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Biomedical subjects

W Xu

Publications and source records attributed to W Xu.

At least 163 records · Page 9Linked to original sources
PubMed2000

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Amino Acid Sequence↗
PubMed2000

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Animals↗
PubMed2000

Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway.

Environmental pH exerts broad control over growth and differentiation, but the molecular responses to external pH changes are poorly understood. Here we have used open reading frame macroarray hybridization to identify alkaline response genes in Saccharomyces cerevisiae. Northern or lacZ fusion assays confirmed the alkaline induction of two ion pump genes (ENA1 and VMA4), several ion limitation genes (CTR3, FRE1, PHO11/12, and PHO84), a siderophore-iron transporter gene (ARN4/ENB1), two transcription factor genes (NRG2 and TIS11), and two predicted membrane protein genes (YAR068W/YHR214W and YOL154W). Unlike ENA1 and SHC1, these new alkaline response genes are not induced by high salinity. The known pH-responsive genes in other fungi depend on the conserved PacC/Rim101p transcription factor, but induction of several of these new genes relied upon Rim101p-independent pH signaling mechanisms. Rim101p-dependent genes were also dependent on Rim13p, a protease required for Rim101p processing. The Rim101p-dependent gene VMA4 is required for growth in alkaline conditions, illustrating how Rim101p may control adaptation. Because Rim101p activates ion pump genes, we tested the role of RIM101 in ion homeostasis and found that RIM101 promotes resistance to elevated cation concentrations. Thus, gene expression surveys can reveal new functions for characterized transcription factors in addition to uncovering physiological responses to environmental conditions.

Base Sequence↗
PubMed2000

Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina.

Before phototransduction, spontaneous activity in the developing mammalian retina is required for the appropriate patterning of retinothalamic connections, and there is growing evidence that this activity influences the development of circuits within the retina itself. We demonstrate here that the neural substrate that generates waves in the mouse retina develops through three distinct stages. First, between embryonic day 16 and birth [postnatal day 0 (P0)], we observed both large, propagating waves inhibited by nicotinic acetylcholine receptor (nAChR) antagonists and small clusters of cells displaying nonpropagating, correlated calcium increases that were independent of nAChR activation. Second, between P0 and P11, we observed only larger propagating waves that were abolished by toxins specific to alpha3 and beta2 subunit-containing nAChRs. Third, between P11 and P14 (eye opening) we observed propagating activity that was abolished by ionotropic glutamate receptor antagonists. The time course of this developmental shift was dramatically altered in retinas from mice lacking the beta2 nAChR subunit or the beta2 and beta4 subunits. These retinas exhibited a novel circuit at P0, no spontaneous correlated activity between P1 and P8, and the premature induction at P8 of an ionotropic glutamate receptor-based circuit. Retinas from postnatal mice lacking the alpha3 nAChR subunit exhibited spontaneous, correlated activity patterns that were similar to those observed in embryonic wild-type mice. In alpha3-/- and beta2-/- mice, the development and distribution of cholinergic neurons and processes and the density of retinal ganglion cells (RGCs) and the gross segregation of their dendrites into ON and OFF sublaminae were normal. However, the refinement of individual RGC dendrites is delayed. These results indicate that retinal waves mediated by nAChRs are involved in, but not required for, the development of neural circuits that define the ON and OFF sublamina of the inner plexiform layer.

Action Potentials↗
PubMed2000

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3.

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.

Affinity Labels↗
PubMed2000

The reduced expression of surrogate light chains in B cell precursors from senescent BALB/c mice is associated with decreased E2A proteins.

Senescent mice exhibit decreased numbers of pre-B cells in the bone marrow. Herein, we show that the molecules, lambda5 and VpreB, which comprise the surrogate light chain component of the pre-B cell receptor, are reduced in pro-B/early pre-B cells derived in vitro from the bone marrow of 18-27 months old BALB/c mice after stimulation with IL-7. Both lambda5 and VpreB expression were decreased at the mRNA level as indicated by semi-quantitative RT-PCR; this suggests that the reduced surrogate light chains seen in senescent B cell precursors result from dysfunctional transcriptional regulation. The transcription of surrogate light chains is regulated, in part, by E2A (E47) gene products. Levels of E2A proteins, including E47, were decreased in senescent B cell precursors by up to 90%. Reduced E2A (E47) expression and subsequent reduced transcription of the surrogate light chain components lambda5 and VpreB may, in part, explain the diminished production of B lineage cells observed in senescence.

Aging↗
PubMed2000

Protein kinase C activation contributes to microvascular barrier dysfunction in the heart at early stages of diabetes.

