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Biomedical subjects

W Zhang

Publications and source records attributed to W Zhang.

At least 379 records · Page 21Linked to original sources
PubMed2000

High-efficiency on-line concentration technique of capillary electrochromatography.

With the coexistence of the mobile phase, the stationary phase, and the electric field in capillary electrochromatography, the chromatographic zone-sharpening effect and field-enhanced sample-stacking technique were utilized to improve detection sensitivity. By the former means, with less organic modifier in the sample solution compared to that in the mobile phase, the concentration factors of neutral solutes benzoin and mephenytion were 134 and 219, respectively. Through the latter one, without electrolyte in the sample solution, the detection sensitivity of propatenene with positive charge was improved by 1600 times. While with the combination of these two methods, improvement of over 17,000 times for the sensitivity of propatenene was obtained. By the combined means, the analysis of basic pharmaceutical compounds at concentrations of nanograms per milliliter by UV detection was realized. In addition, parameters that might affect the efficiency of on-line concentration were studied and equations that described the on-line concentration procedure were deduced.

Journal Article↗
PubMed2000

In vitro and in vivo functional analysis of human T cell lymphotropic virus type 1 pX open reading frames I and II.

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.

Animals↗
PubMed2000

Clotrimazole inhibits lung fibroblast proliferation in vitro: implications for use in the prevention and treatment of obliterative bronchiolitis after lung transplantation.

BACKGROUND: Immunosuppressive therapy has limited activity against the mesenchymal cell proliferation of obliterative bronchiolitis. Clotrimazole (CLT) has been shown to inhibit proliferation in normal and cancer cell lines. Here we investigate whether CLT inhibits the proliferation of lung mesenchymal cells. METHODS: Proliferation of human lung fibroblasts (MRC-5) in the presence of CLT was determined by [3H]thymidine incorporation. Messenger ribonucleic acid (mRNA) expression of platelet-derived growth factor (PDGF)-B and transforming growth factor (TGF)-beta after treatment with CLT was measured by reverse transcriptase-polymerase chain reaction. RESULTS: Treatment of MRC-5 cells with CLT resulted in a significant reduction in proliferation as assessed by DNA incorporation and cell counts compared with dimethylsulfoxide alone. There was no cytotoxic effect associated with CLT treatment. Reverse transcriptase-polymerase chain reaction demonstrated a marked decrease in PDGF-B and TGF-beta mRNA levels in cells treated with CLT compared with those treated with dimethylsulfoxide. CONCLUSION: CLT inhibits proliferation of human lung fibroblasts. This inhibitory effect is associated with decreased levels of PDGF-B and TGF-beta mRNA expression and may have value in the prevention and treatment of obliterative bronchiolitis.

Bronchiolitis Obliterans↗
PubMed2000

Reassembly of phospholipase C-beta2 from separated domains: analysis of basal and G protein-stimulated activities.

Phosphatidylinositol-specific phospholipase C-betas (PLC-betas) are the only PLC isoforms that are regulated by G protein subunits. To further understand the regulation of PLC-beta(2) by G proteins and the functional roles of PLC-beta(2) structural domains, we tested whether the separately expressed amino and carboxyl halves of PLC-beta(2) could associate to form catalytically active enzymes as two polypeptides, and we explored how the complexes thus formed would be regulated by G protein betagamma subunits (Gbetagamma). We expressed cDNA constructs encoding PLC-beta(2) fragments of different lengths in COS-7 cells and demonstrated by coimmunoprecipitation that the coexpressed fragments could assemble and functionally reconstitute an active PLC-beta(2). The pleckstrin homology domain of PLC-beta(2) was required for its targeting to the membrane and for substrate hydrolysis. Reconstituted enzymes that contained the linker region that joins the two catalytic domains were as active or more active than the wild-type PLC-beta(2). When the linker region was removed, basal PLC-beta(2) enzymatic activity was increased further, suggesting that the linker region exerts an inhibitory effect on basal PLC-beta(2) activity. The reconstituted enzymes, like wild-type PLC-beta(2), were activated by Gbetagamma; when the C-terminal region was present in these constructs, they were also activated by Galpha(q). Gbetagamma and Galpha(q) activated these PLC-beta(2) constructs equally in the presence or absence of the linker region. We conclude that the linker region is an inhibitory element in PLC-beta(2) and that Gbetagamma and Galpha(q) do not stimulate PLC-beta(2) through easing the inhibition of enzymatic activity by the linker region.

