Calcium regulation and calcium function in the nucleus of starfish oocytes

Summary

The nucleus (germinal vesicle) of starfish oocytes can be injected in vivo to introduce into it calcium indicators and various effectors or inhibitors of calcium signalling pathways. This is advantageous to the study of the debated problem of nuclear calcium homeostasis, which is related to that of the function of calcium in the nucleus. The work described here has shown that, at variance with other cell types, the nuclear envelope of starfish oocytes is relatively impermeable to calcium and to calcium sensitive dyes. It has also shown that a rise in free nuclear calcium is required for the reinitiation of meiosis induced by 1-methyladenine. Bom inositol 1, 4, 5-trisphosphate (InsP₃) and cyclic ADP-ribose (cADPr) receptors are present and functional in the membrane enveloping the nucleus. The chief processor of the calcium signal, caknodulin, interacts in the nucleus with the heterogeneous ribonucleoprotein particles and could thus play an important role in the processing of pre-mRNA.     

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.     

Subcellular characterization of the primordial germ cell antigen PG2 in adult oocytes

Primordial germ cells represent the founder population for establishing the germ line providing the continuity of life between generations. PG2, a germ cell-specific antigen, is one of the few continuously detectable epitopes in mammalian primordial germ cells and it is dynamically expressed during early post-fertilization development and during postnatal germ cell maturation. Immunoelectron microscopy shows a localization of PG2 in the peri-mitochondrial cytoplasm but its further subcellular or biochemical nature remains elusive. For further characterization of the PG2 epitope we used regular and semi-thin cryosection of ovulated and isolated follicular rabbit oocytes and localized all mitochondria with the help of the constitutive mitochondrial antigen MTC02 in double immunofluorescence stainings. Semi-thin cryosections of ovulated oocytes revealed a general close co-localization of both antibody reactions at the level of single mitochondria. In centrifuged follicular oocytes both antigens co-sedimented almost completely indicating a topographical association of the epitopes on the basis of a strong interaction of PG2 with mitochondria. To begin to characterize the germ cell epitope biochemically we treated oocyte cryosections either with acetone to reduce lipids or with N-glycosidase F to remove N-linked glycosylations before the immuoreaction. Neither treatment affected the antibody characteristics, which suggests that the PG2 epitope is most probably a protein. Because of the close interaction of PG2 with the mitochondria we speculate that PG2 is involved in the change of the mitochondrial morphology typically observed during differentiation of germ cells.     

Coated Vesicles From Locust Oocytes: Isolation and Characterization

Summary

This report demonstrates for the first time the isolation of coated vesicles from insect oocytes. Coated vesicles were purified from oocytes of Locusta migratoria by differential centrifugation and sucrose density centrifugation. The coated vesicles were characterized by electron microscopy, SDS-PAGE and scanning densitometry. Like coated vesicles isolated from pig brain and chicken oocytes, the coated vesicles from locust oocytes contained clathrin as the major protein component. Apart from clathrin, another major protein characteristic of coated vesicles had a molecular weight of about 115,000, and in addition, several minor unidentified bands were identified.     

A GFP-tagged Muscleblind C protein isoform reporter construct

Drosophila muscleblind (mbl), the ortholog of human Muscleblind-like 1 (MBNL1) gene involved in Myotonic Dystrophy (DM), gives raise to protein isoforms MblA to G. The specific functions and subcellular distribution of isoforms are still largely unknown. To overcome the lack of isoform-specific antibodies we generated transgenic flies that express a GFP:MblC fusion protein under the control of the Gal4/UAS system. The reporter fusion protein was able to functionally complement mbl loss of function mutations, demonstrating activity, and accumulated predominantly in adult muscle nuclei. The fluorescent nature of the reporter makes it appropriate for live imaging detection of MblC protein isoform.     

Monoclonal antibodies against unfertilized zona-free mouse oocytes: Characterization and effects on fertilization

A panel of anti-oocyte antibodies was raised against unfertilized zona-free mouse oocytes by intrasplenic immunization and checked for their effects on in vitro fertilization. Four antibodies decreased the fertilization rate from about 90% in controls to 8% (B5-2 F7), 12% (A2-2 A7), 13% (4-G1), and 25% (A2-2 F2), when the sperm cell concentration was 1 × 10⁵ to 1 × 10⁶. Antigen localization: All the antibodies labelled components in the cell membrane of zona-free oocytes as demonstrated by indirect immunofluorescence and/or by complement-mediated oocyte lysis. In various patterns, the ooplasm and zona pellucida were also labelled with different intensities. Western blotting: A2-2 A7 and A2-2 F2 recognized a protein with a molecular weight of approximately 65 kDa, while antibody B5-2 F7 bound a 97 kDa protein. Complement activation and complement-mediated oocyte lysis: Systemically injected antibodies, C3 and C4 were detected on zona-free oocytes recovered from the mouse oviduct indicating the activation of C3 and C4 by antigen-antibody complexes. The recovered oocytes were not damaged, suggesting a presence of complement-regulating factors. In vitro, however, a large number of zona-free oocytes preincubated with antibodies were lysed or protruded ooplasma vesicles in complement-active serum. Stage, tissue, and species specificity: None of the antibodies, except A2-2 A7, showed a positive immunolabelling to the pronuclear stage. Antibodies 4-G1 and A2-2 F2 cross-reacted with the ovarian oocytes. No antibodies bound to any of the tissues tested, indicating that the corresponding antigen epitopes are not commonly expressed. A2-2 A7, A2-2 F2, and B5-2 F7 cross-reacted with hamster and human unfertilized oocytes, suggesting the presence of developmentally conserved molecules and the possibility to apply these antibodies in hamster and human in vitro fertilization. It is concluded that the approach used could be a useful strategy in searching for anti-fertilization antibodies for human contraception. © 1996 Wiley-Liss, Inc.     

