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Biomedical subjects

Wei Yan

Publications and source records attributed to Wei Yan.

At least 55 records · Page 3Linked to original sources
PubMed2004

Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice.

We identified a testis-specific gene encoding a protein containing a BTB/POZ domain and six kelch repeats, which we named kelch homolog 10 (KLHL10). KLHL10 displays high evolutionary conservation in mammals, as evidenced by 98.7% amino acid identity between mouse and human KLHL10. KLHL10 is exclusively expressed in the cytoplasm of elongating and elongated spermatids (steps 9-16). We generated a Klhl10 null allele in 129S6/SvEv mouse embryonic stem cells, and obtained 47 chimeras from six independent embryonic stem cell lines. Whereas low-percentage male chimeras only produce C57BL/6J offspring, high-percentage chimeric and heterozygous males were completely infertile because of disrupted spermiogenesis characterized by asynchronous spermatid maturation, degeneration of late spermatids, sloughing of postmeiotic germ cells from the seminiferous epithelium, and marked reduction in the numbers of late spermatids. Our data demonstrate that, like protamine-1 and -2, both alleles of Klhl10 are required for male fertility and that haploinsufficiency caused by a mutation in one allele of Klhl10 prevents genetic transmission of both mutant and WT alleles.

Amino Acid Sequence↗
PubMed2004

Identification and characterization of evolutionarily conserved pufferfish, zebrafish, and frog orthologs of GASZ.

We previously identified Gasz (a germ cell-specific gene encoding a protein containing four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper) in six mammalian species. Here, we report GASZ orthologs in pufferfish (Fugu rubripes), zebrafish (Danio verio), and frog (Xenopus laevis). Sequences of the three Gasz cDNAs were determined by database mining and 5'- and 3'-rapid amplification of cDNA ends (RACE) followed by sequencing. The three orthologous vertebrate genes encode proteins structurally similar to mammalian GASZ and contain the characteristic four ankyrin repeats (ANKs) and sterile-alpha motif (SAM). Their ANK and SAM domains share 55- 74% and 38-55% amino acid identity with those in human GASZ, respectively. Similar to human and mouse Gasz genes, pufferfish Gasz is composed of 13 exons, spanning approximately 12 kilobases, and flanked by Cftr at its 5'-end and Wnt2 at its 3'-end. Northern and Western blot analyses detect frog Gasz expression only in testis and ovary. In situ hybridization and immunohistochemical analyses show that frog Gasz mRNA and protein expression is confined to pachytene spermatocytes in the testis and to oocytes in the ovary. In frog oocytes, GASZ protein appears to localize to a cytoplasmic structure resembling the Balbiani body, a postulated mRNA transport organizer in the cytoplasm. The high evolutionary conservation and germ cell specificity suggest that GASZ plays an essential role in gametogenesis. The data presented here are important for future studies of the physiological roles of GASZ using fish and amphibians as animal models.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Tektin3 encodes an evolutionarily conserved putative testicular microtubules-related protein expressed preferentially in male germ cells.

Tektins are microtubule-associated cytoskeletal proteins that are expressed primarily in the male germ cell-lineage in centrioles and basal bodies and within ciliary and flagellar doublet microtubules. They are proposed to be important for axonemal architecture and microtubule stability in the sperm tail and in other ciliated and flagellar structures. Using an in silico (electronic database) subtractive approach to identify germ cell-specific genes in vertebrates, we isolated a new member of the Tektin gene family from mice, Tektin3. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses confirm that Tektin3 encodes a 1.7 kb transcript detectable preferentially in the testes of adult mice. In situ hybridization analysis in the testes revealed Tektin3 mRNA expression exclusively in late pachytene spermatocytes and early round spermatids. The putative TEKTIN3 protein shares 83.5% overall sequence identity with the human ortholog and includes a fully conserved carboxy terminal nonapeptide signature sequence present in all TEKTIN family members. In addition, using database mining, highly conserved TEKTIN3 orthologs were identified in puffer fish (Fugu rubripes) and rats (Rattus norvegicus) which shared 60.9 and 91.4% identity, respectively, with mouse TEKTIN3. The Tekt3 gene maps to murine chromosome 11 in a region that is syntenic to the human 17p12 chromosomal region containing the human TEKTIN3 gene. Our studies demonstrate that TEKTIN3 is a novel evolutionarily conserved male germ cell-enriched protein and suggest that it might perform important roles in male reproductive development and physiology.

