Biomedical subjects

X Yu

Publications and source records attributed to X Yu.

At least 109 records · Page 6Linked to original sources

PubMed2000

Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts.

Erythropoietin (Epo) is required for the production of mature red blood cells. The requirement for Epo and its receptor (EpoR) for normal heart development and the response of vascular endothelium and cells of neural origin to Epo provide evidence that the function of Epo as a growth factor or cytokine to protect cells from apoptosis extends beyond the hematopoietic lineage. We now report that the EpoR is expressed on myoblasts and can mediate a biological response of these cells to treatment with Epo. Primary murine satellite cells and myoblast C2C12 cells, both of which express endogenous EpoR, exhibit a proliferative response to Epo and a marked decrease in terminal differentiation to form myotubes. We also observed that Epo stimulation activates Jak2/Stat5 signal transduction and increases cytoplasmic calcium, which is dependent on tyrosine phosphorylation. In erythroid progenitor cells, Epo stimulates induction of transcription factor GATA-1 and EpoR; in C2C12 cells, GATA-3 and EpoR expression are induced. The decrease in differentiation of C2C12 cells is concomitant with an increase in Myf-5 and MyoD expression and inhibition of myogenin induction during differentiation, altering the pattern of expression of the MyoD family of transcription factors during muscle differentiation. These data suggest that, rather than acting in an instructive or specific mode for differentiation, Epo can stimulate proliferation of myoblasts to expand the progenitor population during differentiation and may have a potential role in muscle development or repair.

Animals↗

PubMed2000

Ischemic inactivation of G protein-coupled receptor kinase and altered desensitization of canine cardiac beta-adrenergic receptors.

BACKGROUND: G protein-coupled receptor kinases (GRKs) modulate myocardial beta-adrenergic receptor (betaAR) signaling. We examined whether GRK activity was altered 6, 24, and 96 hours after left anterior descending coronary artery ligation (LAD CAL) in the dog. METHODS AND RESULTS: GRK activity was measured in arrhythmogenic subepicardial border zone (EBZ) tissue overlying the infarct and from nonischemic remote-site (RS) subepicardial tissue from the same animal. GRK activity in the ischemic EBZ was 15% of RS (P:=0.03, n=6) 24 hours after CAL and appeared to start as early as 6 hours through 96 hours. GRK activity and immunoblot data demonstrated a marked decrease of GRK2 but not GRK5 at 24 hours. EBZ tissue exhibited high-affinity binding for (-)-isoproterenol (K:(i) of 0. 076+/-0.026 nmol/L [SEM]) at 24 hours, which was not significantly different from control tissue from nonoperated animals (1.2+/-0.8 nmol/L, P:>0.05, n=6). A significantly lower K:(i) of 13.8+/-2.8 nmol/L (P:<0.001, n=6) was observed for RS taken from the ischemic animals. This was reflected by a 4-fold increase in the EC(50) of isoproterenol-stimulated adenylyl cyclase activity from 18 nmol/L in EBZ tissue to 73 nmol/L in RS (P:<0.05, n=4). CONCLUSIONS: There is a selective decrease in GRK2 activity and a loss of the ability of the arrhythmia-prone EBZ tissue to desensitize to beta-adrenergic stimulation 24 hours after CAL. This correlates temporally with a second (late) peak in sudden cardiac death previously observed between 6 and 24 hours in dog and rat models of myocardial infarction.

Adenylyl Cyclases↗

PubMed2000

Characterization of the promoter of human leukocyte-specific transcript 1. A small gene with a complex pattern of alternative transcripts.

