Localization of yolk proteins and their possible precursors using polyclonal and monoclonal antibodies, in Helix aspersa.

Three major yolk proteins of 140, 100, 80 KD and a faint band of 440 KD were determined by gradient gel electrophoresis in the mature eggs of Helix aspersa. Polyclonal and monoclonal antibodies were raised against mature oocyte extracts. The binding sites of these rabbit and hybridoma antibodies with the different yolk protein components were identified with a combination of WESTERN blotting, ELISA, immunofluorescence and immunogold staining. All these techniques demonstrated materials immunologically similar to vitellins in the hemolymph and in the glandular cells of the digestive gland. The data suggest that, for its vitellogenesis, the garden-snail utilizes a heterosynthetic mechanism similar to that known in oviparous animals. The vitellogenins would be produced by the digestive gland.     

Isolation and Characterization of Goldfish cdc2, a Catalytic Component of Maturation-Promoting Factor

We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.     

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.     

Monoclonal antibodies as probes for processing of yolk protein in the mosquito; production and characterization

We have produced a library of monoclonal antibodies against yolk proteins of the mosquito Aedes aegypti. After the initial screening, 45 hybridoma cell lines were selected and cloned. Immunoblot analysis revealed three groups of monoclonal antibodies. One group recognized a 200-kDa polypeptide, the second a 68-kDa, and the third both of these polypeptides. While the affinity of binding by different antibodies varied widely, all monoclonal antibodies recognized these polypeptides only in extracts from vitellogenic fat bodies and ovaries. The antibodies were further characterized by video-enhanced immunofluorescence, which also showed that both yolk polypeptides originated in the fat body and accumulated in the oöcytes. The immunolocalization in trophocytes of the fat body suggested that monoclonal antibodies may recognize different stages of the secretory pathway of yolk polypeptides. Similar analysis of oöcytes indicated that our panel of antibodies recognizes different steps of processing of both 200-kDa and 68-kDa polypeptides, beginning with internalization by the oöcyte and ending with the final crystalline form in mature yolk bodies.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Cell type-dependent collagen-type recognition by cell receptors.

Affinity chromatography of a number of cell types on collagens I and III reveals three proteins with M(R)of 250, 170 and 140 kDa. These proteins are able to discriminate between types I and III, but not types III and IV. Collagen-type recognition is therefore characteristic for cells of connective tissue origin. Polyclonal antibodies (Ab) raised against 170 and 140 kDa polypeptides and used in immunofluorescence show membrane localisation for both, with their distribution being similar to each other and to the distribution of the integrin beta1 chain. Ab p140 and commercial monoclonal antibodies against alpha(2)chain stain a band of the same molecular mass as from purified collagen binding proteins from liver cells, indicating that the 140 kDa protein is probably the alpha(2)integrin chain. The alpha(2)chain containing integrins are therefore able to discriminate collagen types I and III and collagen type recognition by this receptor is cell-type dependent.     

Vitellogenesis in Bufo arenarum: Identification,characterization and immunolocalization of high molecular mass lipovitellin during oogenesis

Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).     

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.     

Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes

 All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopus laevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200–180 kDa, 140–120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39–48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs. Received: 5 January 1999 / Accepted: 12 February 1999     

C-terminal truncated forms of Met, the hepatocyte growth factor receptor.

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. p190MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p50 alpha) and 145 kDa (p145 beta). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (p140MET) is membrane bound and is composed of an alpha chain (p50 alpha) and an 85-kDa C-terminal truncated beta chain (p85 beta). The second protein (p130MET) is released in the culture supernatant and consists of an alpha chain (p50 alpha) and a 75-kDa C-terminal truncated beta chain (p75 beta). Both truncated forms lack the tyrosine kinase domain. p140MET and p130MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. p140MET is preferentially localized at the cell surface, where it is present in roughly half the amount of p190MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.     

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.     

