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X Yang

Publications and source records attributed to X Yang.

At least 829 records · Page 46Linked to original sources
PubMed1996

Genomic structure of the human bradykinin B1 receptor gene and preliminary characterization of its regulatory regions.

We report on the isolation of the human BK B1 receptor gene from a human lung fibroblast genomic library. The gene is present as a single copy, spans more than 10 kilobases and includes three exons interrupted by two introns. The relative position of the exons was mapped using RNase protection assay, and genomic DNA and cDNA sequence matches. Exon and intron-exon boundaries, and 5', 3' flanking regions were sequenced. While the 5' untranslated region is distributed on all three exons, the coding region is located entirely on the third exon. The exon-intron junction sequences are highly conserved. Primer extension analysis mapped the transcriptional initiation site 21 base pairs downstream of a TATA sequence. Two Aluc sequences and two distinct functional promoters were found in the human BK B1 receptor gene.

Base Sequence↗
PubMed1996

Phosphorylation of Vif and its role in HIV-1 replication.

Vif is a 23-kDa protein encoded by human immunodeficiency virus, type 1 (HIV-1) which is important for virion infectivity. Here, we describe the phosphorylation of HIV-1 Vif and its role in HIV-1 replication. In vivo studies demonstrated that Vif is highly phosphorylated on serine and threonine residues. To identify phosphorylation sites and characterize the Vif kinase(s), Vif was expressed in Escherichia coli and purified for use as a substrate in in vitro kinase assays. The purified Vif protein was phosphorylated in vitro on serine and threonine residues by a kinase(s) present in both cytosol and membrane fractions. Phosphorylation of Vif was stimulated by phorbol 12-myristate 13-acetate and inhibited by staurosporine and hypericin, a drug with potent anti-HIV activity. The Vif kinase(s) was resistant to inhibitors of protein kinase C, cAMP-dependent kinase, and cGMP-dependent kinase, suggesting that it is distinct from these enzymes. To identify the phosphorylation sites, 32P-labeled Vif was digested by V8 protease and the peptides were resolved by reverse-phase high performance liquid chromatography. Radioactive peptide sequencing identified three phosphorylation sites within the C terminus, Ser144, Thr155, and Thr188. Two-dimensional tryptic phosphopeptide mapping indicated that these sites are also phosphorylated in vivo. Both Ser144 and Thr188 are contained in the recognition motifs (R/KXXS*/T* and R/KXXXS*/T*) used by serine/threonine protein kinases such as cGMP-dependent kinase and PKC. Ser144 is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins. Mutation of Ser144 to alanine resulted in loss of Vif activity and >90% inhibition of HIV-1 replication. These studies suggest that phosphorylation of Vif by a serine/threonine protein kinase(s) plays an important role in regulating HIV-1 replication and infectivity.

Alkaloids↗
PubMed1996

Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis.

The mechanism by which tolerance is induced via systemic administration of high doses of aqueous antigen has been analyzed by using mice transgenic for a T-cell receptor specific for the influenza virus hemagglutinin (HA) peptide comprising amino acids 126-138. After intravenous injection of 750 (but not 75) micrograms of HA peptide, a state of hyporesponsiveness was rapidly induced. In the thymus, in situ apoptosis in the cortex and at the corticomedullary junction was responsible for a synchronous and massive deletion of CD4+ CD8+ thymocytes. In secondary lymphoid organs, HA-reactive T cells were initially activated but were hyporesponsive at the single cell level. After 3 days, however, those cells were rapidly deleted, at least partially, through an apoptotic process. Therefore, both thymic and peripheral apoptosis, in addition to T-cell receptor desensitization, contribute to high-dose tolerance.

Amino Acid Sequence↗
PubMed1996

Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression.

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.

Animals↗
PubMed1996

An altered form of apolipoprotein H binds hepatitis B virus surface antigen most efficiently.

Using recombinant (r)HBsAg as a ligand, we previously found a 46-kDa human plasma protein capable of specific binding, and identified this protein as apolipoprotein H (apo H). Apo H is able to bind to rHBsAg containing only the small S protein, in both ligand blot and enzyme immunoassay systems (H. Mehdi, M.J. Kaplan, F.Y. Anlar, X. Yang, R. Bayer, K. Sutherland, and M.E. Peeples, J. Virol. 68, 2415-2424, 1994). Apo H is a plasma glycoprotein, some of which is associated with lipoproteins, particularly chylomicrons and high-density lipoproteins (HDL). During normal lipid trafficking in the bloodstream, chylomicrons and HDL are targeted to the hepatocyte, the primary host cell for HBV, for degradation. In this report the method of apo H presentation was examined. rHBsAg bound to apo H very poorly if the apo H was coated directly on a microtiter well, or if it was presented in a soluble form. Binding was 100-fold more efficient when apo H was presented as a complex with monoclonal antibody (MAb) P2D4. These results suggest that binding to this MAb alters apo H, making it highly reactive with rHBsAg. Apo H binding to rHBsAg is not dependent on divalent cations and is optimal at pH 6.5-8.0. Removal of lipids from rHBsAg resulted in denaturation, preventing analysis of binding activity. Removal of sialic acid or complete removal of N-linked carbohydrates from apo H did not change its ability to bind rHBsAg, indicating that apo H carbohydrates are not involved in rHBsAg binding. Likewise, chemical modification of the arginine residues of apo H had no effect on binding. However, chemical modification of as few as three of the 29 lysine residues of apo H destroyed binding, indicating that one or a few lysines in apo H are involved in rHBsAg binding.