Catalytic Domain↗
PubMed2000

Nonstoichiometric interfaces and Al2O3 adhesion with Al and Ag.

We have determined the relative stability of stoichiometric, oxygen-rich, and aluminum-rich Al/Al 2O (3) and Ag/Al 2O (3) interfaces from first principles. Stable structures vary significantly with oxygen chemical potentials. Computed works of adhesion agree reasonably well with sessile drop experimental values, including correlation with measured oxygen chemisorption effects on Ag. The ordering of predicted bond energies of the interfaces, ceramics, and metals seems consistent with monotonic and fatigue fracture experiments.

Journal Article↗
PubMed2000

The spinal 5-HT system contributes to the generation of fictive locomotion in lamprey.

Activation of NMDA receptors evokes sustained fictive locomotion in the isolated spinal cord of the sea lamprey Petromyzon marinus (P. marinus), but in the river lamprey Lampetra fluviatilis (L. fluviatilis) the ventral root activity is often irregular. A previous study showed that the number of 5-HT immunoreactive fibres, neurones and varicosities are much lower in the spinal cord of L. fluviatilis than in P. marinus. To further analyse the underlying mechanisms, the present study investigated the role of the 5-HT system in stabilising fictive locomotion. In P. marinus a blockade of 5-HT1A receptors by spiperone reversibly increased the frequency and the coefficient of variation. This implies that there is an endogenous release of 5-HT during fictive locomotion that is important for the generation of locomotor activity. In L. fluviatilis bath applied NMDA or D-glutamate evoked in most cases irregular activity. An addition of 5-HT (0.5-2 microM) rapidly stabilised the burst generation and led to a sustained fictive locomotion. In a split-bath configuration, NMDA administered to the rostral part of the spinal cord in P. marinus evoked fictive locomotion in both the rostral part and the first few segments of the caudal part. When spiperone was added to the caudal part, the burst activity changed into tonic activity within 10 min. Taken together, these results indicate that activity in the intrinsic 5-HT system in the lamprey spinal locomotor network contributes significantly to the rhythm generation. The quantitative differences with regard to the 5-HT plexus between P. marinus and L. fluviatilis may account for the observed discrepancy between the two species.

Animals↗
PubMed2000

Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A.

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.

Botulinum Toxins, Type A↗
PubMed2000

Interaction of CR6 (GADD45gamma ) with proliferating cell nuclear antigen impedes negative growth control.

GADD45, MyD118, and CR6 (also termed GADD45alpha, beta, and gamma) comprise a family of genes that encode for related proteins playing important roles in negative growth control, including growth suppression. Data accumulated suggest that MyD118/GADD45/CR6 serve similar but not identical functions along different apoptotic and growth suppressive pathways. It is also apparent that individual members of the MyD118/GADD45/CR6 family are differentially induced by a variety of genetic and environmental stress agents. The MyD118, CR6, and GADD45 proteins were shown to predominantly localize within the cell nucleus. Recently, we have shown that both MyD118 and GADD45 interact with proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA replication, DNA repair, and cell cycle progression, as well as with the universal cyclin-dependent kinase inhibitor p21. In this work we show that also CR6 interacts with PCNA and p21. Moreover, it is shown that CR6 interacts with PCNA via a domain that also mediates interaction of both GADD45 and MyD118 with PCNA. Importantly, evidence has been obtained that interaction of CR6 with PCNA impedes the function of this protein in negative growth control, similar to observations reported for MyD118 and GADD45.

Antigens, Differentiation↗
PubMed2000

Nutrition, HIV, and drug abuse: the molecular basis of a unique role for selenium.