The functional disturbance of microvasculature is recognized as an initiating mechanism that underlies the development of various diabetic complications. Although a causal relationship between microvascular leakage and tissue damage has been well documented in diabetic kidneys and eyes, there is a lack of information regarding the barrier function of coronary exchange vessels in the disease state. The aim of the present study was to evaluate the permeability property of coronary microvessels during the early development of experimental diabetes with a focus on the protein kinase C (PKC)-dependent signaling mechanism. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused porcine coronary venules. The administration of high concentrations of D-glucose induced a dose-dependent increase in the Pa value, which was prevented by blockage of PKC with its selective inhibitors bisindolylmaleimide and Goe 6976. More importantly, an elevated basal permeability to albumin was observed in coronary venules at the early onset of streptozotocin-induced diabetes. The hyperpermeability was corrected with bisindolylmaleimide and the selective PKCbeta inhibitor hispidin. Concomitantly, protein kinase assay showed a high PKC activity in isolated diabetic venules. Immunoblot analysis of the diabetic heart revealed a significant subcellular translocation of PKCbetaII and PKCepsilon from the cytosol to the membrane, indicating that the specific activity of these isoforms was preferentially elevated. The results suggest that endothelial barrier dysfunction attributed to the activation of PKC occurs at the coronary exchange vessels in early diabetes.

Albumins↗
PubMed2000

Lifetime-based pH sensor system based on a polymer-supported ruthenium(II) complex

A new luminescence lifetime-based pH sensor system is described. The system is based on [Ru(Ph2phen)2DCbpy]2+ (DCbpy = 4,4'-dicarboxy-2,2'-bipyridine) immobilized in a mixed domain network copolymer utilizing hydrophobic regions in a hydrophilic, water-swellable, poly(ethylene oxide) matrix. The metal complex binds irreversibly to the hydrophobic domains leaving the pH-sensing COOHs projecting into an aqueous-rich poly(ethylene oxide) region. The complex shows a strong pH dependence of its lifetime (3-4-fold) and provides a usable pH range of about 3-5. The long (approximately 1 micros) excited-state lifetime and visible absorption of the sensor simplifies measurements. A model for the combined pH and oxygen-quenching sensitivity of the complex is provided; this allows use of the pH system over a wide range of oxygen concentrations. The combined polymer sensor is easy to prepare and requires no covalent chemistry. Further, the polymers enhance the luminescence of the complex and minimize interference from oxygen quenching.

Journal Article↗
PubMed2000

Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA.

Unlike cellular mRNA, retroviral mRNA bypasses the tight coupling of the splicing and nuclear export steps to allow the export of intron-containing viral RNA transcripts to the cytoplasm. Two distinct nuclear export pathways for retroviral mRNA have been described: a CRM-1 dependent pathway mediated by the HIV-1 Rev protein and the Rev Response Element (RRE), and a CRM-1 independent pathway mediated by the Constitutive Transport Element (CTE) of type D retroviruses. Two CTE-binding proteins, RNA helicase A (RHA) and Tap, have been implicated in the nuclear export of CTE-containing RNA. Recently, we reported that expression of RRE-containing RNA could also be mediated by a cellular protein, Sam68, independently of Rev. Here we report evidence that Sam68, RHA and Tap cooperate in the nuclear export of both CTE- and RRE-containing RNA. RHA binds to Sam68 and to Tap both in vivo and in vitro. Over-expression of Sam68 activates both RRE- and CTE-regulated reporter gene expression in human cells and in quail cells in the presence of human Tap. This activation was competitively inhibited by the nuclear transport domain (NTD) of RHA and a transdominant negative mutant of Tap. Conversely, the activation of CTE by Tap in quail cells was inhibited by a transdominant mutant of Sam68 and NTD. We propose that both HIV and type D retroviruses may access the same constitutive RNA nuclear export pathway involving RHA, Tap and Sam68, even though HIV also utilizes the Rev protein for more efficient nuclear export. it is likely that this constitutive export pathway is also used by cellular mRNA, but at a different interface with the splicing process.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes.

BACKGROUND-Catecholamine-induced apoptosis is mediated by activation of the beta-adrenergic signaling pathway. We tested the hypothesis that beta(1)- and beta(2)-adrenergic receptor (AR) subtypes differentially affect apoptosis in adult rat ventricular myocytes in vitro. METHODS AND RESULTS-Myocytes were first exposed to norepinephrine (NE) alone (10 mcmol/L) or NE+atenolol (AT) (10 mcmol/L) for 12 hours. AT, a beta(1)-selective AR antagonist, abolished the NE-induced increase in nick end-labeling (TUNEL)-positive cells compared with control (NE, 33+/-3% versus control, 3+/-1%, P<0.0001; NE+AT, 4+/-2% versus control, 3+/-1%, P=0. 98). Annexin V staining, DNA laddering, and caspase activity determinations corroborated these results. Subsequent experiments under prazosin treatment established the apoptosis dose-response curves for the increasingly beta(2)-selective AR agonists isoproterenol (ISO) (beta(1) approximately beta(2)) and albuterol (ALB) (beta(2)>beta(1)). ISO and ALB induced significantly less apoptosis than NE (beta(1)>beta(2)) at equimolar concentrations as assessed by TUNEL staining [1 mcmol/L: NE (8+/-2%) approximately ISO (7+/-1%)>ALB (2+/-1%); 10 mcmol/L: NE (35+/-2%)>ISO (23+/-1%)>ALB (3+/-1%); 100 mcmol/L: NE (50+/-2%)>ISO (29+/-2%)>ALB (14+/-1%), P<0.0001 except for NE versus ISO at 1 mcmol/L with P=0.62]. ALB-induced apoptosis at 100 mcmol/L was abolished by AT (10 mcmol/L), indicating a beta(1)AR-mediated effect. Importantly, ICI 118551 (0.1 mcmol/L), a highly selective beta(2)AR antagonist, did not decrease the percentage of NE-, ISO-, and ALB-induced apoptosis. Reverse transcription-polymerase chain reaction studies revealed that AT completely reversed the beta-adrenergic signaling-induced changes in the Bcl-2-to-Bax ratio. CONCLUSIONS-These observations provide evidence that beta AR-mediated apoptotic death signaling is largely dissociated from beta(2)ARs and selectively mediated by beta(1)ARs in adult rat ventricular myocytes.

