention. To better understand the 'blueprint' of butyrate's chemopreventive role in this disease, we performed 2-dimensional difference gel electrophoresis (2-D DIGE) of butyrate-treated HCT-116 colorectal cancer cells after pre-fractionation using heparin affinity chromatography. A combination of this enrichment step with overlapping narrow range IPGs (pH 4-7 and pH 6-11) in 2-D DIGE resulted in the detection of 46 differentially expressed spots. Twenty-four of these were identified by MS analyses, and 5 spots were found to be heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Three isoforms of 38 kDa were down-regulated while two with Mr approximately 26 kDa were up-regulated. These represent phosphorylated isoforms of hnRNP A1 as verified by immunoblotting with anti-phosphotyrosine and anti-phosphoserine antibodies. Using 2-DE, subcellular fractionation and western blot analysis, we further showed that full-length hnRNP A1 underwent down-regulation, cleavage and cytoplasmic retention upon butyrate treatment. These indicate that modulations of hnRNP A1 may play a significant role in the mediation of growth arrest and apoptosis by butyrate. 相似文献
4.
Martin F. Brown Jacqueline S. Partin Christopher E. Killian William J. Lennarz 《Development, growth & differentiation》1995,37(1):69-78
When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine-linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca2+ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two-dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti-SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti-SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130. 相似文献
5.
Kohji Ikenishi Tetsuya S. Tanaka Tohru Komiya 《Development, growth & differentiation》1996,38(5):527-535
In order to know when the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila , the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting. 相似文献
6.
Localization of antigens homologous to excretory-secretory proteins in developing embryos ofSetaria digitata has been carried out by indirect fluorescent antibody test, [14C] labelling studies and Western blotting. Indirect fluorescent antibody test showed binding of excretory-secretory antibodies
at perivitelline space. The fluorescent antibody binding was almost absent at small morulae stage and increasing in intensity
in the successive developmental stages with maximum at coiled microfilaria stage. Hatched microfilaria did not show the presence
of antigens by immunofluorescence. Immuno-complex of excretory-secretory antiserum against “amniotic fluid” collected from
developing embryos ofSetaria digitata labelled with [14C] amino acids showed highest radioactivity at coiled and tadpole stages and differed significantly from small morulae, big
morulae and hatched microfilaria. Immunoblot analysis of amniotic fluid showed two proteins, 16.5 and 11 kDa, to be highly
antigenic. The antigenic protein (11 kDa) content as seen by immuno blotting increased during embryogenesis and decreased
at the stage of hatching. 相似文献
7.
Sahagun Ruiz A Waghela SD Romany MM Holman PJ Melendy D Cruz D Wagner GG 《International journal for parasitology》2000,30(1):59-64
Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen. 相似文献
8.
J F Angulo P L Moreau R Maunoury J Laporte A M Hill R Bertolotti R Devoret 《Mutation research》1989,217(2):123-134
A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos. 相似文献
9.
Robert S. Winning Niels C. Bols John J. Heikkila 《Differentiation; research in biological diversity》1991,46(3):167-172
Tunicamycin treatment of Xenopus laevis embryos enhanced the synthesis of a specific set of polypeptides with molecular masses of 98, 78, 59 and 58 kDa. The 78-kDa polypeptide was tentatively identified as glucose-regulated protein (GRP) 78 on the basis of molecular mass, pl (5.2), and tunicamycin inducibility, which took place upon treating embryos after the midblastula transition (MBT). The synthesis of a polypeptide with this electrophoretic mobility was detected but was not tunicamycin-inducible at stages prior to the MBT. GRP78 mRNA was detectable before the MBT but was not inducible by tunicamycin until the tailbud stage. A comparison of tunicamycin-induced polypeptide synthesis in Xenopus embryos, A6 cell line, and white blood cells by 2D-PAGE and fluorography revealed three spots in the GRP78 region of the gel. One was observed in both embryos and adult cells; another was adult-specific; and the third one was possibly an embryo-specific form. These results suggest that GRP78 synthesis might undergo a switch from an embryonic to an adult pattern during Xenopus development. 相似文献
10.