HIV-infected injection drug users (IDUs) often suffer from serious nutritional deficiencies. This is a concern because plasma levels of micronutrients such as vitamin B12, zinc, and selenium have been correlated with mortality risk in HIV-positive populations. Injection drug use also increases lipid peroxidation and other indicators of oxidative stress, which, combined with antioxidant deficiencies, can stimulate HIV-1 replication through activation of NF-kappaB transcription factors, while weakening immune defenses. As detailed herein, these prooxidant stimuli can also increase the pathogenic effects of HIV-1 by another mechanism, involving viral selenoproteins. Overlapping the envelope coding region, HIV-1 encodes a truncated glutathione peroxidase (GPx) gene (see #6 in reference list). Sequence analysis and molecular modeling show that this viral GPx (vGPx) module has highly significant structural similarity to known mammalian GPx, with conservation of the catalytic triad of selenocysteine (Sec), glutamine, and tryptophan. In addition to other functions, HIV-1 vGPx may serve as a negative regulator of proviral transcription, by acting as an NF-kappaB inhibitor (a known property of cellular GPx). Another potential selenoprotein coding function of HIV-1 is associated with the 3' end of the nef gene, which terminates in a conserved UGA (potential Sec) codon in the context of a sequence (Cys-Sec) identical to the C-terminal redox center of thioredoxin reductase, another cellular regulator of NF-kappaB. Thus, in combination with known cellular mechanisms involving Se, viral selenoproteins may represent a unique mechanism by which HIV-1 monitors and exploits an essential micronutrient to optimize its replication relative to the host.

Amino Acid Sequence↗
PubMed2000

Tamoxifen-induced enhancement of calcium signaling in glioma and MCF-7 breast cancer cells.

The antiestrogen tamoxifen is commonly used to treat breast cancer, but it also has therapeutic activity in several other types of cancer. Many of these tumors, including malignant gliomas, are estrogen receptor negative. Nonetheless, high concentrations of tamoxifen can directly reduce cell proliferation in some of these tumors and induce apoptosis. In this study, the role of tamoxifen in calcium signaling and calcium-induced cell death was studied in both malignant glioma cell lines and MCF-7 breast cancer cells. Tamoxifen potently increased the spatial expansion of calcium waves by 30-150% while significantly enhancing and prolonging agonist-induced calcium elevations. Furthermore, tamoxifen pretreatment accelerated calcium ionophore-induced death by more than 20 min, suggesting that tamoxifen lowered cellular resistance to calcium loads. In contrast to its potentiating of calcium signaling in tumors, tamoxifen had no significant effect on calcium signaling in cultures of primary astrocytes from either human or rat brain. This study demonstrates the existence of calcium signaling in breast cancer and glioma cells and identifies tamoxifen as a potential modulator of tumor-associated calcium signaling.

Adenosine Triphosphate↗
PubMed2000

Inhibiting proteasomes in human HepG2 and LNCaP cells increases endogenous androgen receptor levels.

Treating HepG2 cells with MG132 for 4 h to inhibit proteasomal activity increased androgen receptor immunoreactivity in two major bands with molecular weights of 102 and 110 kDa by 77% each (P < 0. 05). MG132 treatment also increased the overall level of polyubiquitinated proteins between 66 and 220 kDa by 140% (P < 0.05). Antiubiquitin immunoreactivity comigrating with the androgen receptor bands was also increased by MG132 treatment. Two other proteasome inhibitors, lactacystin and epoxomycin, caused similar increases in the androgen receptor in HepG2 cells. Proteosome-inhibition studies conducted in LNCaP cells also showed that the two major androgen receptor bands with molecular weights of 102 and 110 kDa were increased by 85 and 115%, respectively (P < 0. 05 for both) by MG132 treatment. Overall levels of polyubiquitinated proteins with molecular weights between 66 and 220 kDa increased 365%. Ubiquitin immunoreactivity comigrating with the androgen receptor bands was also significantly increased. Thus inhibiting proteasomes in two human androgen-responsive cell lines increases endogenous androgen receptor levels as well as androgen receptor-associated ubiquitin-modified immunoreactivity. The regulation of steady-state levels of endogenous androgen receptor by proteasomal degradation could be involved in its rapid turnover in the absence of ligand and would provide a mechanism for limiting androgen responses. A PEST sequence similar to one in the vitamin D receptor is present in the hinge region of all known mammalian androgen receptors, suggesting that it may function in proteasome-mediated androgen receptor turnover.