Apolipoproteins↗
PubMed1996

Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice.

The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.

Aging↗
PubMed1996

Twenty-four-hour intermittent perfusion storage of the isolated rat heart. II. Perfusion pressure and functional preservation.

This study investigated the effect of intermittent perfusion (IP) pressures on functional preservation of the cold-stored isolated rat heart. The heart was flushed with cardioplegic solution #11 with ethylene diamine-tetraacetic acid and 2, 3-butanedione monoxime (CP-11EB), and stored at 0 degree C for 24 hr. During storage, the heart was intermittently perfused for 3 min at 10 and 17 hr of storage with 25 degrees C oxygenated CP-11EB. The IP pressures studied were: 20, 30, 40, 50, 60, 70, 80, and 100 mm Hg. Poststorage recovery of function was assessed with 30 min of working reperfusion. Control function of the unstored heart was: aortic flow (AF), 51.3 +/- 2.2 ml/min; coronary flow (CF), 25.8 +/- 1.2 ml/min; cardiac output (CO), 77.7 +/- 2.3 ml/min; work, 92.0 +/- 4.9 g-m/min; and coronary vascular resistance (CVR), 2.64 +/- .21 mm Hg-min/ml. Functional return in the 30,40,50,60,70, and 80 mm Hg groups was similar. Recovery in a representative group (60 mm Hg) was: AF, 52 +/- 3%; CF, 41 +/- 2%; CO, 48 +/- 3%; work, 42 +/- 3%; and CVR, 225 +/- 10% of the control. The total IP volume in this group was 71.0 +/- 1.7 ml; total lactate dehydrogenase (LDH) release during IP was 8.62 +/- .37 units/g dry; and end-storage tissue lactate was 52.6 +/- 6.4 mumole/g dry. As IP pressure decreased to 20 mm Hg, cardiac functional recovery declined significantly to AF, 35 +/- 6%; CF, 33 +/- 2%; CO, 34 +/- 5%; and work, 27 = 4% of control (P < .05 vs. 60 mm Hg group). The total IP volume decreased to 10.1 +/- .9 ml, tissue lactate content rose to 87.1 +/- 10.9 (P < .05 vs. 60 mm Hg group), but LDH release fell to 5.59 +/- .46 (P < .05 vs. 60 and 100 mm Hg groups). When IP pressure increased to 100 mm Hg, cardiac function was depressed with AF recovered to 25 +/- 3%; CF, 30 +/- 3%; CO, 26 +/- 3%; work, 21 +/- 3%; and CVR, 294 +/- 26% of control (P < .05 vs. 60 mm Hg group). IP volume increased to 100.7 +/- 2.4 ml; total LDH release increased to 19.1 +/- 1.2 (P < .05 vs. 60 mm Hg group), although tissue lactate (58.9 +/- 3.9) was not different from the 60 mm Hg group. End-storage tissue wet/dry weight ratio and myocardial adenosine triphosphate content were not different among the 20, 60, and 100 mm Hg groups. In conclusion, IP has a broad pressure optimum ranging from 30 to 80 mm Hg for 24-hr hypothermic preservation of the rat cardiac explant.

Adenosine Triphosphate↗
PubMed1996

Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation.

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year-old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction-independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.

Adenine Nucleotides↗
PubMed1996

Malignant transformation of HPV 16-immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis.

A number of epidemiological studies indicate that cigarette smokers are at increased risk of developing cervical cancer. However, convincing biological evidence is lacking. This report examines the biological and cellular role of human papillomavirus (HPV) type 16 and cigarette smoke in multistage cervical carcinogenesis. Two lines of HPV 16-immortalized human endocervical cells (HEN-16 and HEN-16-2) generated from primary cells (HEN) were treated with cigarette smoke condensate (CSC). CSC-treated, but not untreated, HEN-16 and HEN-16-2 formed tumors that were invasive squamous cell carcinomas in nude mice. The tumors were used to initiate 2 tumor lines of cells (HEN-16T and HEN-16-2T, respectively). Cells of both tumor lines, compared with HEN, HEN-16 and HEN-16-2, featured: (a) tumorigenicity, (b) distinct morphologies in monolayer and organotypic (raft) cultures, (c) faster growth in serum plus high calcium levels after immortalization and after transformation, (d) higher saturation density and (e) anchorage-independent growth. Our results provide unique direct in vitro evidence that cigarette smoke causes cancer in HPV-containing cervices.

