Biomedical subjects

W Zhang

Publications and source records attributed to W Zhang.

At least 163 records · Page 9Linked to original sources

PubMed2001

Inhibition of pancreatic protein secretion by ghrelin in the rat.

1. The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. 2. In vivo, pancreatic protein output stimulated by CCK-8 (400 pmol kg(-1) h(-1)) was dose-dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg(-1) h(-1)) by 45 +/- 8 and 84 +/- 7 %, respectively. 3. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg(-1) h(-1)) significantly inhibited CCK-stimulated pancreatic protein secretion by 75 +/- 18 %. 4. Infusion of ghrelin (12 nmol kg(-1) h(-1)) abolished pancreatic protein secretion caused by the central vagal stimulant 2-deoxy-D-glucose (75 mg kg(-1)), whereas bethanechol-stimulated pancreatic protein output was inhibited by only 59 +/- 7 %. 5. In vitro, ghrelin (10(-11)-10(-7) M) produced no change in basal amylase release from dispersed, purified acinar cells. Co-incubation of ghrelin (10(-11)-10(-7) M) with CCK-8 (10(-10) M) demonstrated no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, ghrelin (10(-9)-10(-7) M) dose-dependently inhibited amylase release from pancreatic lobules exposed to 75 mM potassium. 6. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.

Animals↗

PubMed2001

Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis.

4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C4 of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 microM(-1) x s(-1)) and 4-HBA-CoA (33 and 11 microM(-1) x s(-1)) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) and 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and mutant dehalogenase are approximately 200 s(-1) while the rate constants for EAr formation are 40 and 0.3 s(-1), respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.

Binding Sites↗

PubMed2001

Escaping the stem cell compartment: sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations.

Once mutated, a single cell must expand into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogen's continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the p53 tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280-320 nm UV radiation (UVB), sunlight's principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets. p53-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 +/- 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare "imprisoned clones" increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a p53-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the p53-mutant keratinocyte to colonize without incurring an additional mutation.

Animals↗

PubMed2001

Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.

Acetylcholine↗

PubMed2001

The pathway of leukemic cell death caused by glucocorticoid receptor fragment 465*.

The truncated glucocorticoid receptor mutant gene 465* codes for a protein that is interrupted by a frame-shift mutation in the second zinc finger of the natural DNA binding domain. Thus, 465* represents the natural amino acid sequence 1-465 followed by 21 novel amino acids starting at position 466. The entire ligand binding domain is missing. Prior studies have shown that transient transfection of the glucocorticoid-resistant leukemic T-cell clone ICR-27 with a plasmid expressing 465* rapidly reduces the number of viable cells. This response does not require activation by a steroid, and a hybrid protein consisting of green fluorescent protein fused to 465* is found primarily in the cytoplasm. In the present study, we present evidence that the decrease in cell number is due to a form of cell death that bears many of the classic characteristics of apoptosis. Expression of the 465* protein can be detected a few hours after electroporation and is followed by activation of caspase-3 as well as reduction of the mitochondrial inner transmembrane potential. The caspase-3 inhibitor ZVAD-fmk blocks 465*-dependent cell death when added acutely after electroporation, but fails to do so later. We conclude that the novel 465* gene causes cell death by apoptosis.

Apoptosis↗

PubMed2001

[Effects of inorganic arsenicals on the methylation of p16 gene CpG islands and the expression of p16 gene in BEP2D cells].

OBJECTIVE: To study the effects of inorganic arsenicals on the methylation of p16 gene CpG islands and the expression of p16 gene in BEP2D cells. METHODS: The methylation of p16 gene CpG islands and the expression of p16 gene in BEP2D cells were measured by methylation-specific PCR (MSP) and RT-PCR methods. RESULTS: (1) The exposure of the BEP2D cells to sodium arsenite (0.016 approximately 2 micromol/L) or high-density sodium arsenate (80 approximately 160 micromol/L), but not low-density sodium arsenate (20 approximately 40 micromol/L), produced significant hypermethylation of p16 gene CpG islands in BEP2D cells. (2) The expressions of p16 gene in the groups of sodium arsenite and sodium arsenate at the test were lower than that of the control group, especially in the groups of sodium arsenite. CONCLUSIONS: Inorganic arsenicals alter the methylation patterns and the expression of p16 gene in BEP2D cells, which suggests that the hypermethylation of p16 gene CpG islands may be one of the mechanisms of carcinogenesis of inorganic arsenicals.