The gene for the human leukocyte-specific transcript 1 (LST1) encodes a small protein that modulates immune responses and cellular morphogenesis. The LST1 transcripts are expressed at high levels in dendritic cells. Because of the complex splicing pattern, use of alternative 5'-untranslated exons, and a biologically interesting pattern of expression of LST1 mRNA, we studied the human LST1 gene promoter and regulatory elements. We identified an additional upstream 5'-untranslated exon in U937 monocytic cells. Transient transfection studies demonstrated that the combination of regions from -1363 to -621 with -112 to -54, relative to the translation start codon, produced the highest level of transcripts from among the various constructs tested, but the pattern of transcripts produced was only a subset of those produced from the endogenous gene. DNase I footprinting analysis and electrophoretic mobility shift assays showed that oligonucleotide probes corresponding to three regions, -1171 to -1142 (BI), -1136 to -1111 (BII), and -783 to -751 (BIV), bound proteins in U937 nuclear extracts. Competition and supershift electrophoretic mobility shift assay did not identify any known transcription factors responsible for BII probe binding. These studies suggest that a novel DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA.

Alternative Splicing↗

PubMed2000

Production and processing of erythropoietin receptor transcripts in brain.

The expression of erythropoietin receptor (EpoR) in brain and neuronal cells, and hypoxia-responsive production of erythropoietin (Epo) in the brain suggests that the function of Epo as a survival or viability factor may extend beyond hematopoietic tissue and erythroid progenitor cells. Epo, produced by astrocytes and neurons, can be induced by hypoxia by severalfold, and in animal models Epo administration is neuroprotective to ischemic challenge. We characterized the human EpoR transcript in brain and neuronal cells to determine its contribution in regulating the Epo response in brain. Screening of a human brain cDNA library and quantitative analysis of EpoR transcripts indicate that the EpoR gene locus is transcriptionally active in brain. In addition to the proximal promoter that is active in hematopoietic cells, a significant proportion of transcripts originates far upstream from the EpoR coding region. Unlike erythroid cells with efficient splicing of EpoR transcripts to its mature form, brain EpoR transcripts are inefficiently or alternately processed with a bias towards the 3' coding region. In human EpoR transgenic mice, anemic stress induces expression of the transgene and endogenous EpoR gene in hematopoietic tissue and brain. In culture of neuronal cells, hypoxia induces EpoR expression and increases sensitivity to Epo. Induction of EpoR expression appears to be a consequence of increased transcription from the upstream region and proximal promoter, and a shift towards increased processing efficiency. These data suggest that in contrast to erythropoiesis where erythroid progenitor cells express high levels of EpoR and are directly responsive to Epo stimulation, the neuroprotective effect of Epo and its receptor may require two molecular events: the induction of Epo production by hypoxia and an increase in EpoR expression in neuronal cells resulting in increased sensitivity to Epo.

Adult↗

PubMed2000

The polarity and magnitude of ambient charge influences three-dimensional neurite extension from DRGs.

Sulfated proteoglycans have inhibitory effects on neurite extension, and the negative charge of the glycosaminoglycan side chains may be involved in the inhibitory process. The main goal of this study is to investigate the effects of charge on three-dimensional neurite extension. Various concentrations of dermatan sulfate (DS), a chondroitin sulfate glycosaminoglycan, and consequently, various degrees of negative charge were presented on three-dimensional agarose hydrogels and the effect of charge on neurite extension from primary neurons was investigated. Dose-response experiments were also performed with the polycationic (positively charged) polysaccharide chitosan covalently coupled to agarose. The amount of DS or chitosan coupled to the agarose gel was quantified via metachromatic dye or Fourier transform infrared spectroscopy methods, respectively. The length of embryonic day 9 (E9) chick dorsal root ganglia neurites extended through charged agarose gels is dependent on the polarity and quantity of ambient charge. The inhibitory effects of the sulfated DS and the enhancing effects of the polycationic chitosan on neurite extension decrease as the amount of DS or chitosan coupled to agarose is decreased. These findings indicate that primary neural process extension is influenced by the polarity of ambient charge in a dose-responsive manner.

Animals↗

PubMed2000

Three-dimensional reconstruction of transcription termination factor rho: orientation of the N-terminal domain and visualization of an RNA-binding site.

The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid. Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring. We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring. An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring. Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases.

Binding Sites↗

PubMed2000

Nuclear localization and cell cycle-specific expression of CtIP, a protein that associates with the BRCA1 tumor suppressor.

