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Biomedical subjects

F Wang

Publications and source records attributed to F Wang.

At least 253 records · Page 14Linked to original sources

Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals.

A novel and efficient method for the production of enantiomerically pure (R)-(-)-hydroxycarboxylic acids by in vivo depolymerization of microbial polyester polyhydroxyalkanoates (PHAs) was developed. Using this method, several model compounds, (R)-(-)-3-hydroxyalkanoic acids, consisting of 4 to 12 carbon atoms, and (R)-(-)-3-hydroxy-5-phenylvaleric acid, could be prepared. In particular, (R)-(-)-3-hydroxybutyric acid could be efficiently prepared by this method. By providing the environmental condition in which cells possess high activity of intracellular PHA depolymerase and low activity of (R)-(-)-3-hydroxybutyric acid dehydrogenase, (R)-(-)-3-hydroxybutyric acid could be produced with a yield of 96% in only 30 min by in vivo depolymerization of polyhydroxybutyrate (PHB) accumulated in Alcaligenes latus.

Bacteria↗

Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells.

bcl-2 gene expression is induced by 17beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ER(alpha)) did not bind [(32)P]bcl-2j, whereas Sp1 protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Sp1 protein. Subsequent studies confirmed that transactivation by E2 was dependent on ER(alpha)/Sp1 interactions with both GC-rich sites, and this was confirmed by in vitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Sp1 or ER(alpha) protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif. These data coupled with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element. Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.

Activating Transcription Factor 1↗

Identification of glucagon-like peptide-2 (GLP-2)-activated signaling pathways in baby hamster kidney fibroblasts expressing the rat GLP-2 receptor.

Glucagon-like peptide-2 (GLP-2) promotes the expansion of the intestinal epithelium through stimulation of the GLP-2 receptor, a recently identified member of the glucagon-secretin G protein-coupled receptor superfamily. Although activation of G protein-coupled receptors may lead to stimulation of cell growth, the mechanisms transducing the GLP-2 signal to mitogenic proliferation remain unknown. We now report studies of GLP-2R signaling in baby hamster kidney (BHK) cells expressing a transfected rat GLP-2 receptor (BHK-GLP-2R cells). GLP-2, but not glucagon or GLP-1, increased the levels of cAMP and activated both cAMP-response element- and AP-1-dependent transcriptional activity in a dose-dependent manner. The activation of AP-1-luciferase activity was protein kinase A (PKA) -dependent and markedly diminished in the presence of a dominant negative inhibitor of PKA. Although GLP-2 stimulated the expression of c-fos, c-jun, junB, and zif268, and transiently increased p70 S6 kinase in quiescent BHK-GLP-2R cells, GLP-2 also inhibited extracellular signal-regulated kinase 1/2 and reduced serum-stimulated Elk-1 activity. Furthermore, no rise in intracellular calcium was observed following GLP-2 exposure in BHK-GLP-2R cells. Although GLP-2 stimulated both cAMP accumulation and cell proliferation, 8-bromo-cyclic AMP alone did not promote cell proliferation. These findings suggest that the GLP-2R may be coupled to activation of mitogenic signaling in heterologous cell types independent of PKA via as yet unidentified downstream mediators of GLP-2 action in vivo.

Animals↗

Genomic structure, chromosomal mapping, and promoter region analysis of murine uridine phosphorylase gene.

Uridine phosphorylase (UPase) plays an important role in the activation of 5-fluorouracil and in the regulation of tissue and plasma concentration of uridine, a potential biochemical modulator of 5-fluorouracil therapy. UPase expression is affected by the c-H-ras oncogene and various cytokines through unknown mechanisms. To understand its expression and regulation, we cloned the murine UPase gene, defined its genomic organization, determined its 5'- and 3'-end flanking sequences, and evaluated the promoter activity. The UPase gene contains nine exons and eight introns, spanning a total of approximately 18.0 kb. Its promoter lacks canonical TATA and CCAAT boxes, although a CAATAAAAA TATA-like box is seen from -41 to -49. Furthermore, IFN regulatory factor 1, c/v-Myb, and p53 binding sites are present in the promoter region, indicating that UPase expression may be directly regulated by cytokines and oncogene products. The 1.2-kb flanking fragment showed promoter activity driving the expression of the luciferase gene in various mammalian cells. A TGGGG repeat sequence is seen in the 3'-end flanking region. This element is considered to be a potential recombination consensus hot spot that may contribute to the encoding of different UPase isoforms present in different tissues, both normal and neoplastic.

3' Untranslated Regions↗

[Effects of long-term exposure to lower concentration of carbon disulfide on cardiovascular system of workers in a viscose rayon factory].

An epidemiological cross-sectional study was conducted on 99 workers exposed to lower concentration of carbon disulfide in a viscose rayon factory for a long time and 28 non-exposed ones in the same factory. Blood pressure, serum lipoproteins and electrocardiograph were detected. The results showed that neither diastolic blood pressure nor systolic one between exposed group and control group was obviously different All indicators of serum lipids and lipoproteins were not significantly different except the lipoprotein(a) of exposed group was significantly higher than that of control group, and the risk of abnormal electrocardiogram in the workers exposed to carbon disulfide was 1.1 times of those non-exposed ones but with no statistical significance. The results suggest that lipoprotein(a) can be used as a sensitive indicator for the cardiotoxic effects of carbon disulfide.

