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S Y Miao

Publications and source records attributed to S Y Miao.

At least 19 recordsLinked to original sources
PubMed2001

Extracellular domain of YWK-II, a human sperm transmembrane protein, interacts with rat Müllerian-inhibiting substance.

The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues. Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility. In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component. Müllerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein. The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays. The recombinant Müllerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane. The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Müllerian-inhibiting substance.

Amino Acid Sequence↗
PubMed2001

[Determination of the binding site of testis-specific nucleoporin BS-63 to transportin (karopherin beta 2) and the proof of their combination in vitro].

OBJECTIVE: To locate the binding site of testis-specific nucleoporin BS-63 to transportin (karopherin beta 2) and confirm their combination in vitro. METHODS: Constructed different fragments of C terminal BS-63 was employed to localize the binding site of the testis-specific nucleoporin BS-63 to transportin by yeast two-hybrid system technique pull-down test was used to identify the interaction between the purified expressed fragments of BS-63 0.6 K and transportin in vitro. RESULTS: BS-63 binding site to transportin was shortened from 1.6 kb to 0.6 kb which included a Ran binding domain (RanBD). SDS-PAGE and Western blot tests confirmed the recombinant purified protein coded by 0.6 kb fragment of BS-63 cDNA could interact with transportin in vitro. CONCLUSIONS: In germ cells, the function of the testis-specific nucleoporin BS-63 localized at cytoplasmic side of NPC importing cargoes into nuclear may be accomplished by transportin cooperation.

Binding Sites↗
PubMed1998

Gene encoding a human testis Sertoli cell component related to LIM domain protein.

The cDNA (HED-2) encoding a 20 kDa protein found in mammalian epididymal fluid was isolated from a human testis expression library. It is composed of 1908 bp, containing a reading frame of 1479 bp, coding a polypeptide consisting of 493 amino acids, and assigned the accession number: U15158 by GenBank (Biochem. Mol. Biol. Int. 34, 1131-1136, 1994). HED-2 has 99% identity with the zyxin gene in amino acid sequence, a component of cell junction matrix and a member of the LIM domain protein family. Northern blot analysis of RNAs prepared from various human tissues showed that the HED-2 gene was expressed in all tissues analyzed. Sertoli cells of human testis expressed the gene as determined by an in situ hybridization method. The present study shows that the HED-2 gene is a member of the LIM domain protein family.

Amino Acid Sequence↗
PubMed1996

Expression and characterization of a novel human sperm membrane protein.

A cDNA fragment (HSD-1) coding for part of a human sperm membrane protein (hSMP-1) was previously isolated from a human testis cDNA expression library, with the serum from an infertile patient used as a probe. By rescreening human testis cDNA libraries with the HSD-1 insert and using rapid amplification of cDNA ends, the complete cDNA of 2482 bp was identified and sequenced. An open reading frame of 1572 bp encodes 523 amino acid residues with a computed molecular mass of 55.08 kDa. This protein sequence does not match any other sequence in the databases, indicating that it represents a novel sperm antigen. Northern blot analysis of human and rat testis poly(A) mRNA detected a band of approximately 2.5 kb in both species. Reverse transcriptase polymerase chain reaction analysis showed that hSMP-1 mRNA was present in human testis but was not in either kidney or liver. When the cDNA was expressed in Escherichia coli under the control of the T7 promoter, the expressed protein accumulated to a level of about 50% of the total cellular protein. The expressed protein, which contained an N-terminal poly(his) sequence tag, was purified by chromatography on an nitrilo-tri-acetic acid affinity resin. Approximately 10 mg of pure protein was obtained from a 500-ml culture, purified, and used as antigen to generate a polyclonal antiserum in rabbits. Western blot analysis of human sperm extracts showed a single specific band at 55.5 kDa. Immunofluorescence data showed that hSMP-1 was localized to the head of human sperm. The fluorescent staining formed a cap-shaped pattern that was similar in morphology to the human sperm acrosome. The availability of large amounts of recombinant hSMP-1 and its antiserum will facilitate studies on the function and expression of the protein during spermatogenesis and the assessment of its potential value as a contraceptive immunogen.

