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X Wang

Publications and source records attributed to X Wang.

At least 325 records · Page 18Linked to original sources
PubMed2001

Allosteric regulation of CCR5 by guanine nucleotides and HIV-1 envelope.

The chemokine receptor CCR5 is the principal coreceptor for R5 (macrophage-tropic) strains of HIV-1. CCR5 uses G-proteins as transducing elements. Here we report the biochemical consequences of the interaction between CCR5 and G-proteins. Macrophage inflammatory protein-1beta (MIP-1beta) binding to CCR5 was potently and specifically inhibited by guanine nucleotides. The molecular mechanism of this inhibitory effect was shown to be a dose-dependent reduction in MIP-1beta receptors. We also show that the MIP-1beta binding site is allosterically regulated by monovalent cations and that binding of this endogenous agonist is highly temperature sensitive and dependent on divalent cations, characteristic of a G-protein-coupled receptor(GPCR). HIV-1 envelope glycoprotein decreased the affinity of CCR5 for MIP-1beta but also altered the kinetics of MIP-1beta binding to CCR5, proving that it interacts with a distinct, but allosterically coupled binding site. The findings described herein contribute to our understanding of how CCR5 interacts with chemokines and HIV-1 envelope.

Allosteric Regulation↗
PubMed2001

Quantitative quality control in microarray image processing and data acquisition.

A new integrated image analysis package with quantitative quality control schemes is described for cDNA microarray technology. The package employs an iterative algorithm that utilizes both intensity characteristics and spatial information of the spots on a microarray image for signal-background segmentation and defines five quality scores for each spot to record irregularities in spot intensity, size and background noise levels. A composite score q(com) is defined based on these individual scores to give an overall assessment of spot quality. Using q(com) we demonstrate that the inherent variability in intensity ratio measurements is closely correlated with spot quality, namely spots with higher quality give less variable measurements and vice versa. In addition, gauging data by q(com) can improve data reliability dramatically and efficiently. We further show that the variability in ratio measurements drops exponentially with increasing q(com) and, for the majority of spots at the high quality end, this improvement is mainly due to an improvement in correlation between the two dyes. Based on these studies, we discuss the potential of quantitative quality control for microarray data and the possibility of filtering and normalizing microarray data using a quality metrics-dependent scheme.

Algorithms↗
PubMed2001

Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals.

We describe imaging the luminance of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in several mouse organs including the peritoneal cavity, stomach, small intestine, and colon. Instantaneous real-time images of the infectious process were acquired by using a color charge-coupled device video camera by simply illuminating mice at 490 nm. Most techniques for imaging the interior of intact animals may require the administration of exogenous substrates, anesthesia, or contrasting substances and require very long data collection times. In contrast, the whole-body fluorescence imaging described here is fast and requires no extraneous agents. The progress of Escherichia coli-GFP through the mouse gastrointestinal tract after gavage was followed in real-time by whole-body imaging. Bacteria, seen first in the stomach, migrated into the small intestine and subsequently into the colon, an observation confirmed by intravital direct imaging. An i.p. infection was established by i.p. injection of E. coli-GFP. The development of infection over 6 h and its regression after kanamycin treatment were visualized by whole-body imaging. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, and the spatial migration of the infectious agents.

Administration, Oral↗
PubMed2001

Multiple transcription-factor genes are early targets of phytochrome A signaling.

The phytochrome family of sensory photoreceptors directs adaptational changes in gene expression in response to environmental light signals. Using oligonucleotide microarrays to measure expression profiles in wild-type and phytochrome A (phyA) null-mutant Arabidopsis seedlings, we have shown that 10% of the genes represented on the array are regulated by phyA in response to a continuous far-red light signal. Strikingly, 44% of the genes responding to the signal within 1 h are predicted to encode multiple classes of transcriptional regulators. Together with previous data, this observation suggests that phyA may regulate seedling photomorphogenesis by direct targeting of light signals to the promoters of genes encoding a master set of diverse transcriptional regulators, responsible in turn for orchestrating the expression of multiple downstream target genes in various branches of a phyA-regulated transcriptional network.

Arabidopsis↗
PubMed2001

Recombinant vascular endothelial growth factor secreted from tissue-engineered bioartificial muscles promotes localized angiogenesis.