Arsenic↗

PubMed2001

Allosteric interaction of minor groove binding ligands with UL9-DNA complexes.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

Allosteric Regulation↗

PubMed2001

Mimicking oxide surfaces: different types of defects and ligand coordination at well defined positions of a molybdenum oxide based nanocluster.

The mixed valence cluster anion of the compound (NH4)32[MoVI110MoV28O416H6(H2O)58(CH3CO2)6].xH2O 1 (x approximately 250), synthesized under one-pot conditions, contains well-defined different types of defects--missing groups compared to the complete parent (Mo154) type cluster with full D7d symmetry--and acetate ligand coordination; this proves that the giant-wheel type anion can be considered as an object with a variety of nanoscale structural features ("nanostructured landscape") allowing reactions at a variety of well defined centers.

Journal Article↗

PubMed2001

Sources of nonlinearity in cDNA microarray expression measurements.

BACKGROUND: A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides. RESULTS: We uncovered two sources of nonlinearity. The first, which led to discrepancies in analysis affecting the fluorescent signals, was signal quenching associated with excessive dye concentrations. The second, affecting the radioactive signals, was a nonlinear transformation of the raw data introduced by the scanner. Correction for this transformation was made by some, but not all, image-quantification software packages. CONCLUSIONS: The second type of nonlinearity is more troublesome, because it could not have been predicted a priori. Both types of nonlinearities were detected by simple dilution series, which we recommend as a quality-control step.

Calibration↗

PubMed2001

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8(+) T cells.

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR alpha- or beta-chain of a VSV8 (unmodified)/H-2K(b)-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3alpha and beta loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3beta residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jbeta region, while the Jalpha region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3alpha and beta loops to allow interaction with a haptenated peptide.

Animals↗

PubMed2001

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I.

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.

Amino Acid Sequence↗

PubMed2001

The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.

We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.

Animals↗

PubMed2001

Overexpression of the thymosin beta-10 gene in human ovarian cancer cells disrupts F-actin stress fiber and leads to apoptosis.

To understand the molecular changes during ovarian cancer development, we profiled differentially expressed genes in five paired normal and cancerous ovarian tissues. Among the genes that showed differential expression, thymosin beta-10 expression was decreased in four of five cancer tissues. The decreased level of expression was confirmed by Northern. To investigate the gene's functional role in ovarian cancers, we constructed an adenovirus vector expressing thymosin beta-10 and used it to infect ovarian cancer cell lines PA-I and SKOV3. The infected cells showed disrupted F-actin stress fibers, markedly decreased cell growth, and a high rate of apoptosis. Thus, because loss of thymosin beta-10 expression may contribute to the development of a subset of ovarian cancers, restoration of thymosin beta-10 expression may be a new strategy for ovarian cancer treatment.

Actins↗

PubMed2001

Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells.

Although the nongenomic effects of glucocorticoids have been well acknowledged, its precise intracellular signal transduction pathway remains to be elucidated. The present study using Western immunoblot and protein kinase activity assay, for the first time, showed that corticosterone (B) can induce a rapid activation of Erk1/2 mitogen-activated protein kinase (MAPK) in PC12 cells. The dose-response curve was bell shaped, with the maximal activation at 10(-9) M in 15 min. The results from immunofluorescence staining also revealed that the activated Erk1/2 MAPK was translocated from cytoplasm to nucleus of PC12 cells in 15 min. Activation of Erk1/2 MAPK by B was apparently not mediated by the classical cytosolic steroid receptors, for B-BSA can induce the phosphorylation of Erk1/2 MAPK, but the antagonist (RU38486) cannot block the phosphorylation of Erk1/2 MAPK induced by B. Phosphorylation of Erk1/2 MAPK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway did not involve the tyrosine kinase activity. On the other hand, protein kinase C activator (PMA) can activate and protein kinase C inhibitor (Gö6976) can block the activation of Erk1/2 MAPK induced by B. Taken together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate Erk1/2 MAPK through protein kinase C alpha in PC12 cells.

