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Biomedical subjects

X Yang

Publications and source records attributed to X Yang.

At least 397 records · Page 22Linked to original sources
PubMed2000

Dynamic changes of cumulus-oocyte cell communication during in vitro maturation of porcine oocytes.

Oocyte maturation is a key issue of current animal biotechnology. This study was designed to examine the morphodynamics of the cumulus-oocyte association during oocyte maturation. Porcine cumulus-oocyte complexes were recovered from slaughterhouse ovaries; matured in vitro for 0, 24, 36, and 44 h; and evaluated by scanning electron microscopy either combined or not combined with the osmium-dimethyl sulfoxide-osmium maceration (ODO) method. The cytoskeleton distribution was also observed by fluorescence staining. Prior to maturation culture (0 h), the spherical cumulus cells were tightly clustered around the oocyte, with narrow intercellular spaces. They showed active secretion at 36 h and were fully expanded at 44 h of culture. The ODO methods revealed that the cumulus cells projected numerous long and thin transzonal projections at 0 h, but these were largely disconnected at 44 h. The outer surface of the zona pellucida showed a meshwork surface regardless of time of incubation, whereas the inner surface changed from a fine fibrous surface to a spongy surface that was coated with mucin. The vitelline surface changed from a sparse distribution of short microvilli (MV) to a dense distribution of well-developed MV. Fluorescence staining showed that the cumulus cell projections consisted mainly of microfilaments, which were abundant at the germinal vesicle and metaphase-I (M-I) stages (0-24 h) but which were decreased in number at the M-II stage (36-44 h). We conclude that the cumulus-oocyte transzonal projections became disconnected between the M-I and M-II stages as a result of cumulus expansion. The cumulus-cumulus communications, however, remained intact at these stages, although the biological functions of these communications were not clear.

Animals↗
PubMed2000

Telomerase activity in bovine embryos during early development.

The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.

Animals↗
PubMed2000

Differential effects of omega-conotoxin GVIA, tetrodotoxin and prolonged cold storage on purinergic and adrenergic transmission in isolated canine splenic artery.

UNLABELLED: Double-peaked vasoconstrictions (biphases of vasoconstrictions) were readily induced in the conditions of 30 s trains of pulses at 1 Hz in the isolated, perfused canine splenic artery. P2X purinoceptors have previously been shown to be involved mainfy in the first-peaked response and alpha1-adrenoceptors mostly in the second. The treatment with 10 nM omega-conotoxin GVIA (omega-CTX) produced a parallel inhibitory effect on the first- and second-peaked vasoconstrictor responses to nerve stimulation. A submaximal concentration of tetrodotoxin (TTX) (3 nM) did not affect the first peak of constriction, but strongly inhibited the second peak, although a larger dose of TTX (30 nM) abolished either the first- or second-peaked response. On the other hand, after cold storage at 4 degrees C for 7 days, the first-peaked vasoconstriction markedly decreased, whereas the second-peaked response was not significantly modified. IN CONCLUSION: (1) omega-CTX-sensitive calcium channels may produce a parallel modulation of purinergic and adrenergic components of sympathetic cotransmission; (2) TTX-sensitive sodium channels may have a more important role in controlling the adrenergic rather than purinergic transmission; and (3) the function of purinergic transmission of sympathetic nerve might be affected more strongly than that of adrenergic transmission in the cold-stored canine splenic artery.

Adenosine Triphosphate↗
PubMed2000

Decline of physical activity from youth to young adulthood in Finland.

PURPOSE: As a part of a nation-level research program, Cardiovascular Risk in Young Finns, the aim of this study was to analyze age-related decline of physical activity among Finnish young people. METHODS: The number of subjects at the beginning of the study in 1980 was 2309, representing both genders and ages 9, 12, 15, and 18. The follow-up measurements were repeated in 1983, 1986, and 1989. Thus, the data cover ages from 9 to 27. To measure physical activity, a short self-report questionnaire was administrated in connection with medical examination. The questionnaire consisted of items concerning frequency and intensity of physical activity, way of spending leisure-time, and participation in organized sports. Using five items an index of physical activity was calculated. RESULTS: The results showed a remarkable decline after the age of 12 in frequency of physical activity and sport participation, in particular. The self-reported intensity of physical activity increased with age. In younger age groups, the boys were more active than girls according to all variables, but the decline of activity was steeper among male than female subjects, and after the age of 15 the female subjects participated in physical activity more frequently than male subjects. CONCLUSION: Reasons for the decline need to be further studied, and interventions are needed to assist young people in maintaining healthful levels of physical activity.

Adolescent↗
PubMed2000

Hexakis(1H-imidazole-kappaN3)nickel(II) bis[O,O'-diisopropyl dithiophosphate(1-)].

In the title complex, [Ni(Im)(6)]((i)Pr-dtp)(2) or [Ni(C(3)H(4)N(2))(6)](C(6)H(14)O(2)PS(2))(2), the coordination around the Ni atom, located on an inversion centre, is octhahedral with all positions being occupied by tertiary N atoms of the imidazole moieties. Hydrogen bonds link the anions and cations into a two-dimensional network in the bc plane.

Imidazoles↗
PubMed2000

Role of presenilin-1 in murine neural development.

Our previous studies showed that presenilin-1 (PS1) is required for murine neural and skeletal development. Here we report that the reduction in the neural progenitor cells observed in the PS1-/- mouse brain is due to premature differentiation of progenitor cells, rather than to increased apoptotic cell death or decreased cell proliferation. In the ventricular zone of PS1-/- mice, expression of the Notch1 downstream effector gene Hes5 is reduced, and expression of the Notch1 ligand Dll1 is elevated, indicating reduced Notch signaling. These results provide direct evidence that PS1 is involved in the regulation of neurogenesis and Notch signaling during development.

Animals↗
PubMed2000

Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297.

In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526-1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.

Amino Acid Sequence↗
PubMed2000

Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses.

We previously reported that DNA vaccination was able to elicit cellular immune responses and partial protection against Chlamydia trachomatis infection. However, DNA immunization alone did not generate immune responses or protection as great as that induced by using live organisms. In this study, we evaluated the immunologic effects of a combinational vaccination approach using C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) DNA priming followed by boosting with immune-stimulating complexes (ISCOM) of MOMP protein (MOMP ISCOM) for protection of BALB/c mice against MoPn lung infection. Substantially better protection to challenge infection was observed in mice given combinational vaccination compared with mice given MOMP ISCOM immunization alone, and the protection approximated that induced by live organisms. Enhanced protection was correlated with stronger delayed-type hypersensitivity, higher levels of gamma interferon production, and increased immunoglobulin A antibody responses in lung homogenates. The results indicate that DNA priming followed by ISCOM protein boosting may be useful in designing a fully protective chlamydial vaccine.

Animals↗
PubMed2000

Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks.

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.

Adhesins, Bacterial↗
PubMed2000

Recombinant chimeric Borrelia proteins for diagnosis of Lyme disease.

Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens of Borrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P<0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.

Antibodies, Bacterial↗
PubMed2000

Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution.

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.

Amino Acid Sequence↗
PubMed2000

Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.

Amino Acid Sequence↗
PubMed2000

LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene.

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4(R24C) oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.

Animals↗
PubMed2000

Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3' cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3' single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3'-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

Hepacivirus↗
PubMed2000

Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo.

Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP(5) and InsP(6). These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP(5) and InsP(6) is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

3T3 Cells↗
PubMed2000

Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover.

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA(2)-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data demonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3'UTR in this regulation.

3' Untranslated Regions↗