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1.
A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc. 相似文献
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研究了在大肠杆菌体内表达了以组蛋白C 末端为骨架的融合蛋白HNHG[H(HA2 0 )N(NLS)H(组蛋白H10 的C末端 97个氨基酸 )G(GE7) ],并发现质粒DNA与其形成复合体后 ,在电镜下出现与体内染色体相同的凝聚、螺旋现象。由于HNHG的这种有效地结合质粒、并使之凝聚的特性 ,有望使之成为新兴的能将外源基因导入体内的载体系统。 相似文献
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Jihye Kim Jun Tae Kwon Juri Jeong Jaehwan Kim Seong Hyeon Hong Jinyoung Kim Zee Yong Park Kyung Hwun Chung Edward M Eddy Chunghee Cho 《EMBO reports》2018,19(9)
Spermatogenesis is a tightly regulated process involving germ cell‐specific and germ cell‐predominant genes. Here we investigate a novel germ cell‐specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9‐mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z‐line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L‐PKA complex in maintaining the stability of the sperm head‐tail junction, thereby revealing a new molecular basis for sperm head‐tail integrity. 相似文献
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For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody. 相似文献
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Marianne Klint Karin Sege Bengt Curman Leif Plen Per A. Peterson 《Molecular reproduction and development》1985,11(4):335-348
Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins. 相似文献
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We use a coarse-grained protein model to characterize the critical nucleus, structural stability, and fibril elongation propensity of Aβ1-40 oligomers for the C2x and C2z quaternary forms proposed by solid-state NMR. By estimating equilibrium populations of structurally stable and unstable protofibrils, we determine the shift in the dominant population from free monomer to ordered fibril at a critical nucleus of ten chains for the C2x and C2z forms. We find that a minimum assembly of 16 monomer chains is necessary to mimic a mature fibril, and show that its structural stability correlates with a plateau in the hydrophobic residue density and a decrease in the likelihood of losing hydrophobic interactions by rotating the fibril subunits. While Aβ1-40 protofibrils show similar structural stability for both C2x and C2z quaternary structures, we find that the fibril elongation propensity is greater for the C2z form relative to the C2x form. We attribute the increased propensity for elongation of the C2z form as being due to a stagger in the interdigitation of the N-terminal and C-terminal β-strands, resulting in structural asymmetry in the presented fibril ends that decreases the amount of incorrect addition to the N terminus on one end. We show that because different combinations of stagger and quaternary structure affect the structural symmetry of the fibril end, we propose that differences in quaternary structures will affect directional growth patterns and possibly different morphologies in the mature fiber. 相似文献
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Abstract: A novel splicing form of βA4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP695 , APP751 , or APP770 ) either with (N-APP) or without (L-APP; leukocytederived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP695 without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP695 of the brain and influence the proteolytic processing of APP. 相似文献
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cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-λgt11 expression library using antibodies to human sperm surface antigens belonging to 14–18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46–54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH2-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1λT vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14–18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism. Mol. Reprod. Dev. 48:449–457, 1997. © 1997 Wiley-Liss, Inc. 相似文献
11.
Marisabel Mourelle Irma Vargas Alberto Darszon 《Molecular reproduction and development》1984,9(1):87-97
The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I125-labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15-fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg++ and Na+ stimulated their cyclase activity about sevenfold at 2.5 mM Mn++ and 3.2 mM ATP. At this ATP to Mn++ ratio, high concentrations of Ca++ have a small stimulatory effect. 相似文献
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Chang Shin Yoon Eun Gyu Lee Young Seek Lee Il Yup Chung 《Biotechnology and Bioprocess Engineering》1997,2(2):86-89
Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical
usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector
in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature
EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate
proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology
and information for the production of recombinant EGF. 相似文献
13.
Mona Harnasch Sandra Grau Christian Behrends Simon L. Dove Ann Hochschild Maria-karnina Iskandar 《Molecular membrane biology》2013,30(6):373-383
An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of γ-secretase are discussed. 相似文献
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Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids--(1)A D T I V A V E L D T Y P N(14)--correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A. 相似文献
15.
Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization
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Simona Pipolo Rossella Puglisi Valentina Mularoni Valentina Esposito Andrea Fuso Marco Lucarelli Maria T. Fiorenza Franco Mangia Carla Boitani 《Journal of cellular physiology》2018,233(4):3093-3104
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The sperm tail of bug insects has 9 + 9 + 2 flagellar axonemes and two mitochondrial derivatives showing two to three crystalline inclusions in their matrix. During spermiogenesis, the axoneme is surrounded by a membrane cistern which, at sperm maturity, reduces to two short cisterns on the opposite sides of the axoneme adhering to the mitochondrial derivatives. Filamentous bridges connect the intertubular material of the axoneme to these cisterns. Such bridges, which represent a peculiar feature of bug insects, are resistant to detergent treatment, whereas part of the intertubular material and the inner content of microtubular doublets are affected by the treatment. After freeze‐fracture replicas, at the insertion of the bridges to the cisternal membrane, the P‐face of this membrane shows a characteristic ribbon consisting of four rows of 11 ± 1 nm staggered intramembrane particles, 13 ± 2 nm apart along each row. The bridges could be able to maintain the axoneme in the proper position during flagellar beating avoiding distortion affecting sperm motility. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc. 相似文献
19.
Hans-Jürg Monstein 《Bioscience reports》1990,10(5):461-467
A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/ME. coli vector. The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg6-Phe2 antibody, directed against the C-terminal part of the enkephalin A prohormone. The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrat to identify endoproteolytic processing activities. 相似文献
20.
To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures. 相似文献