Inhibition of in vitro fertilization by mouse anti-mouse sperm sera and preliminary antigen identification

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

结核分枝杆菌抗体检测蛋白芯片的制备及应用

目的:用重组结核分枝杆菌ESAT6、CFP10、M16和M38抗原制备相应的抗体检测蛋白芯片。方法:将制备的结核分枝杆菌抗原ESAT6、CFP10、M16和M38及购买的LAM抗原点于醛基化修饰的玻片上,制备成结核抗体检测蛋白芯片;使用该芯片对130例临床结核病患者和50例健康体检者血液样品进行检测,分析其敏感性和特异性,以及5项结核抗体的构成比。结果:结核杆菌抗体检测蛋白芯片的敏感性为90.8%(118/130),特异性为90%(45/50),LAM的检出率最高为91.5%。结论:用ESAT6、CFP10、M16和M38及LAM抗原制备的结核杆菌抗体检测蛋白芯片用于结核病辅助诊断的敏感性和特异性较高,可用于结核分枝杆菌抗体检测蛋白芯片试剂盒的开发。     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5 raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶ - Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel -strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5.     

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall ofHistoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.Abbreviations CHHA Cystine-heart-hemoglobin agar - PBS phosphate buffered saline - Ig immunoglobulin - TBS Tris buffered saline - DAB 3,3-diaminobenzidine tetrachloride - FITC fluorescein isothiocyanate - M199 tissue culture medium 199, according to Morgan et al. (1950)     

Connective tissue influences on the expression of epithelial cell-surface antigens

Summary Adult mice were found to show regional variation in the epithelial expression of some molecules of the blood-group antigen series. To investigate connective tissue influences on such differences, heterotypic recombinants of epithelia and connective tissues from various regions were prepared and examined using monoclonal antibodies directed against bloodgroup antigens H and Le^y. The results indicate that epithelia may maintain a preexisting regionally specific pattern following recombination but that, in some recombinant matches, the connective tissue is capable of signalling redirection of the pattern of expression towards that typical of the epithelium with which it is normally associated.This work was supported by NIH-NIDR RO1-DEO-5190     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.