Amino Acid Sequence↗
PubMed1994

Taxol inhibits progression of congenital polycystic kidney disease.

Polycystic kidney diseases (PKD) are the most common hereditary diseases of the human kidney and account for ten per cent of patients requiring renal transplantation or dialysis. Renal cyst formation has been attributed to enhanced cell proliferation, unbalanced cell death, abnormal targeting of membrane proteins, aberrant kidney development and tubular obstruction, but there is no treatment that blocks the formation and enlargement of renal cysts. We have now developed an in vitro model of spontaneous cyst formation that distinguishes polycystic kidney epithelium from its normal counterpart. Inhibitors of DNA, RNA and protein synthesis did not prevent in vitro cyst formation, but this was reversibly inhibited by ouabain, amiloride and the microtubule-specific agents colchicine, vinblastine and taxol. The cpk mouse is a well-characterized recessive PKD model and we find that cpk/cpk mice develop PKD and die from uraemia by 4-5 weeks of age, but when treated weekly with taxol they survive for more than 200 days with minimal loss of renal function, show limited collecting-dust cyst enlargement, and attain adult size. Our results indicate that the microtubule cytoskeleton has a central role in the pathogenesis of PKD in cpk mice and that taxol may also be useful in treating human PKD.

Animals↗
PubMed1994

Gene encoding a mammalian epididymal protein.

Polyclonal antibodies raised against a 20 kD epididymal protein (EP20) were used to isolate the cDNA from a human testis lambda gt11 expression library. The nucleotide sequence of the cDNA consisted of 1908 base pairs (bp) containing an open reading frame composed of 1479 bp encoding a polypeptide of 493 amino acid residues. The nucleotide sequence of EP-20 cDNA had 97% identities (282/288) with ESTO 0991 Homo Sapiens cDNA clone HHC M14 in the reverse orientation. The HHC M14 sequence corresponded to a segment in the non-translatable 3'end of EP-20 cDNA. The amino acid sequence of the deduced polypeptide showed no homology with reported polypeptides. The epididymal protein may be involved in sperm maturation and/or capacitation.

Amino Acid Sequence↗
PubMed1994

Calpastatin gene in human testis.

Antibodies present in a serum obtained from an infertile woman interacted with a 17.5 kD glycoprotein (BS-17 component) extracted from human sperm by Western blot. Polyclonal antibodies were raised against the BS-17 component and used to identify positive staining clones from a human testis lambda gt11 expression library. The cDNA encoding the BS-17 component was isolated and its nucleotide sequence determined. The BS-17 cDNA contained 758 nucleotides with an open reading frame of 558 nucleotides encoding a polypeptide consisting of 186 amino acids. The BS-17 cDNA showed 99.7% homology in 758 nucleotides overlap with the 3' terminus of the gene coding calpastatin and 99.5% identity in 186 amino acid overlap with the carboxyl terminus of calpastatin. The BS-17 component of human sperm corresponds to the carboxyl terminus of calpastatin. This conclusion is supported by the finding that the polyclonal antibodies also interacted with a 84 kD protein corresponding to the M(r) of calpastatin.

Amino Acid Sequence↗
PubMed1992

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.

Animals↗
PubMed1992

Isolation and sequencing of the cDNA encoding the 75-kD human sperm protein related to infertility.

Serum was obtained from an infertile woman (IS) inducing head-to-head agglutination of human sperm and was used to screen a human testis lambda gt11 cDNA library. A plaque producing the interacting antigen was located. The recombinant lambda gt11 was isolated and cut with EcoRI releasing a 0.7-kb cDNA. Using the 0.7-kb cDNA as a probe, a larger cDNA of 2.4 kb was isolated and its nucleotide sequence determined. It was composed of 2 427 nucleotides with an open reading frame of 1584 nucleotides encoding 528 amino acid residues. The specific antisperm antibody was isolated from IS by epitope selection, using positive plaques of E. coli Y1090. The epitope-selected antibodies interacted with a 75-kD human sperm protein and with a polypeptide in the form of a beta-galactosidase fusion protein in the recombinant lysate of E. coli Y1089, determined by immunoblot. The fusion protein was purified by affinity chromatography on an anti-beta-galactosidase-Sepharose column. It is proposed that production of anti-75-kD antibodies may be the underlying cause of the infertility.