Antibodies Directed against Functional Sites on the Acetylcholine Receptor from Torpedo Marmorata

Abstract

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata have been obtained by the following two procedures: (i) Our library of monoclonal antibodies raised against the whole receptor protein was screened for antibodies competing with cholinergic agonists, antagonists and local anesthetics for receptor binding, (ii) antibodies were raised against short peptides matching the sequence of predetermined sites on the receptor protein. In this way, a topographic map of the functional sites on the receptor surface can be constructed.     

Vitellogenin uptake and vitellin localization in insect follicles examined using monoclonal antibodies and confocal scanning microscopy

Summary

Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.     

Rapid Purification of Annexin V from Human Placenta by Affinity Chromatography

Abstract

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

Hélène Charniaux-Cotton (1918–1986)

Summary

1-Methyladenine (1-MA) secreted from the follicle cells is the biological signal for meiosis reinitiation of starfish oocytes. The signal of-1-MA is transduced into cytoplasmic formation of maturation-promoting factor (MPF) that eventually induces a germinal vesicle breakdown (GVBD). Microinjection of pertussis toxin (PTX) inhibited 1-MA-induced GVBD in Asterina pectinifera and Asterina (Patina) miniata. PTX-inhibition of GVBD was rescued by the injection of MPF into PTX-preinjected oocytes. Most of the PTX- and MPF-double injected eggs were fertilized and underwent cleavage, suggesting the presence of a GTP-binding protein (G protein) specific for 1-MA signal transduction. Indeed, plasma membrane preparations of A. pectinifera oocytes contained a G protein consisting of 39-kDa α, 37-kDa β, and 8-kDa γ subunits. The α subunit contained a site for ADP-ribosylation catalyzed by PTX. It was also recognized by antibodies specific for a common GTP-binding site of mammalian α subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G protein (G_i) α subunits. Its gene was 74% and 83.7% identical to the rat G_i-2α gene in nucleotide and deduced amino acid sequences, respectively. The 39-kDa α subunit shared the common GTP-binding site of mammalian G protein α subunits and the PTX-catalyzed ADP-ribosylation site of mammalian G_i α subunits as expected from the immunoreactivity. The oocyte membranes had apparently two forms of 1-MA receptors with high and low affinities. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. The 39-kDa α subunit of starfish G protein was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. These results indicate that in starfish oocyte membranes, 1-MA receptors are functionally coupled with the 39-kDa PTX-substrate G protein that transduces the signal into the formation of a cytoplasmic factor (MPF) and eventually into the reinitiation of meiosis.     

Design and characterization of a germ-line targeting soluble,native-like,trimeric HIV-1 Env lacking key glycans from the V1V2-loop

BackgroundThe HIV-1 envelope glycoprotein (Env) is the primary target for broadly neutralizing antibodies (bNAbs) which can block infection. The current design strategy of soluble forms of Env in native-like trimeric conformation induces neutralizing antibodies with minimal breadth and potency. Extensive shielding by N-glycans on the surface of the HIV-1 Env acts as an immune evasion mechanism by restricting B cell recognition of conserved neutralizing determinants. An alternate approach is to design Env protein with glycan deletion to expose the protein surface.MethodsA stable native-like trimeric Env with glycan holes at potentially immunogenic locations is expected to elicit better induction of germ-line B-cells due to exposure of the immunogenic regions. However, the extent and consequences of glycan removal from the trimer apex that form an important epitope is not explored. In this work, we have designed a construct with glycans deleted from the trimer apex of an Indian clade C origin Env that has previously been characterized for immunogenicity, to understand the impact of deglycosylation on the structural and functional integrity as well as on the antibody binding properties.ResultsThe V1V2 glycan-deleted protein maintains native-like trimeric conformation with improved accessibility of the V1V2-directed germ-line antibodies. Furthermore, we showed that the protein binds specifically to quaternary conformation-dependent bnAbs but minimally to non-neutralizing antibodies.ConclusionsThis study provide an important design aspect of HIV-1 Env-based immunogens with glycan holes in the apex region that could be useful in eliciting apex directed antibodies in immunization studies.     