Reuter A Fortunato D Garoffo LP Napolitano L Scheurer S Giuffrida MG Vieths S Conti A 《Proteomics》2005,5(1):282-289
Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy. 相似文献
11.
Yamaguchi A Katsu Y Matsuyama M Yoshikuni M Nagahama Y 《European journal of cell biology》2006,85(6):501-517
The nuclear membranes surrounding fish and frog oocyte germinal vesicles (GVs) are supported by the lamina, an internal, mesh-like structure that consists of the protein lamin B3. The mechanisms by which lamin B3 is transported into GVs and is assembled to form the nuclear lamina are not well understood. In this study, we developed a heterogeneous microinjection system in which wild-type or mutated goldfish GV lamin B3 (GFLB3) was expressed in Escherichia coli, biotinylated, and microinjected into Xenopus oocytes. The localization of the biotinylated GFLB3 was visualized by fluorescence confocal microscopy. The results of these experiments indicated that the N-terminal domain plays important roles in both nuclear transport and assembly of lamin B3 to form the nuclear lamina. The N-terminal domain includes a major consensus phosphoacceptor site for the p34(cdc2) kinase at amino acid residue Ser-28. To investigate nuclear lamin phosphorylation, we generated a monoclonal antibody (C7B8D) against Ser-28-phosphorylated GFLB3. Two-dimensional (2-D) electrophoresis of GV protein revealed two major spots of lamin B3 with different isoelectric points (5.9 and 6.1). The C7B8D antibody recognized the pI-5.9 spot but not the pI-6.1 spot. The former spot disappeared when the native lamina was incubated with lambda phage protein phosphatase (lambda-PP), indicating that a portion of the lamin protein was already phosphorylated in the goldfish GV-stage oocytes. GFLB3 that had been microinjected into Xenopus oocytes was also phosphorylated in Xenopus GV lamina, as judged by Western blotting with C7B8D. Thus, lamin phosphorylation appears to occur prior to oocyte maturation in vivo in both these species. Taken together, our results suggest that the balance between phosphorylation by interphase lamin kinases and dephosphorylation by phosphatases regulates the conformational changes in the lamin B3 N-terminal head domain that in turn regulates the continual in vivo rearrangement and remodeling of the oocyte lamina. 相似文献
12.
Sparganosis is caused by invasion of Spirometra plerocercoid into various tissues/organs. Subcutaneous sparganosis can be diagnosed and treated by worm removal, while visceral/cerebral sparganosis is not easy to diagnose. The diagnosis depends largely on the detection of specific antibodies circulating in the patients' sera. Previous studies demonstrated that 31 and 36kDa proteins of the sparganum invoked specific and sensitive antibody responses, but also showed cross reactions with cysticercosis sera. We enriched protein fractions containing 31-36kDa through gel filtration and examined immune recognition pattern against the patient sera by 2-dimensional electrophoresis (2-DE) followed by immunoblotting. Serum samples from sparganosis patients recognized 8-10 protein spots of 31 and 36kDa with different isoelectric point (pI) values with variable combinations, in which four spots of 31kDa with pIs 3.4, 3.9, 4.0 and 4.1, and one 36kDa spot (pI 3.5) appeared to be specifically reactive. One 31kDa protein spot with pI 3.3 and two spots of 36kDa with pIs 3.3 and 3.5 reacted crossly with neurocysticercosis sera. Neither sera from patients with other parasitic infections nor those from healthy controls showed positive reaction. Two-DE/immunoblot analysis might be highly available in differential serodiagnosis of human sparganosis. 相似文献
13.