Amino Acid Sequence↗
PubMed2004

Chemical probes and tandem mass spectrometry: a strategy for the quantitative analysis of proteomes and subproteomes.

Quantitative proteome profiling using mass spectrometry and stable isotope dilution is being widely applied for the functional analysis of biological systems and for the detection of clinical, diagnostic or prognostic marker proteins. Because of the enormous complexity of proteomes, their comprehensive analysis is unlikely to be routinely achieved in the near future. However, in recent years, significant progress has been achieved focusing quantitative proteomic analyses on specific protein classes or subproteomes that are rich in biologically or clinically important information. Such projects typically combine the use of chemical probes that are specific for a targeted group of proteins and may contain stable isotope signatures for accurate quantification with automated tandem mass spectrometry and bioinformatics tools for data analysis. In this review, we summarize technical and conceptual advances in quantitative subproteome profiling based on tandem mass spectrometry and chemical probes.

Amino Acids↗
PubMed2004

Static compression induces zonal-specific changes in gene expression for extracellular matrix and cytoskeletal proteins in intervertebral disc cells in vitro.

Compressive stimuli associated with weight-bearing and loading of the intervertebral disc are believed to be important regulators of disc cell metabolism. In this study, changes in gene expression levels for extracellular matrix and cytoskeletal proteins were quantified in disc cells in an alginate culture system subjected to static unconfined compression (25% compressive strain) after different time periods (2, 18 and 30 h). Differences in gene expression were observed between anulus fibrosus and nucleus pulposus cells following static compression for the matrix proteins studied here. Anulus fibrosus cells responded to mechanical deformation at the 30-h time point, with increasing gene expression for types I and II collagen, aggrecan, biglycan, decorin and lumican. In contrast, nucleus pulposus cells were not responsive to mechanical loading with changes in gene expression for these matrix proteins at any time. Our results also show that anulus fibrosus cells, but not nucleus pulposus cells, responded to static compression with increased expression of vimentin mRNA as well as increased polymerization of vimentin subunits. The results of the current study illustrate that fibrochondrocytes of the anulus fibrosus may regulate biosynthesis at the transcriptional level following mechanical deformation in an alginate construct. In contrast, the biological response of nucleus pulposus cells to these same stimuli is not detectable. These differences may be attributed to the presence of a notochordal cell population in the immature nucleus pulposus studied here, with a more diffuse and stiff cytoskeleton that may restrict deformations or shape changes upon compressive loading.

Animals↗
PubMed2004

Restricted germ cell expression of a gene encoding a novel mammalian HORMA domain-containing protein.

HORMA-domain containing proteins are involved in cell cycle regulation by interactions with chromatin. Through an in silico subtractive screen for mouse genes preferentially expressed in newborn ovaries, we identified a gene that would encode a novel mammalian HORMA domain-containing protein termed Newborn Ovary HORMA protein (Nohma). Reverse transcription polymerase chain reaction and Northern blot analysis have demonstrated a 1.7 kb transcript that is expressed exclusively in germ cells in both male and female mouse gonads. Nohma expression can be detected postnatally in male, and prenatally in female gonads, but shows a sexually dimorphic expression in adult gonads. The Nohma transcript is abundant in the adult testis but limited in the adult ovary. The mouse Nohma gene is comprised of 15 exons and maps to chromosome 3. A human ortholog was also identified. The expression pattern of Nohma suggests that it may be a critical regulatory protein in germ cell meiosis.

Amino Acid Sequence↗
PubMed2004

Time-dependent response of laminated isotropic strips with viscoelastic interfaces.

The two dimensional problem of simply supported laminated isotropic strips with viscoelastic interfaces and under static loading was studied. Exact solution was derived based on the exact elasticity equation and the Kevin-Voigt viscoelastic interfacial model. Numerical computations were performed for a strip consisting of three layers of equal thickness. Results indicated that the response of the laminate was very sensitive to the presence of viscoelastic interfaces.