可口革囊星虫体腔中卵细胞大小组成的周年变化

在浙江温岭沿海逐月采样,显微观察了可口革囊星虫体腔中卵细胞大小组成的周年变化。3—5月,卵径30~90μm的卵细胞占大部分。6月,卵径70~130μm的卵细胞占多数。7月,大部分卵细胞卵径达100μm以上,其中130~150μm的卵细胞约占1/3。8月,卵细胞卵径均在110μm以上,其中130~150μm的卵细胞约占2/3。9月,仍多数卵细胞卵径在110μm以上,130~150μm的卵细胞约占1/3。10月,以110~140μm的卵细胞占多,40μm以下的卵细胞较少,大卵细胞开始退化。11月至翌年2月,随着大型卵细胞的逐渐退化,小型及中型卵细胞逐渐增多,2月仍可见少量未退化的大型卵细胞。分析认为,可口革囊星虫在浙江温岭的繁殖期主要在7—9月。     

Brain-derived neurotrophic factor induces cell surface expression of short-form tenascin R complex in hippocampal postsynapses

Brain-derived neurotrophic factor (BDNF) is involved in hippocampal functions such as learning and memory and it plays a crucial role in regulating synaptic plasticity. To investigate potential mechanisms by which BDNF participates in neuronal communication through postsynaptic membrane proteins, we generated monoclonal antibodies against the synaptoneurosomal particulate fraction of mouse brain. One of the monoclonal antibodies, termed mAb#27, was found to be useful for analyzing BDNF-induced externalization of synaptoneurosomal membrane proteins of the hippocampus. In dissociated neuronal cultures, BDNF stimulation increased mAb#27 immunoprecipitates of biotin-labeled proteins with apparent masses, 55kDa, 80kDa, 100kDa, 130kDa, 140kDa and 160kDa. The mAb#27 recognition molecules were located in specific hippocampal regions of the brain and at postsynaptic sites in cultured cells. Proteomic studies of the mAb#27 immunocomplex identified newly derived short forms of tenascin R (TNR) as the mAb#27 recognition molecule. Contactin 1, prostaglandin regulatory-like protein and GABA A receptor subunit beta3 were identified as TNR-associated proteins. These proteins were recruited to mAb#27 when BDNF was applied to cells in culture. Each molecules identified in the present study contributes to the postsynaptic plasticity or the active cycle of cellular vesicle membranes. The formation of the TNR complex may serve as an underlying basis for synaptic plasticity in the hippocampus. Our results demonstrate that BDNF plays a role in external molecular dynamics and is likely to regulate synaptic functions such as the enhancement of neuronal excitability through GABAergic neurons.     

Monoclonal antibodies as probes for processing of the mosquito yolk protein; a high-resolution immunolocalization of secretory and accumulative pathways

A library of monoclonal antibodies (mAB) directed against yolk polypeptides of the mosquito Aedes aegypti was utilized to visualize the secretory pathway of these polypeptides in the fat body and their accumulative pathway in developing oocytes. Single and double immunolabelling using mABs and colloidal gold of different sizes confirmed biochemical observation that 200 +/- 5 and 65 +/- 3 kDa polypeptides represent subunits of the yolk protein. This immunocytochemical analysis showed that, in trophocytes of the fat body, both the subunits of the yolk protein were routed simultaneously through the Golgi complex into secretory granules and were subsequently secreted. The yolk protein subunits were also directed together through all the steps of the accumulative pathway in the oocyte. Double immunogold labelling revealed that the subunits were present together during their binding to the oocyte membrane, transportation into and accumulation in the transitional yolk body, and, finally, crystallization in the mature yolk body. Electron microscopical immunocytochemistry also confirmed immunofluorescent data and showed that mABs directed against different steps in the biosynthetic processing of the yolk protein in the fat body, as well as in its accumulative pathway in oocytes.     

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.     

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.     

Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture.

An analysis of the binding properties of a collection of monoclonal antibodies to African swine fever virus particles showed that virus field isolates passaged in porcine macrophages changed antigenically more than a strain of a cell-adapted virus passaged in Vero cells. From seven clones isolated from the spleen of a field-infected pig, we found four clones that had the same antigenic properties, one clone that had large changes in proteins p150 and p27 and small changes in proteins p37 and p14, and two clones that had minor changes in proteins p150 and p27, respectively. An analysis of the binding properties of the monoclonal antibodies to 23 field isolates from Africa, Europe, and America showed that the African isolates differed among themselves more than the European and the American isolates; in this study we found changes in 8 of the 10 virus proteins tested. The most variable proteins in the African isolates were p150, p27, p14, and p12. In contrast to the African isolates, protein p12 from the non-African viruses did not change. The clustering of the field virus isolates in six antigenic homology groups indicated the existence of a complex variety of African swine fever virus serotypes.