Cell Line↗
PubMed2000

Determination of pharmacokinetics of 8-chloroadenosine and its two major metabolites in dogs by high-performance liquid chromatography.

High-performance liquid chromatography was used to measure concentration of 8-chloroadenosine (8-Cl-A) and its two major metabolites 8-chloroadenine (8-Cl-Ad) and 8-chloroinosine (8-Cl-I), and their pharmacokinetics in dogs. 8-Cl-A and its metabolites in serum were treated by deproteinization with acetonitrile, then organic impurities were extracted with dichloromethane, followed by centrifuged and direct injection of the supernatant into the liquid chromatograph. After intravenous injection of 8-Cl-A (30 mg/kg), the parent drug and 8-Cl-I were not detected, but the other metabolite, 8-Cl-Ad, was found at a high concentration for 240 min in dog serum. The main pharmacokinetic parameters of 8-Cl-Ad, t1/2beta and AUC, were 69.30 min and 580 microg min/ml. Our finding indicates that in dogs 8-Cl-A is rapidly metabolized and forms its major metabolites, 8-Cl-Ad and 8-Cl-I. 8-Cl-Ad appeared in many tissues, but 8-Cl-A and 8-Cl-I did not. The concentration of 8-Cl-Ad in dog tissues was highest in the liver and spleen, intermediate in the kidney, intestine, and lowest in the bone marrow, heart, and lungs. However, it was not detected in some liposoluble tissues such as the testes, brain, or uterus. Our study provides useful information for clinical experiment.

2-Chloroadenosine↗
PubMed2000

Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation.

Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCgamma-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCgamma-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Shear modulation force microscopy study of near surface glass transition temperatures

We report results of glass transition (T(g)) measurements for polymer thin films using atomic force microscopy (AFM). The AFM mode, shear modulation force microscopy (SMFM), involves measuring the temperature-dependent shear force on a tip modulated parallel to the sample surface. Using this method we have measured the surface T(g) of thin (17-500 nm) polymer films and found that T(g) is independent of film thickness (t>17 nm), strength of substrate interactions, or even presence of substrate.

Journal Article↗
PubMed2000

Partial deletion of a loop region in the high affinity K+ transporter HKT1 changes ionic permeability leading to increased salt tolerance.

HKT1 is a high affinity K(+) transporter protein that is a member of a large superfamily of transporters found in plants, bacteria, and fungi. These transporters are primarily involved in K(+) uptake and are energized by Na(+) or H(+). HKT1 is energized by Na(+) but also mediates low affinity Na(+) uptake and may therefore be a pathway for Na(+) uptake, which is toxic to plants. The aim of this study was to identify regions of HKT1 that are involved in K(+)/Na(+) selectivity and alter the amino acid composition in those regions to increase the ionic selectivity of the transporter. A highly charged loop was identified, and two deletions were created that resulted in the removal of charged and uncharged amino acids. The functional changes caused by the deletions were studied in yeast and Xenopus oocytes. The deletions improved the K(+)/Na(+) selectivity of the transporter and increased the salt tolerance of the yeast cells in which they were expressed. In light of recent structural models of members of this symporter superfamily, it was necessary to determine the orientation of this highly charged loop. Introduction of an epitope tag allowed us to demonstrate that this loop faces the outside of the membrane where it is likely to facilitate the interaction with cations such as K(+) and Na(+). This study has identified an important structural feature in HKT1 that in part determines its K(+)/Na(+) selectivity. Understanding the structural basis of the functional characteristics in transporters such as HKT1 may have important implications for increasing the salt tolerance of higher plants.

Amino Acid Sequence↗