BACKGROUND: Therapeutic angiogenesis by the administration of recombinant vascular endothelial growth factor (rVEGF) is a novel strategy for the treatment of ischemic disorders. rVEGF has been delivered as a protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. In the present study, we examined a new method for delivery of rVEGF using implantable bioartificial muscle (BAM) tissues made from genetically modified primary skeletal myoblasts. Our goal was to determine whether the rVEGF delivered by this technique promoted controlled angiogenesis in nonischemic and/or ischemic adult mouse tissue. METHODS AND RESULTS: Primary adult mouse myoblasts were retrovirally transduced to secrete human or mouse rVEGF and tissue-engineered into implantable 1x10 to 15-mm BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 290 to 511 ng of bioactive mouse or human VEGF/BAM per day. rVEGF BAMs implanted subcutaneously into syngeneic mice caused a 30-fold increase in the number of CD31-positive capillary cells within the BAM by 1 week compared with control BAMs. Implantation of rVEGF-secreting BAMs into ischemic hindlimbs resulted in a 2- to 3-fold increase in capillary density of neighboring host muscle by 1 week and out to 4 weeks with no evidence of hemangioma formation. CONCLUSIONS: Local delivery of rVEGF from BAMs rapidly increases capillary density both within the BAM itself and in adjacent ischemic muscle tissue. Genetically engineered muscle tissue provides a method for therapeutic protein delivery in a dose-regulated fashion.

Animals↗
PubMed2001

Shedding of membrane type matrix metalloproteinase 5 by a furin-type convertase: a potential mechanism for down-regulation.

The shedding of membrane-associated proteins has been recognized as a regulatory mechanism to either up-regulate or down-regulate cellular functions by releasing membrane-bound growth factors or removing ectodomains of adhesion molecules and receptors. We have reported previously that the ectoenzyme of membrane type matrix metalloproteinase 5 (MT5-MMP) is shed into extracellular milieu (Pei, D. (1999) J. Biol. Chem. 274, 8925-8932). Here we present evidence that MT5-MMP is shed by a furin-type convertase activity in the trans-Golgi network. Among proteinase inhibitors screened, only decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a known inhibitor for furin-type convertases, blocked the shedding of MT5-MMP in a dose-dependent manner. As expected, decanoyl-Arg-Val-Lys-Arg-chloromethylketone also prevented the activation of MT5-MMP, raising the possibility that the observed shedding could be autolytic. However, an active site mutant devoid of any catalytic activity, is also shed efficiently, thus ruling out the autolytic pathway. The shedding cleavage was subsequently mapped to the stem region immediately upstream of the transmembrane domain, where a cryptic furin recognition site, (545)RRKERR, was recognized. Indeed, MT5-MMP and furin are co-localized in the trans-Golgi network and the shed species could be detected inside the cells. Furthermore, deletion mutations removing this cryptic site prevented MT5-MMP from shedding. The resulting mutants express a gain-of-function phenotype by mediating more robust activation of proMMP-2 than the wild type molecule. Thus, shedding provides a potential mechanism to regulate proteolytic activity of membrane-bound MMPs.

Amino Acid Sequence↗
PubMed2001

Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation.

Vascular endothelial cells (ECs) in vivo are subject to different flow conditions due to the variation in vessel geometry. The aim of this study is to elucidate the effects of different flow conditions on EC monolayer migration into a mechanically denuded zone and their underlying mechanisms. Both laminar and disturbed flows significantly enhanced EC migration. EC migration speed was the fastest under laminar flow, which preferentially promoted directional EC migration from the upstream side of the wounded monolayer. C3 exoenzyme (a Rho inhibitor) inhibited EC migration under static and flow conditions, and markedly reduced the effects of flow on EC migration. These results indicate that flow promotes EC migration through the Rho signaling pathway. Genistein (a tyrosine kinase inhibitor) selectively retarded EC migration under disturbed flow, suggesting that tyrosine phosphorylation may play a role in EC migration under disturbed flow. This study has demonstrated that different flow patterns differentially affect EC monolayer migration into the denuded zone involving multiple mechanisms.