The BRCA1 tumor suppressor has been implicated in a diverse spectrum of cellular processes, including transcriptional regulation, DNA repair, and cell cycle checkpoint control. CtIP was recently identified as a protein that associates with BRCA1 and two other nuclear factors, CtBP1 and Rb1. To understand the functions of CtIP, we have evaluated its biological properties with respect to those of BRCA1. Our results show that CtIP, like its associated factors, is predominantly a nuclear protein. A subset of the endogenous pool of CtIP polypeptides exists in a protein complex that includes both BRCA1 and the BRCA1-associated RING domain protein (BARD1). At the protein level, CtIP expression varies with cell cycle progression in a pattern identical to that of BRCA1. Thus, the steady-state levels of CtIP polypeptides, which remain low in resting cells and G(1) cycling cells, increase dramatically as dividing cells traverse the G(1)/S boundary. In contrast to BRCA1, however, the G(1)/S induction of CtIP expression is mediated primarily by post-transcriptional mechanisms. Finally, the interaction between CtIP and BRCA1 is shown to be stable in the face of genotoxic stress elicited by treatment with UV light, adriamycin, or hydrogen peroxide. Together, these results indicate that CtIP can potentially modulate the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control.

Animals↗

PubMed2000

Development of a multi-specificity opsonophagocytic killing assay.

The opsonophagocytic-killing assay (OPKA) is one of the primary surrogate assays for evaluating the pneumococcal capsular polysaccharide-protein conjugates under development as vaccines. Because each vaccine contains seven or more different conjugates, multiple OPKA must be performed on each serum. Moreover, the large number of assays can deplete serum samples from infants. To reduce the amount of serum and effort required to conduct OPKA we developed a multi-specificity OPKA using antibiotic resistant pneumococci. Equal numbers of optochin-resistant serotype 6B and streptomycin-resistant 19F pneumococci were used as the target bacteria. Surviving bacteria of each serotype were enumerated by plating on agar containing the appropriate antibiotic. In an examination of 25 immune sera the results obtained with this new assay correlated well with those obtained when bacterial targets were examined individually. By using additional antibiotic resistance markers, more than two specificities can be examined in a single assay.

Antibodies, Monoclonal↗

PubMed2000

Characterization of the complete transcriptionally active ehrlichia chaffeensis 28 kDa outer membrane protein multigene family.

The 28kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family. The purpose of this study was to determine all the p28 gene sequences and their transcriptional activities. There were 21 members of the p28 multigene family located in a 23kb DNA fragment in the genome of E. chaffeensis. The p28 genes each contained 816-903 nucleotides with intergenic spaces of 10-605 nucleotides. All the genes were complete and were predicted to have a signal sequence. The molecular masses of the mature proteins were predicted to be 28-32kDa. The amino acid sequence identity of the P28 proteins was 20-83%. Ten p28 genes were investigated for transcriptional activity by using RT-PCR amplification of mRNA. Six of 10 tested p28 genes were actively transcribed in cell-culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were divergent among isolates of E. chaffeensis also. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that P28s may be involved in immune avoidance.

Amino Acid Sequence↗

PubMed2000

Distinct effects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca(2+) levels.

These studies describe inhibitory effects of N-acetylcysteine on several biochemical events associated with the activation of extracellular signal-regulated kinases (ERK) by angiotensin II in the cardiac fibroblast and compare these effects with those of the nitric oxide donor, S-nitroso-N-acetylpenicillamine, an agent we showed previously to inhibit angiotensin II-induced ERK activation and the concomitant phosphorylation of proline-rich tyrosine kinase 2 (Wang, D., Yu, X., and Brecher, P. (1999) J. Biol. Chem. 274, 24342-24348). The transactivation of the epidermal growth factor receptor by angiotensin II, a process required for the activation of ERK, was inhibited by N-acetylcysteine but not by nitric oxide. The transactivation of the epidermal growth factor receptor by angiotensin II was shown to be independent of intracellular calcium increases. Nitric oxide, but not N-acetylcysteine, inhibited the angiotensin II-induced increase in intracellular Ca(2+). Neither nitric oxide nor N-acetylcysteine inhibited either phospholipase C activation or inositol triphosphate generation in response to angiotensin II. N-Acetylcysteine did inhibit the phosphorylation of the calcium sensitive tyrosine kinases PYK2 and Src, effects that also occurred using nitric oxide. These studies describe a novel effect of N-acetylcysteine on cross-talk between a G protein-linked receptor and a tyrosine kinase receptor and offer additional molecular insight to explain how N-acetylcysteine and nitric oxide act at different sites and might have an additive effect on specific hormonal responses.