Adult↗

Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by physical interactions showing that AhR-Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Sp1 may also require interactions with AhR-Arnt.

Animals↗

Non-aqueous capillary electrophoresis chiral separations with sulfated beta-cyclodextrin.

This paper reports the application of an anionic cyclodextrin (CD), sulfated beta-cyclodextrin with a degree of substitution of four (beta-CD-(SO4-)4, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between beta-CD-(SO4-)4-analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of beta-CD-(SO4-)4 was needed due to the fact that the electrostatic attraction caused stronger binding between beta-CD-(SO4-)4 and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.

Cyclodextrins↗

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.

Amino Acid Substitution↗

Human prostate cancer expresses the low affinity insulin-like growth factor binding protein IGFBP-rP1.

Many of the alterations in the insulin-like growth factor (IGF) axis in prostatic disease have been associated with changes in the insulin-like growth factor binding proteins (IGFBPs), a multigene family of proteins that are thought to mediate the action of IGFs on target tissues. IGFBP-related protein 1 (rP1), also known as IGFBP-7 or mac25, is a recently described member of the IGFBP family, the biological function of which has yet to be completely ascertained. In this study, we analyzed the localization of IGFBP-rP1 in prostate cancer and benign prostate tissues using immunohistochemistry and a polyclonal antibody, T1A12, that is specific for IGFBP-rP1. The most intense staining was observed in nerves, whereas smooth muscle cells in the prostate stained weakly. Lymphocytes were always negative. When normal prostatic secretory epithelium was present, staining was usually absent. The lining secretory epithelium stained positively in 0 of 12 (0%) cases of benign prostatic hyperplasia, 57 of 63 (90.5%) primary adenocarcinomas, and 7 of 7 (100%) prostate cancer metastases. Prostatic intraepithelial neoplasia showed a similar pattern of staining to that observed for the invasive tumors. Analysis of Northern blots showed that none of the prostate cancer cell lines (LNCaP, C4, C4-2, C4-2B4, 9069E3, DU145, and PC3) expressed IGFBP-rP1 mRNA. This lack of expression was confirmed by immunohistochemistry of s.c.-generated tumor xenografts of LNCaP and C4-2 and by immunoblot on serum-free-conditioned media from all prostatic cell lines. In contrast to these results, tumor xenografts generated by direct intraosseous injection of LNCaP or C4-2 to bone marrow space resulted in tumors that stained positively for IGFBP-rP1. Our results show that IGFBP-rP1 is expressed in both in situ and invasive prostate neoplasms, but not typically in normal secretory or BPH epithelium; furthermore, the expression of IGFBP-rP1 can be induced in human prostate cancer cell lines in vivo on interaction with an appropriate host environment.

Carrier Proteins↗

[Study on the existence, distribution and bioaccumulation of chlorophenols in crucian carps].

Four representative chlorophenols (CPs) and their conjugates in fish tissues were determined by gas chromatography with electron capture detector(GC-ECD). Crucian carps were exposed to water contaminated with CPs or not contaminated water separately at room temperature(22 +/- 5 C) for 96 h. If the contaminated water samples are continuously replaced at 24 h interval, the concentration of CPs can be maintained constant throughout the experiment. The presence of fish will not have influence on the concentration of CPs. The experiment results showed that both free and conjugated CPs existed in fish tissues. The total amount of these chlorophenols in fish tissues is bile > liver and kidney > muscle. The proportion of conjugated CPs increases with the number of chlorine atoms in each compound, nevertheless, the proportion of conjugated CPs in PCP is less than that of TCP. Conjugated CPs in bile are composed of glucuronide and sulfate ester conjugates, and glucuronide conjugate is over 93%. The bio-accumulation of CPs in fish tissues is expressed by bio-concentration factors (BCF). The BCF abstained from free CPs and their sulfate ester conjugate in bile has no correlation with the partition coefficient of 1-octanol/water system(Kow). On the other hand, a good correlation is observed between BCF and Kow abstained from glucuronide conjugate and total amount of CPs(r > 0.96). The bioconcentration factors(BCF) of CPs in bile are 2.0 x 10(3)-6.3 x 10(3).

Animals↗

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.

Animals↗

Tissue structure, nuclear organization, and gene expression in normal and malignant breast.