Arsanto JP Caubit X Rivier F Hugon G Thouveny Y Mornet D 《The International journal of developmental biology》1999,43(1):75-83
The expression patterns of the DMD (Duchenne Muscular Dystrophy) gene products, especially of Dp71 (apodystrophin-1) were investigated by immunofluorescence and immunoblotting in the retina of the Amphibian urodele Pleurodeles waltl. H-5A3 monoclonal antibody (mAb), directed against the C-terminal region of dystrophin/utrophin, and 5F3 mAb, directed against the last 31 amino acids of dystrophin and specific of Dp71, were used. Western blot analyses with H-5A3 mAb revealed distinct dystrophin-family isoforms in adult newt retinal extracts: a doublet 400-420 kDa, Dp260 isoform, a protein at about 120 kDa, and a diffuse zone at 70-80 kDa, which might correspond to Dp71. Reactivity with H-5A3 mAb appeared nearly restricted to the outer plexiform synaptic layer. On the other hand, Dp71-specific 5F3 mAb recognized trhee polypeptide bands at 70-80, 60-65 and 50-55 kDa in adult newt retina corresponding most probably to alternative spliced isoforms of Dp71. In immunohistochemistry by conventional epifluorescence microscopy, 5F3 labeling was mainly observed in the plexiform layers, the outer nuclear layer, and the photoreceptor inner segments, especially at the myoid regions. Analysis by confocal scanning laser microscopy (CSLM) revealed that 5F3 labeling was, in addition, present in the pigmented epithelium and the inner nuclear layer. Furthermore, CSLM showed that 5F3 staining at the myoids was concentrated at discrete domains underneath the plasma membrane. Our findings raised the question concerning the functional significance of Dp71 isoforms, especially at the myoid where Dp71 was detected for the first time, although it occurred here highly expressed. Putative role(s) played in this retinal compartment and other ones by Dp71 and/or other dystrophin isoforms were discussed. 相似文献
14.
Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells 总被引:1,自引:0,他引:1
In order to understand the role of the protein of Xenopus vasa homolog ( Xenopus vasa -like gene 1, XVLG1 ) in germ line cells, an attempt was made to perturb the function of the protein with the anti-vasa antibody 2L-13. The 2L-13 or the control antibody was microinjected with a lineage tracer (FITC-dextran-lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32-cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL-labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL-labeled PGC were not observed in almost all the 2L-13 antibody-injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L-13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage. 相似文献
15.
16.
A Cloeckaert M S Zygmunt P de Wergifosse G Dubray J N Limet 《Journal of general microbiology》1992,138(7):1543-1550
A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
18.
O. Loiseau J. Cottin R. Robert G. Tronchin C. Mahaza J.M. Senet 《FEMS immunology and medical microbiology》1995,11(3):219-230
Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area. 相似文献
19.
Mouse oocytes at the dictyate and metaphase II stages as well as fertilized eggs have been studied by indirect immunofluorescence for the expression of H-2 histocompatibility antigens on surface membranes. Serologically specific reactivity to H-2 antibody was observed as patchy fluorescence distributed over the surface of the oocyte membrane. In contrast, one-cell zygotes exhibited variable reactivity, and early two-cell stages were negative. Absorption studies confirmed the serologic specificity of the reactivity on oocytes, which could be shown to be due to H-2 antibody. The results suggest that fertilization results in altered expression of major histocompatibility complex surface antigens, and confirms earlier studies that cleavage stage mouse embryos are not reactive with H-2 antibody. 相似文献
20.
G Tedeschi G Bernardini M Mortarino A Negri F Ceciliani S Ronchi 《Cell biology international》1998,22(7-8):517-525
Protein expression during the early development of Xenopus has been followed by 2D-polyacrylamide gel electrophoresis (PAGE). The analysis of two-dimensional maps of eggs and embryos at different stages of development has allowed the separation of more than 2000 spots. Identification of numerous polypeptides was obtained in four different ways: (1) immuno-blotting; (2) amino terminal sequence after blotting on to PVDF membranes; (3) comigration; and (4) assignment in comparison with proteins separated by 2D techniques on reference maps such as human liver, red blood cells, plasma and cerebrospinal fluid reported in the Swiss 2D-PAGE Data Base. The maps presented in this report are a step toward the study of the protein expression in Xenopus eggs and embryos and may be a powerful working tool since Xenopus embryos are popular models for the study of development. 相似文献