Acetylcysteine↗

PubMed2000

Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb.

Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression.

Animals↗

PubMed2000

Self-Assembly and Surface Structure of an Amphiphilic Graft Copolymer, Polystyrene-graft-omega-Stearyl-Poly(ethylene oxide).

Surface properties of the polystyrene-graft-omega-stearyl-poly(ethylene oxide) (PS-g-SPEO) have been characterized by X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), contact angle, and spin probe techniques. The XPS results indicate that the surface and bulk composition of the PS-g-SPEO copolymers differ remarkably from each other. The stearyl and EO components enrich at the copolymer/air interfaces due to the self-assembly of the stearyl groups. At the PS-g-SPEO-72.6 surface (the x in PS-g-SPEO-x indicates the bulk density of the SPEO in wt%), the self-assembly of the hydrophobic stearyl groups is strong enough to form a stable liquid crystalline phase as indicated by DSC. At polymer/water interfaces, PS-g-SPEO-72.6 presents a hydrophilic surface with low PEO mobility, whereas PS-g-SPEO-50.6 and PS-g-SPEO-31.0 present the hydrophobic surface with high PEO mobility. The two different types of the surfaces, with different characters in surface energy, surface mobility of PEO, and surface architecture of SPEO, will be quite valuable as models for detecting the synergistic action of the PEO chains and the stearyl groups (specific ligand for albumin binding) in protein solutions. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Speciation of alkyllead and inorganic lead by derivatization with deuterium-labeled sodium tetraethylborate and SPME-GC/MS.

A method for full speciation and determination of alkyllead and inorganic lead(II) in aqueous samples was developed. This was accomplished by in situ derivatization with deuterium-labeled sodium tetraethylborate NaB(C2D5)4 (DSTEB). The derivatization was carried out directly in the aqueous sample and the derivatives were extracted from the headspace by a solid-phase microextraction (SPME) fiber. The extracted analytes were then transferred to a GC/MS or a GC/FID for separation and detection. The research presented demonstrates that SPME and the derivatization reagent DSTEB can be used successfully for the speciation of Pb2+, Pb(CH3)3+, Pb(C2H5)3+, and Pb(C2H5)4 in water samples. All derivatives, Pb(C2D5)4, (CH3)3Pb(C2D5), (C2H5)3Pb(C2D5), and Pb(C2H5)4, are separated using an SBP-5 column. This method was applied to monitor degradation of tetraethyllead in water. This is the first report of ethylation by DSTEB for full speciation of methyllead, ethyllead, and inorganic lead compounds. This approach can be extended to other organometallic compounds as demonstrated for ethyltin speciation. This full speciation method will aid in monitoring occurrence, pathways, toxicity, and biological effects of these compounds in the environment. It is easily adopted for field analysis.

Borates↗

PubMed2000

The role of B7 costimulation in CD4/CD8 T cell homeostasis.

The effect of B7-mediated costimulation on T cell homeostasis was examined in studies of B7-1 (CD80) and B7-2 (CD86) transgenic as well as B7-deficient mice. B7 overexpression in transgenic mice resulted in marked polyclonal peripheral T cell hyperplasia accompanied by skewing toward an increased proportion of CD8 single-positive cells and a decreased proportion of CD4 single-positive cells in thymus and more markedly in peripheral T cells. B7-induced T cell expansion was dependent on both CD28 and TCR expression. Transgenic overexpression of B7-1 or B7-2 resulted in down-regulation of cell surface CD28 on thymocytes and peripheral T cells through a mechanism mediated by intercellular interaction. Mice deficient in B7-1 and B7-2 exhibited changes that were the reciprocal of those observed in B7-overexpressing transgenics: a marked increase in the CD4/CD8 ratio in peripheral T cells and an increase in cell surface CD28 in thymus and peripheral T cells. These reciprocal effects of genetically engineered increase or decrease in B7 expression indicate that B7 costimulation plays a physiological role in the regulation of CD4+ and CD8+ T cell homeostasis.