Actin in oogenesis

Summary

The laser-scanning confocal microscope employed in conjunction with various specific agents and antibodies conjugated to fluorescent dyes reveals details of the actin scaffolding of developing oocytes and the nuclei of attendant cells. The employment of DNase I followed by anti-DNase I antibody has been particularly useful in revealing otherwise cryptic actin-containing structures. The cortical cytoskeleton of developing moth eggs was found to bind both poly (A)+RNA and RNA Pol II. Exposure to cytochalasin D disrupted the actin of the cortex, and at the same time caused redistribution of the proteins and RNA associated with the cytoskeleton. Cytochalasin also had dramatic effects on the structure of nuclei of nurse and follicle cells. Taken in context of the actin network in nuclei uncovered by DNase-anti-DNase treatment, these results suggest that actin plays a major structural and perhaps functional role in insect nuclei.     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

A Case Of Severe Neuropat Hy Associated With Hypertriglyceridemia

ObjectiveTo describe a case of severe neuropathy associated with hypertriglyceridemia.MethodsWe describe the clinical and laboratory findings of the study patient and review the relevant literature.ResultsA 45-year-old woman presented to the emergency department with recurrent abdominal pain and severe peripheral neuropathy. Her laboratory data revealed elevated lipase and a very high triglyceride concentration (> 10 000 mg/dL), consistent with a diagnosis of recurrent hypertriglyceridemia-induced pancreatitis. Workup for peripheral neuropathy showed normal concentrations of thyrotropin, fasting blood glucose, vitamin B₁₂, and creatinine, as well as a normal hemoglobin A_1c level, serum protein electrophoresis, and urine protein electrophoresis. Rapid plasma reagin antibodies, antinuclear antibodies, and lyme antibodies were not detected. In the absence of other identifiable causes, hypertriglyceridemia was deemed the likely etiology of severe neuropathy in this patient.ConclusionsPeripheral nerve conduction abnormalities can be identified in patients with mild hypertriglyceridemia in the absence of symptoms. Early recognition and aggressive management of hypertriglyceridemia may prevent the complications of severe peripheral neuropathy.(Endocr Pract. 2008;14:1020-1022)     

研究报告：金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果。ELISA的本质是抗原与相应抗体之间的特异性相互作用。然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果。本课题组先前应用构象工程方法开发了一种基于金纳米粒子的人工抗体(简称金抗体)。金抗体可以像天然抗体一样特异性地与抗原相互作用,并且具备远优于天然抗体的稳定性。出色的稳定性使金抗体可能成为天然抗体更好的替代物,用于ELISA中。方法经过必要的优化并与辣根过氧化物酶(HRP)耦联后,制得酶标金抗体10HRP-(Au-400P1),然后用酶标金抗体代替天然酶标抗体用于ELISA检测中。结果通过一系列的实验证明,抗溶菌酶金抗体可用于ELISA特异性检测1～16 mg/L范围内的鸡蛋清溶菌酶(HEWL)样品。结论金抗体可以替代天然抗体用于ELISA检测,并具有优于传统ELISA法的检测准确性和一致性。     

金黄色葡萄球菌黏附素ClfA等抗体抑制该菌黏附牛乳腺上皮细胞效果的比较

【目的】为从免疫的角度比较和分析黏附素分子细胞外纤维蛋白原结合蛋白(Extracellular fibrinogen-binding protein,EfB)和纤维连接蛋白结合蛋A(Fibronection binding protein,FnBP)对聚集因子A(Clumping factor A,ClfA)抑制牛源金黄色葡萄球菌(Staphylococcus aureus,SA)黏附牛乳腺原代上皮细胞作用的增强效果。【方法】本试验分离培养牛乳腺上皮细胞并鉴定;将真核重组质粒EfB和FnBPA分别与ClfA组合免疫新西兰大白兔,并利用免疫后抗体体外抑制2株SA分离株侵染牛乳腺上皮细胞,采用平板计数法定量检测与比较不同免疫组合诱导抗体对黏附的抑制作用;对SA和乳腺上皮细胞分别进行荧光染色,观察不同免疫组合诱导的抗体对黏附抑制效果的差异。【结果】成功分离培养了牛乳腺原代上皮细胞;证明了构建的ClfA、FnBPA和EfB真核重组表达质粒均可在细胞中表达,且在免疫实验兔后可诱导特异性抗体的产生;细菌平板计数和荧光染色观察的结果表明,黏附素分子单独和组合免疫的抗体对该菌不同菌株(GY278和GY309)的黏附抑制能力不同,ClfA抗体的黏附抑制能力最强,FnBPA分子A区的黏附抑制能力优于D区。FnBPA、EfB分别与ClfA联合免疫抗体对黏附的抑制程度高于黏附素分子单独免疫组,FnBPA分子A区对ClfA黏附抑制的增强作用优于D区,FnBPA-A区与Efb相比对ClfA的黏附抑制增强差异不显著。【结论】FnBPA-A、FnBPA-D和EfB分别与ClfA联合免疫可不同程度地影响ClfA的黏附抑制效果,该结果为以黏附素分子为靶点的牛乳腺炎疫苗的研究提供了实验数据。