ADP Ribose Transferases↗
PubMed2001

Genetic locus in mice that blocks development of atherosclerosis despite extreme hyperlipidemia.

The genes contributing to the common forms of atherosclerosis are largely unknown. One approach to dissecting complex traits such as atherosclerosis is to use animal models, such as the mouse, to map and characterize the genetic loci involved. We now report the identification of a locus for aortic lesion formation on mouse chromosome 6 that exhibits a highly significant lod score of 6.7 in a genetic cross between the susceptible strain, C57BL/6J, and the resistant strain, CAST/Ei. The locus was confirmed by constructing a congenic strain in which the chromosome 6 segment from CAST/Ei was transferred to a C57BL/6J background in a series of backcrosses. The congenic strain was almost completely resistant to diet-induced atherosclerosis. The chromosome 6 segment was also transferred onto the background of an LDL receptor-null mutation and resulted again in almost complete resistance to aortic lesion formation. This locus also influenced insulin levels but did not affect plasma lipoprotein levels, blood pressure, or body fat. The chromosome 6 gene, which we call Artles (for arterial lesions), did not affect endothelial cell responses to oxidized LDL, but lesion formation was partially reduced through bone marrow transplantation. The locus contains the candidate gene peroxisome proliferator-activated receptor-gamma, and the congenic mice exhibited significantly reduced expression of peroxisome proliferator-activated receptor-gamma.

Animals↗
PubMed2001

M cell-targeted DNA vaccination.

DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein final sigma1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein final sigma1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cell-mediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

Animals↗
PubMed2001

Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified as a common cellular receptor for both viruses, but its biological and pathogenic relevance is uncertain. Knowledge of CAR localization in the human cardiovascular system is limited but important with respect to CAR-dependent viral infections and gene transfer using CAR-dependent viral vectors. METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM]) and normal donor hearts (n=7) were investigated for the expression levels and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3) and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in normal and non-DCM hearts, whereas strong CAR signals occurred at the intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%); these strong signals colocalized with both integrins. In all hearts, CAR was detectable in subendothelial layers of the vessel wall, but not on the luminal endothelial surface, and on interstitial cells. Human CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell contacts, which resembled CAR localization in DCM hearts and resulted in 15-fold increased adenovirus uptake. CONCLUSIONS: Low hCAR abundance may render normal human myocardium resistant to CAR-dependent viruses, whereas re-expression of hCAR, such as that observed in DCM, may be a key determinant of cardiac susceptibility to viral infections. Asymmetric expression of hCAR in the vessel wall may be an important determinant of adenovirus tropism in humans. hCAR subcellular localization in human myocardium and hCAR targeting to cell-cell contacts in cardiomyocyte cultures suggest that hCAR may play a role in cell-cell contact formation.

Adenoviridae↗
PubMed2001

Characterization of a p75(NTR) apoptotic signaling pathway using a novel cellular model.

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.

Apoptosis↗
PubMed2001

[Process of recovery of peristaltic function at anastomotic stoma after dismembering pyeloplasty in rabbits].

