Plate in the zone of oocyte and germinal epithelium contact in Scyphomedusa Aurelia aurita binds antibodies to ZP-domain protein mesoglein

The morphological features of oocyte and germinal epithelium (epithelial wall of germinal sinus) contact area in Scyphomedusa Aurelia aurita are described. Growing oocytes were divided into seven stages based on oocyte size. The structure revealed the area of contact between the oocyte and the germinal epithelium called the contact plate. During oocyte growth, single granules are fused into the homogeneous mass area of oocyte contact with epithelium. Plate components bound antibodies to mesoglein. It was assumed that the plate material contained ZP-domain proteins. Electrophoresis and immunoblotting results show that proteins immunologically similar to mesoglein have higher molecular masses, probably due to post-translation modifications, which are common for extracellular proteins. On the other hand, however, gonad proteins may be other representatives of jellyfish ZP-domain proteins. Further experiments should be conducted to clarify which alternative is true.     

Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan <Emphasis Type="Italic">Aurelia aurita</Emphasis> (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages of oocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the Zona Pellucida type.     

Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan Aurelia aurita (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages ofoocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the zona pellucida type.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis

Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (M_r) 68,000. A rapid extraction procedure using buffer containing 1% ($\text{v}\text{v}$) Triton X-100 and 1.0 m-KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of M_r 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species.For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (L_I) of M_r 72,000 focusing at approximately pH 5.35 and another (L_II) of M_r 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein L_I from Xenopus and, in addition, two other polypeptides (M_r values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9.It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of M_r 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the M_r 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex.     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud

During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex. Thus the fragmentation of the cloud that occurs later in oogenesis is irrelevant to the movement of METRO-localized RNAs and germinal granules. On the basis of these findings, we propose here a revised model of germinal granule and localized RNAs delivery to the oocyte vegetal cortex via the METRO pathway.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10?days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin).Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M_r region as determined by staining with Coomassie blue and labelling with a mixture of ¹⁴C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF³ polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M_rmr) and six polypeptides (IEF 12, 68,000 M_r; IEF 24, 56,000 M_r; IEF 31, 50,000 M_r; IEF 35, 49,000 M_r; IEF 36, 48,500 M_r and IEF 46, 43,500 M_r) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis.Labelling of mitotic and interphase cells with [³⁵S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.     

Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M_r was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M_r was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of ²⁵²Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

FORMATION AND BEHAVIOR OF PINOSOMES IN THE SEA URCHIN OOCYTE DURING OOGENESIS

Formation and behavior of the pinosomes at the surface of the oocyte during oogenesis in the 4 species of sea urchins, Anthocidaris crassispina, Temnopleurus toreumaticus, Mespilia globulus and Pseudocentrotus depressus, were studied. The plasma membrane of the oocyte is almost smooth at the early stage of oogenesis, although a small number of cytoplasmic processes appear on it, facing the germinal epithelium. At the beginning of vitellogenetic stage many processes appear on the whole surface of the oocyte. Near the base of the fully grown process, the pinosome designated as the α-pinosome is formed. The α-pinosome may play a part in maturation of the yolk granule. The processes shorten as a whole at the time of the breakdown of the germinal vesicle. Formation of the pinosome designated as the β-pinosome begins just before vitellogenetic stage and continues during this stage. The β-pinosome may be directly concerned with the formation of cortical granules.     

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

The occurrence of artemocyanin in Branchiopoda (Crustacea)

Artemocyanin, the extracellular hemolymph biliprotein of Artemia, is demonstrated in the fairy shrimp Streptocephalus, the clam shrimp Leptestheria and the water flea Daphnia. Artemocyanins can be purified from hemolymph as intact polypeptides (M_r 170–190,000), but are degraded upon homogenization of the whole animal by partial proteolysis to polypeptides with M_r 102,000 and 85,000. The aminoterminal sequence of the intact artemocyanin polypeptide was determined, but no clear-cut relationships with arthropod biliproteins or other protein families could be demonstrated.     

Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.