Cell Line, Transformed↗
PubMed1996

Resistance to retinoic acid and altered cytokeratin expression of human papillomavirus type 16-immortalized endocervical cells after tumorigenesis.

Human papillomaviruses (HPVs) and cigarette smoking are epidemiologically associated with cervical cancer. We recently found that HEN-16 and HEN-16-2 HPV type 16-immortalized endocervical cells form tumors after treatment with cigarette smoke condensate and derived 2 tumor cell line cultures, HEN-16T and HEN-16-2T, respectively. Here, we examine the molecular pathologic effect of tumorigenesis. HEN-16T and HEN-16-2T exhibit unchanged status and expression of integrated HPV 16 DNA. However, the expression of the cytokeratin CK7 and CK13 endocervical cell markers is more homogeneous in monolayer and organotypic raft cultures after tumorigenesis. For the effect of retinoic acid on monolayers for growth inhibition, HEN-16T were significantly less sensitive than the normal and immortalized non-tumorigenic cells. HEN-16-2T were completely resistant. Moreover, the rafts from both tumorigenic cell line cultures were resistant to retinoic acid and continued to display thick rafts and homogeneous severe dysplasia/carcinoma in situ. In contrast, the non-malignant HEN-16 and HEN-16-2 rafts were thinner, and treatment with retinoic acid blocked the formation of severe dysplasia, reconstructing an epithelium resembling that of the normal endocervix. Our results support the significance of non-viral factors in the mechanism by which cigarette smoking induces tumorigenesis in the late stages of HPV-initiated progression to cervical cancer. Importantly, our data indicate that the sensitivity to retinoic acid of the HPV-containing endocervical cells is lost following tumorigenesis in vitro and possibly in women.

Cell Division↗
PubMed1996

Cell fate determination in the vertebrate retina.

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.

Animals↗
PubMed1996

Estradiol and antiestrogens regulate a growth inhibitory insulin-like growth factor binding protein 3 autocrine loop in human breast cancer cells.

MCF-7 human breast cancer cells are commonly used to model tissues responsive to estrogens and antiestrogens. We examined the effects of estradiol and the antiestrogen ICI 182780 on MCF-7 cell proliferation and insulin-like growth factor binding protein 3 (IGFBP-3) gene expression. ICI 182780-induced growth inhibition was associated with increased transcription of the IG-FBP-3 gene, increased IGFBP-3 mRNA abundance, and increased IGFBP-3 protein accumulation in the conditioned medium. The growth stimulatory effect of estradiol was associated with opposite effects, and the correlation between cellular proliferation and IGFBP-3 mRNA abundance was strong (r = -0.91). Recombinant IGFBP-3 inhibited basal and estradiol-stimulated MCF-7 cell proliferation, and an IGFBP-3 antisense oligodeoxynucleotide abolished antiestrogen-induced growth inhibition. These results provide evidence for an estradiol and antiestrogen-regulated IGFBP-3 growth inhibitory autocrine pathway in MCF-7 cells.

Breast Neoplasms↗
PubMed1996

Activation of single whisker barrel in rat brain localized by functional magnetic resonance imaging.

The previously established cortical representation of rat whiskers in layer IV of the cortex contains distinct cylindrical columns of cellular aggregates, which are termed barrels and correlate in a one-to-one relation to whiskers on the contralateral rat face. In the present study, functional magnetic resonance imaging (fMRI) of the rat brain was used to map whisker barrel activation during mechanical up-down movement (+/- 2.5 mm amplitude at 8 Hz) of single/multiple whisker(s). Multislice gradient echo fMRI experiments were performed at 7 T with in-plane image resolution of 220 x 220 microns, slice thickness of 1 mm, and echo time of 16 ms. Highly significant (P < 0.001) and localized contralateral regions of activation were observed upon stimulation of single/multiple whisker(s). In all experiments (n = 10), the locations of activation relative to bregma and midline were highly correlated with the neuroanatomical position of the corresponding whisker barrels, and the results were reproducible intra- and interanimal. Our results indicate that fMRI based on blood oxygenation level-dependent image contrast has the sensitivity to depict activation of a single whisker barrel in the rat brain. This noninvasive technique will supplement existing methods in the study of rat barrel cortex and should be particularly useful for the long-term investigations of central nervous system in the same animal.

Animals↗
PubMed1996

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea.

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.

Alkylating Agents↗
PubMed1996

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of bovine oocytes.

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4-24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [35S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation.

Animals↗