Amino Acid Sequence↗
PubMed1990

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.

Animals↗
PubMed1990

Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid residues, Arg-Lys-Arg, was found instead of Lys-Lys-Lys at the probable membrane-cytoplasmic junction that may be a unique property of sperm membrane proteins.

Amino Acid Sequence↗
PubMed1986

Isolation and characterization of a rabbit sperm tail protein.

Rabbit sperm tails were obtained by a nitrogen cavitation procedure and separated by discontinuous sucrose gradient centrifugation. Proteins were extracted from sperm tails with 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and separative disk electrophoresis and DEAE-trisacryl column chromatography. A protein with an estimated mol wt of 20.1 +/- 1.1 kD was isolated and found to be homogeneous by SDS-PAGE and designated as rSMP-B. The isoelectric point of rSMP-B was in the range of pH 4.4-4.7. The amino acid composition was determined, and glycine was identified as the N-terminal residue. Antisera were raised against purified rSMP-B. Using a peroxidase-antiperoxidase method, the rSMP-B antigen was located on the surface of the midpiece and tail of the sperm. Testis sections showed intense staining of late spermatids located within the seminiferous tubules. Adult male rabbits were inoculated with rSMP-B protein and Freund's adjuvant. The testis and epididymis of the immunized animals showed absence of sperm. The immunolocalization findings and the immunization data suggest that rSMP-B is formed in late spermatid and that it is an essential structural component of sperm.

Amino Acids↗
PubMedDate not supplied

Immune responses in rats following oral immunization with attenuated Salmonella typhimurium expressing human sperm antigen.

The HSD-I gene codes a human sperm membrane protein (hSMP-1) and has been assigned the accession number U12978. The gene is located on human chromosome 9, region p12-p13. When the 1.7-kb cDNA of HSD-I was digested sequentially with EcoRI, BamHI, and HindIII, a 550-bp cDNA fragment was formed, which codes for the extracellular domain. This fragment was cloned into the asd+ vector pYA3149 to construct pYA3149R. The recombinant plasmid was used to transform an avirulent deltacva, deltacrp, deltaasd vaccine strain of Salmonella typhimurium chi4550. The hSMP-1 component was localized on the surface of the head of mature rat spermatozoa by an immunofluorescence technique using polyclonal anti-hSMP-1 antibodies. Since rat sperm contain hSMP-1, this rodent can be used to assay the immunogenicity of pYA3149R. Female Wistar rats were immunized by oral administration of the recombinant Salmonella. Anti-hSMP-1 antibodies in blood and vaginal washes of immunized animals were determined. Both body fluids contained significant amounts of the antibodies, showing that the recombinant Salmonella is an effective oral immunogen in rats.

Administration, Oral↗
PubMedDate not supplied

Human sperm membrane protein (hSMP-1): a developmental testis-specific component during germ cell differentiation.

Serum was obtained from an infertile woman having antibodies with sperm agglutinating activity. The antibodies interacted with a human sperm membrane protein (hSMP-1) with an estimated Mr of 55 kD. The gene (HSD-1) coding hSMP-1 was isolated from a human testis cDNA expression library and assigned the accession number U12978. The cDNA was conjugated to a prokaryotic expression vector to construct the recombinant vector, pRSET-HSD-I, which was expressed in Escherichia coli. The recombinant hSMP-1 was isolated and used to immunize rabbits to raise polyclonal antibodies. Usingan immunocytochemical technique, hSMP-1 protein was immunolocalized in germ cells of human testis at all stages of spermatogenesis. mRNAs were prepared from 16 different human tissues and analyzed by Northern blot using HSD-1 as probe. A positive reaction was elicited only with testis mRNA. The present findings suggest that the expression of hSMP-1 gene is testis-specific and occurs during the early stages of germ cell differentiation. In a comparative study, the location of the hSMP-I protein in sperm and in germ cells of the seminiferous tubules of rats was determined. The target antigen was immunolocated on the head and tail of rat sperm and in late spermatids and spermatozoa of rat testis. These results suggest that, in the rat, the HSD-1 gene is expressed during spermiogenesis.

Animals↗