Adrenergic beta-Antagonists↗
PubMed2000

Cloning and characterization of the NADH pyrophosphatases from Caenorhabditis elegans and Saccharomyces cerevisiae, members of a Nudix hydrolase subfamily.

Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli. The purified enzymes are homodimers of 39.1 and 43. 5 kDa, respectively, are activated by Mg(2+) and Mn(2+), and are 30 to 50 times more active on NADH than on NAD(+). They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E. coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

Administration, Oral↗
PubMed2000

ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3.

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.

ADAM Proteins↗
PubMed2000

Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis.

P0, the major peripheral nervous system (PNS) myelin protein, is a member of the immunoglobulin supergene family of membrane proteins and can mediate homotypic adhesion. P0 is an essential structural component of PNS myelin; mice in which P0 expression has been eliminated by homologous recombination (P0-/-) develop a severe dysmyelinating neuropathy with predominantly uncompacted myelin. Although P0 is thought to play a role in myelin compaction by promoting adhesion between adjacent extracellular myelin wraps, as an adhesion molecule it could also have a regulatory function. Consistent with this hypothesis, Schwann cells in adult P0-/- mice display a novel molecular phenotype: PMP22 expression is down-regulated, MAG and PLP expression are up-regulated, and MBP expression is unchanged. As in quaking viable mutant mice (qk(v)), which have uncompacted myelin morphologically similar to that found in P0-/- mice, neither the qKI-6 or qKI-7 proteins are expressed in P0-/- peripheral nerve. In addition to these changes in gene expression in the P0 knockout, PLP/DM-20 accumulates in the endoplasmic reticulum of P0-/- Schwann cells, whereas MAG accumulates in redundant loops of uncompacted myelin, not at nodes of Ranvier or Schmidt-Lantermann incisures. Taken together, these results demonstrate that P0 is involved, either directly or indirectly, in the regulation of both myelin gene expression and myelin morphogenesis.

Animals↗
PubMed2000

Evidence that fertilization activates starfish eggs by sequential activation of a Src-like kinase and phospholipase cgamma.

Recent evidence has indicated a requirement for a Src family kinase in initiating Ca(2+) release at fertilization in starfish eggs (Giusti, A. F., Carroll, D. J., Abassi, Y. A., Terasaki, M., Foltz, K. R., and Jaffe, L. A. (1999) J. Biol. Chem. 274, 29318-29322). We now show that injection of Src protein into starfish eggs initiates Ca(2+) release and DNA synthesis, as occur at fertilization. These responses depend on the phosphorylation state of the Src protein; only the kinase active form is effective. Like Ca(2+) release at fertilization, the Ca(2+) release in response to Src protein injection is inhibited by prior injection of the SH2 domains of phospholipase Cgamma. These findings support the conclusion that in starfish, sperm-egg interaction causes egg activation by sequential activation of a Src-like kinase and phospholipase Cgamma. Injection of the SH2 domain of Src, which inhibits Ca(2+) release at fertilization, does not inhibit Ca(2+) release caused by Src protein injection. This indicates that the requirement for a Src SH2 domain interaction is upstream of Src activation in the pathway leading to Ca(2+) release at fertilization.

Animals↗
PubMed2000

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

Cell Nucleus↗
PubMed2000

Slow death of postnatal hippocampal neurons by GABA(A) receptor overactivation.

Neurotransmitters can have both toxic and trophic functions in addition to their role in neural signaling. Surprisingly, chronic blockade of GABA(A) receptor activity for 5-8 d in vitro enhanced survival of hippocampal neurons, suggesting that GABA(A) receptor overactivation may be neurotoxic. Potentiating GABA(A) receptor activity by chronic treatment with the endogenous neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one caused massive cell loss over 1 week in culture. Other potentiators of GABA(A) receptors, including benzodiazepines, mimicked the cell loss, suggesting that potentiating endogenous GABA activity is sufficient to produce neuronal death. Neurosteroid-treated neurons had lower resting intracellular calcium levels than control cells and produced smaller calcium rises in response to depolarizing challenges. Manipulating intracellular calcium levels with chronic elevated extracellular potassium or with the calcium channel agonist Bay K 8644 protected neurons. The results may have implications for the mechanisms of programmed cell death in the developing CNS as well as implications for the long-term consequences of chronic GABAmimetic drug use during development.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