Abatacept↗

PubMed2000

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Adult↗

PubMed2000

Targeted drug delivery to C6 glioma by transferrin-coupled liposomes.

Recent advances in liposome technology have shown promise relative to the introduction of chemotherapeutic agents with reduced toxicity, extended longevity, and potential for cell-specific targeting. In this study we report the engineering of a liposomal delivery system for the chemotherapeutic drug doxorubicin. The system was targeted specifically to C6 glioma in vitro by coupling transferrin to the distal ends of liposomal polyethylene glycol (PEG) chains. The transferrin receptor is overexpressed on glioma, with the extent of overexpression correlated to the severity of the tumor. Significantly increased gliomal doxorubicin uptake was achieved by drug encapsulation within transferrin-coupled liposomes compared to other liposome populations. Doxorubicin encapsulated within transferrin-coupled liposomes exhibited 70% of free doxorubicin uptake as compared to 54, 14, and 34% for non-PEG, PEG, and albumin-coupled PEG liposomes, respectively. Competitive binding assays support the receptor-mediated mechanism of targeting. The addition of one microM free transferrin reduced the uptake of doxorubicin encapsulated within transferrin-coupled liposomes by 30%.

Animals↗

PubMed2000

Copper induces apoptosis in BA/F3beta cells: Bax, reactive oxygen species, and NFkappaB are involved.

Copper, an essential trace element, can be toxic to some cells when present in excess. But thorough investigations into the cytotoxicity of copper and subsequent molecular mechanisms are rare, although the cytotoxicity of copper has been applied to cancer chemotherapy. The present study demonstrates that Cu(2+) inhibits [(3)H] thymidine incorporation in mouse pro-B cell line BA/F3beta and induces apoptosis. Apoptosis was mainly judged by morphology of cells, quantification of subdiploid DNA contents by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. The apoptotic effect is dose and time dependent. Western blotting shows Bax is upregulated by Cu(2+). Bcl-2 overexpression can partially inhibit this apoptosis. Moreover, Cu(2+) increases the production of reactive oxygen species (ROS) in a dose-dependent manner. The antioxidant N-acetylcysteine (NAC) not only significantly inhibited copper-induced apoptosis but also totally blocked generation of ROS, while Bcl-2 overexpression has no effect on the generation of ROS. Furthermore, our results show that NFkappaB is downregulated by Cu(2+). Bcl-2 overexpression or NAC can sustain the activity of NFkappaB. These data indicate that Cu(2+) might induce apoptosis in BA/F3beta cells via upregulation of Bax and ROS and subsequent inactivation of NFkappaB.

Acetylcysteine↗

PubMed2000

High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent.

In this study, the sensitivity of a novel fibrin-targeted contrast agent for fibrin detection was defined in vitro on human thrombus. The contrast agent was a lipid-encapsulated perfluorocarbon nanoparticle with numerous Gd-DTPA complexes incorporated into the outer surface. After binding to fibrin clots, scanning electron microscopy of treated clots revealed dense accumulation of nanoparticles on the clot surfaces. Fibrin clots with sizes ranging from 0.5-7.0 mm were imaged at 4.7 T with or without treatment with the targeted contrast agent. Regardless of sizes, untreated clots were not detectable by T(1)-weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T(1) and T(2) relaxation times (20-40%) were measured relative to the surrounding media and the control clots. These results suggest the potential for sensitive and specific detection of microthrombi that form on the intimal surfaces of unstable atherosclerotic plaque.

Antibodies, Monoclonal↗