Anisotropy↗
PubMed2004

[Factors related to false positive results of treadmill electrocardiogram test for the detection of coronary heart disease].

OBJECTIVE: To investigate the clinical factors related to false positive results of electrocardiogram treadmill exercise test (TET) for the detection of coronary heart disease (CHD). METHODS: 258 patients with chest pain undergoing TET and coronary angiography (CAG) were on rolled. The case history and various clinical parameters were collected. (1) The results of TET and CAG were compared to evaluate the sensitivity, specificity and accuracy of TET; (2) Clinical informations of the true positive group and the false positive group were compared and a statistic analysis was carried out. RESULTS: The sensitivity, specificity and accuracy of TET for diagnosing CHD was 77.3%, 65.9%, and 69.8% respectively. The number of female in the false positive group was larger than that in the true positive group; The number of patients with typical chest pain, hyperlipidemic history, smoking history and family history of CHD was smaller than that in the true positive group. In the false positive group, the change of ST segment was mainly in II, III, aVF. The number of patients with change of more than 0.2 mV in ST and with angina induced during exertion was lower in the false positive group than in the true positive group. The difference was significant. CONCLUSION: Factors including sex, typical chest pain, risk factors of CHD, the location and extent of ECG changes and angina induced by exertion are closely related to false positive results of TET.

Adult↗
PubMed2003

Gene expression at early stages of Brassica napus seed development as revealed by transcript profiling of seed-abundant cDNAs.

Approximately 5000 plaques derived from a Brassica napus L. (canola) seed-cDNA library representing 15 days after pollination (DAP) were differentially screened for highly expressed genes at the early stages of seed development. Analysis of 104 differentially expressed sequence tags revealed 54 unique genes, of which 33 had putative homologues described in Arabidopsis thaliana (L.) Heynh. or B. napus. These encoded diverse proteins, ranging from proteins of unknown function to metabolic enzymes and proteins associated with cell structure and development. Twenty-five genes were only expressed in seeds, and 11 of these started to express as early as 5 or 10 DAP. The majority of the seed-specific genes that are expressed at early stages of seed development encoded proteins with high similarity to hypothetical Arabidopsis proteins. Tissue-specificity determined by Northern analysis revealed that four seed-specific genes were expressed only in seed coats and another five in both embryos and seed coats. Analysis of transcript profiles of seed-abundant as well as seed-specific genes, and their expression patterns, implies that the B. napus seed is undergoing an active cell proliferation during 10-20 DAP, while establishing metabolic networks for subsequent seed maturation.

Arabidopsis↗
PubMed2003

HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis.

Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines. It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis. Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human. Both mouse and human HILS1 genes are located in intron 8 of the alpha-sarcoglycan genes. HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15). HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin. Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones. Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.

Amino Acid Sequence↗
PubMed2003

Amplified voltammetric detection of DNA hybridization via oxidation of ferrocene caps on gold nanoparticle/streptavidin conjugates.

Gold nanoparticle/streptavidin conjugates covered with 6-ferrocenylhexanethiol were attached onto a biotinylated DNA detection probe of a sandwich DNA complex. Due to the elasticity of the DNA strands, the ferrocene caps on gold nanoparticle/streptavidin conjugates are positioned in close proximity to the underlying electrode modified with a mixed DNA capture probe/hexanethiol self-assembled monolayer and can undergo reversible electron-transfer reactions. A detection level, down to 2.0 pM (10 amol for the 5 microL of sample needed) for oligodeoxynucleotide samples was obtained. The amplification of the voltammetric signals was attributed to the attachment of a large number of redox (ferrocene) markers per DNA duplex formed. The ferrocene oxidation current increased with the target concentration and began to level off at a target concentration of 10 nM. An Excellent linearity was found within the range between 6.9 and 150.0 pM and reasonable relative standard deviations (between 3.0 and 13.0%) were obtained. The amenability of this method to the analyses of polynucleotides (i.e., PCR products of the pre-S gene of hepatitis B virus in serum samples) was also demonstrated. The method is shown to be simple, selective, reproducible, and cost-effective and does not require labeling of the DNA targets.

DNA↗
PubMed2003

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control.

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.

Animals↗
PubMed2003

Previously uncharacterized roles of platelet-activating factor acetylhydrolase 1b complex in mouse spermatogenesis.