Active Transport, Cell Nucleus↗

PubMed2001

Inflammatory gene transcription in human astrocytes exposed to hypoxia: roles of the nuclear factor-kappaB and autocrine stimulation.

Mechanisms of hypoxia-induced activation of nuclear factor-kappaB (NF-kappaB) and inflammatory genes were investigated in fetal human astrocytes in culture. Astrocytes were subjected to interleukin-1beta (IL-1beta; 50-100 u/ml; 4-24 h), or to a 4-h hypoxia (<2% O2) followed by a 4-24-h reoxygenation. NF-kappaB binding and transcriptional activity increased up to 10-fold in astrocytes exposed to IL-1beta, and up to 3-fold in astrocytes subjected to hypoxia followed by reoxygenation. Both IL-1beta- mRNAs and proteins hypoxia-induced NF-kappaB activation were blocked by the proteasome inhibitor, MG-132. MG-132 inhibited IL-1beta-induced up-regulation of IL-1beta and IL-8 mRNA and protein but increased hypoxia-stimulated expression/release of IL-1beta and IL-8. IL-1 receptor antagonist (IL-1Ra) blocked both hypoxic astrocyte-conditioned media-induced NF-kappaB activation and the expression/release of IL-1beta and IL-8. Astrocytes subjected to hypoxia in the presence of IL-1Ra failed to activate NF-kappaB, but expressed elevated levels of IL-1beta and IL-8. The data suggest that hypoxia/reoxygenation-induced up-regulation of IL-1beta and IL-8 in human astrocytes has two components, a NF-kappaB independent up-regulation during hypoxia, followed by amplification through autocrine IL-1beta-induced NF-kappaB activation during reoxygenation.

Astrocytes↗

PubMed2001

Pressure-driven laminar flow in tangential microchannels: an elastomeric microfluidic switch.

This paper describes laminar fluid flow through a three-dimensional elastomeric microstructure formed by two microfluidic channels, fabricated in layers that contact one another face-to-face (typically at a 90 degree angle), with the fluid flows in tangential contact. There are two ways to control fluid flow through these tangentially connected microchannels. First, the flow profiles through the crossings are sensitive to the aspect ratio of the channels; the flow can be controlled by applying external pressure and changing this aspect ratio. Second, the flow direction of an individual laminar stream in multiphase laminar flow depends on the lateral position of the stream within the channel; this position can be controlled by injecting additional streams of fluid into the channel. We describe two microfluidic switches based on these two ways for controlling fluid flow through tangential microchannels and present theoretical arguments that explain the observed dependence of the flow profiles on the aspect ratio of the channels.

Journal Article↗

PubMed2001

Orthogonal, convergent syntheses of dendrimers based on melamine with one or two unique surface sites for manipulation.

An orthogonal, convergent route for the introduction of substoichiometric numbers of latent surface sites into dendrimers based on melamine is used to prepare targets that display one or two Boc-protected amines on the periphery. Asymmetry is the result of the stepwise incorporation of functionalized and unfunctionalized dendrons onto the triazine cores, a highly selective process due to the different reactivities of the substituted triazines. The routes to the dendrons rely on iterative reactions of the growing dendrons with triazine cores and diamine linkers. p-Aminobenzylamine is used as a linking group to avoid functional group interconversions or protecting group manipulations. Addition of the benzylamine group to the monochlorotriazine of the dendron proceeds cleanly leaving a less reactive aniline for subsequent reaction with trichlorotriazine. The routes to these targets proceed in 5 or 6 linear steps (11 or 12 total steps) in 40% overall yield. The unique surface sites can be deprotected and subjected to additional chemistries. Reaction of the monofunctionalized dendrimer with trichlorotriazine yields the desired dimer, a molecule whose increased size is evident from light scattering and tapping mode atomic force microscopy, and corroborated with computation.

Journal Article↗

PubMed2001

Ferromagnetism in a lattice of Bose-Einstein condensates.

We show that an ensemble of spinor Bose-Einstein condensates confined in a one-dimensional optical lattice can undergo a ferromagnetic phase transition and spontaneous magnetization arises due to the magnetic dipole-dipole interaction. This phenomenon is analogous to ferromagnetism in solid state physics, but occurs with bosons instead of fermions.

Journal Article↗