OBJECTIVE: To study the process of recovery of peristaltic function at anastomotic stoma after dismembering pyeloplasty in rabbits. METHODS: An animal model of obstructive hydronephrosis was made with 32 New Zealand rabbits. These rabbits were divided into four groups to be examined within the first, second, third, and fourth week respectively after a dismembering pyeloplasty was performed on them, eight rabbits in each group. Thirty-two normal New Zealand rabbits were divided into four corresponding control groups as controls. Pressures of the renal pelvis and upper ureter were measured, electromyogram was recorded, and histological examination was made to the tissue of anastomotic stoma before and after the operation. RESULTS: In the normal control rabbits, the resting pressure was (10.8 +/- 1.3) mm Hg, contraction amplitude was (1.6 +/- 0.4) mm Hg, pulse amplitude of EMG was (0.39 +/- 0.10) mV, and pulse frequency was (5.4 +/- 1.0)/min in the renal pelvis; and the resting pressure was (11.7 +/- 1.7) mm Hg, contraction amplitude was (1.8 +/- 0.5) mm Hg, pulse amplitude of EMG was (0.38 +/- 0.08) mV, pulse frequency was (5.3 +/- 0.9)/min in the upper ureter. Within the first week after operation, the resting pressure of renal pelvis was (14.4 +/- 1.3) mm Hg, significant higher than normal (P < 0.05), the contraction amplitude of renal pelvis was (1.1 +/- 0.4) mm Hg, lower than normal (P < 0.05); the pulse amplitudes of EMG in renal pelvis (0.19 +/- 0.05) mV and in upper ureter (0.26 +/- 0.04) mV were lower than normal (P < 0.05). A thin layer of mucosa was seen covering the anastomotic stoma. In the second week after operation, no statistical difference was observed in resting pressure, contraction amplitude, amplitude of EMG, and frequency of EMG in renal pelvis between the experimental rabbits and the controls (P > 0.05). Newborn smooth muscle cells were seen running through the anastomotic stoma, and close contact was observed among the cells. Within the third week after operation, the pressures and EMG in renal pelvis and in upper ureter were not significantly different from those in normal rabbits (P > 0.05). Nerve endings could be seen near the anastomotic stoma. Within the fourth week after operation, newborn muscle cells became mature and arranged regularly, and contacted each other closely. CONCLUSION: Within the first week after the dismembering pyeloplasty, no peristalsis runs through the anastomotic stoma, and the renal pelvic resting pressure increases. Within the second week after the operation, the newborn smooth muscle cells at the anastomotic stoma begin to exercise their contractive and conductive functions. Peristalsis begins to run through the anastomotic stoma. Within the third after the operation, the peristaltic function basically recovers to normal.

Anastomosis, Surgical↗
PubMed2001

Expression of R120G-alphaB-crystallin causes aberrant desmin and alphaB-crystallin aggregation and cardiomyopathy in mice.

Upregulation of alphaB-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased approximately 104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy.

Animals↗
PubMed2001

Mutations in the GDD motif of rubella virus putative RNA-dependent RNA polymerase affect virus replication.

Rubella virus (RV) nonstructural proteins are translated as a p200 polyprotein that undergoes proteolytic cleavage into p150 and p90. From conserved amino acid sequence motifs in polypeptides, p90 has been proposed to be the RV RNA-dependent RNA polymerase (RdRp). To test whether the conserved GDD motif is involved in RdRp catalytic activity, three different alanine substitutions were introduced into it. Substitution of glycine by alanine (G1966A) resulted in impaired virus infectivity. Alteration of either aspartate residue completely abolished virus replication. A fully infectious variant was isolated from the G1966A mutant. Sequencing analysis showed that the alanine residue substituted in G1966A mutant had reverted to glycine in this variant. Complementation experiments were carried out to rescue the replication-defective RNA carrying G1966A, D1967A, or D1968A mutations. The defective RNA with G1966A mutation in p90 replicated efficiently when the helper genome that supplied a wild-type p90 was provided in trans. However, the replication-defective RNA with D1967A or D1968A was not rescued by supplementation of p90 in trans. Our studies support the idea that the GDD motif is critical for RV replication and p90 function as RV RdRp.

Amino Acid Substitution↗
PubMed2001

Mitochondrial endonuclease G is important for apoptosis in C. elegans.

Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

Amino Acid Sequence↗
PubMed2001

Endonuclease G is an apoptotic DNase when released from mitochondria.

Nucleosomal fragmentation of DNA is a hallmark of apoptosis (programmed cell death), and results from the activation of nucleases in cells undergoing apoptosis. One such nuclease, DNA fragmentation factor (DFF, a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD)), is capable of inducing DNA fragmentation and chromatin condensation after cleavage by caspase-3 (refs 2,3,4). However, although transgenic mice lacking DFF45 or its caspase cleavage site have significantly reduced DNA fragmentation, these mice still show residual DNA fragmentation and are phenotypically normal. Here we report the identification and characterization of another nuclease that is specifically activated by apoptotic stimuli and is able to induce nucleosomal fragmentation of DNA in fibroblast cells from embryonic mice lacking DFF. This nuclease is endonuclease G (endoG), a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis. Once released from mitochondria, endoG cleaves chromatin DNA into nucleosomal fragments independently of caspases. Therefore, endoG represents a caspase-independent apoptotic pathway initiated from the mitochondria.

Animals↗