Platelet-activating factor (PAF) has been shown to affect sperm motility and acrosomal function, thereby altering fertility. PAF acetylhydrolase 1b (PAFAH1B) hydrolyzes PAF and is composed of three subunits [the lissencephaly (LIS1) protein and alpha1 and alpha2 subunits] and structurally resembles a GTP-hydrolyzing protein. Besides the brain, transcripts for Lis1, alpha1, and alpha2 are localized to meiotic and early haploid germ cells. Here, we report disruptions of the alpha2 (Pafah1b2) and alpha1 (Pafah1b3) genes in mice. Male mice homozygous null for alpha2(alpha2-/-) are infertile, and spermatogenesis is disrupted at mid- or late pachytene stages of meiosis or early spermiogenesis. Whereas mice homozygous mutant for alpha1(alpha1-/-) have normal fertility and normal spermatogenesis, those with disruptions of both alpha1 and alpha2 (alpha1-/-alpha2-/-) manifest an earlier disturbance of spermatogenesis with an onset at preleptotene or leptotene stages of meiosis. Testicular Lis1 protein levels are up-regulated in the alpha2-/- and alpha1-/-alpha2-/- mice. Lowering Lis1 levels by inactivating one allele of Lis1 in alpha2 null or alpha1/alpha 2 null genetic backgrounds (i.e., alpha2-/-Lis1+/- or alpha1-/-alpha2-/-Lis1+/- mice) restored spermatogenesis and male fertility. Our data provide evidence for unique roles of the PAFAH1B complex and, particularly, the lissencephaly protein Lis1 in spermatogenesis.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2003

RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway.

Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation. Ret Finger Protein-Like 4 (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, was discovered during an in silico search for germ cell-specific genes. To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein-protein interaction assays. Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage. We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit beta type 1, ubiquitin B, as well as a degradation target protein, cyclin B1. Coimmunoprecipitation analyses of in vitro translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase. The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis.

Animals↗
PubMed2003

Apatite deposition on thermally and anodically oxidized titanium surfaces in a simulated body fluid.

By application of a special specimen set-up, thermally oxidized titanium specimen pairs were found able to deposit apatite on the contact surfaces after soaking for 7 days in the simulated body fluid (SBF) of Kokubo's recipe. The specimens oxidized at 400 degrees C and 500 degrees C showed the highest ability of apatite deposition. Both increase and decrease in oxidation temperature from this range caused the apatite deposition ability to decrease. The specimen without treatment failed to deposit any apatite. Specimens anodically oxidized in electrolytes of H(3)PO(4), H(2)SO(4) and acetic acid exhibited very low ability of apatite deposition. Furthermore, the specimen thermally oxidized at 400 degrees C was even able to help the surfaces of PTFE and silicone deposit apatite in the PTFE-Ti and silicone-Ti pairs. This in vitro experimental results indicated that the difference in apatite deposition among various titanium oxides does exist and can be distinguished by applying the present specimen set-up. The mechanism of the apatite deposition on the contact surfaces was discussed in relation to the passive dissolution of titanium in SBF. The release of titanium hydroxide and OH(-) ions from the titanium surfaces and their accumulation inside the confined space between the two contact surfaces were suggested to be responsible for the apatite deposition.

Adsorption↗
PubMed2003

Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis.

To discover germ cell-specific genes, we used in silico subtraction and identified testis expressed gene 14 (Tex14). Mouse Tex14 contains an open reading frame encoding a 1450-amino-acid protein, which shares 64% amino acid identity with the predicted human TEX14 protein. The predicted TEX14 amino acid sequence consists of three ankyrin repeats, a protein kinase domain, and a leucine zipper dimerization motif. Northern blot analysis and in situ hybridization show that Tex14 mRNA is expressed specifically in the testis, with highest levels observed in pachytene, diplotene, and meiotically dividing spermatocytes. Two 5' splice variants of mouse Tex14 were discovered by sequencing 5'-RACE polymerase chain reaction products. TEX14 is predicted to be localized to the nucleus, suggesting that it may play a key role in regulating gene expression or modulating nuclear events during mammalian spermatogenesis.

Amino Acid Sequence↗