Because every cell within the body has the same genetic information, a significant problem in biology is to understand how cells within a tissue express genes selectively. A sophisticated network of physical and biochemical signals converge in a highly orchestrated manner to bring about the exquisite regulation that governs gene expression in diverse tissues. Thus, the ultimate decision of a cell to proliferate, express tissue-specific genes, or apoptose must be a coordinated response to its adhesive, growth factor, and hormonal milieu. The unifying hypothesis examined in this overview is that the unit of function in higher organisms is neither the genome nor the cell alone but the complex, three-dimensional tissue. This is because there are bidirectional connections between the components of the cellular microenvironment (growth factors, hormones, and extracellular matrix) and the nucleus. These connections are made via membrane-bound receptors and transmitted to the nucleus, where the signals result in modifications to the nuclear matrix and chromatin structure and lead to selective gene expression. Thus, cells need to be studied "in context", i.e., within a proper tissue structure, if one is to understand the bidirectional pathways that connect the cellular microenvironment and the genome. In the last decades, we have used well-characterized human and mouse mammary cell lines in "designer microenvironments" to create an appropriate context to study tissue-specific gene expression. The use of a three-dimensional culture assay, developed with reconstituted basement membrane, has allowed us to distinguish normal and malignant human breast cells easily and rapidly. Whereas normal cells become growth arrested and form organized "acini," tumor cells continue to grow, pile up, and in general fail to respond to extracellular matrix and microenvironmental cues. By correcting the extracellular matrix-receptor (integrin) signaling and balance, we have been able to revert the malignant phenotype when a human breast tumor cell is cultured in, or on, a basement membrane. Most recently, we have shown that whereas beta1 integrin and epidermal growth factor receptor signal transduction pathways are integrated reciprocally in three-dimensional cultures, on tissue culture plastic (two-dimensional monolayers), these are not coordinated. Finally, we have demonstrated that, rather than passively reflecting changes in gene expression, nuclear organization itself can modulate cellular and tissue phenotype. We conclude that the structure of the tissue is dominant over the genome, and that we may need a new paradigm for how epithelial-specific genes are regulated in vivo. We also argue that unless the structure of the tissue is critically altered, malignancy will not progress, even in the presence of multiple chromosomal mutations.

Animals↗

[Preheating decrease the sensitivity of K562 cell to chemotherapeutic drugs].

Hyperthermia is a powerful tool for enhancing the effect of many chemical drugs on killing cells, but too many times of hyperthermia could decrease the effect and increase the ability of tumor cell against chemical drugs. Heat stress protein 70(HSP70) mRNA of K562 cell was analyzed by RT-PCR, and the chemosensitivity of K562 cell was tested by MTT method before and after heat-exposure. The results showed that HSP70 mRNA increased greatly after K562 cells exposed to 40 degrees C for 30 min and then it went to the top after 120 min heat-exposure. The sensitivity of K562 cell to adriamycin (ADM) and cyclophosphamide (CTX) in a usual dosage (1/250 of one dosage in clinical use) was in middle degree before exposed to heat, and the sensitivity dramatically went down after K562 cell being heat-stressed. The sensitivity of heat-stressed tumor cells to chemotherapeutic drugs was decreased, which may come from the increased expression of stress proteins in cells.

Antineoplastic Agents↗

[Effect of antioxidant vitamins on lipid peroxide injury of aortic endothelial cells induced by oxidatively modified low-density lipoprotein in vitro].

The effect of antioxidant vitamins including vitamin E, vitamin C and beta-carotene on lipid peroxidation injury of aortic endothelial cells induced by oxidatively modified low density lipoprotein (oxLDL) were observed. Bovine aortic endothelial cells were incubated in culture media with antioxidant vitamins for 12 hours, and then with oxLDL (0.1 g/L) for 24 hours respectively. The results showed that the malondialdehyde (MDA) content in culture media of the groups with oxLDL was significantly higher than those of groups with vitamin E, vitamin C and beta-carotene alone. The number of HL60 monocyte cells adhered to bovine aortic endothelial cells was obviously higher in oxLDL groups than those in groups with only antioxidant vitamins. This study indicated that antioxidant vitamins are protective on vascular from injury induced by oxLDL, including decreasing lipid peroxidation and reducing adherence of monocyte cells on endothelia and seemed to be promising in the prevention of atherosclerosis.

Animals↗

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Breast Neoplasms↗

Common and specific determinants for fibroblast growth factors in the ectodomain of the receptor kinase complex.

The assembly and activation of oligomeric complexes of FGF, the transmembrane receptor kinase (FGFR), and heparan sulfate transmit intracellular signals regulating growth and function of cells. An understanding of the structural relationships between the three subunits and their redundancy and specificity is essential for understanding the ubiquitous FGF signaling system in health and disease. Previously, we reported that a primary heparin or heparan sulfate binding site resides in a distinct sequence in immunoglobulin (Ig)-like module II of the three modules of FGFR. Here we report that in the absence of flanking sequences, isolated Ig module II of FGFR1 supports the binding of FGF-1, FGF-2, and FGF-7 in respective order of affinity. None of the three FGFs detectably bind Ig module I or the IIIb and IIIc splice variants of Ig module III in the absence of flanking sequences. Ig module I and the C-terminus of Ig module III are dispensable for high-affinity binding of FGF-1, FGF-2, and FGF-7. Alterations in highly conserved Ig module II in the heparin binding domain and substitution of individual sequence domains spanning the entire sequence of Ig module II with those from Ig module I obliterated FGF binding. Addition of a specific number of FGFR sequences to the C-terminus of Ig module II resulted in a gain in affinity for FGF-7. Several site-specific alterations in the C-terminus of full-length FGFR1IIIc, an isoform that otherwise absolutely rejects FGF-7, resulted in gain of FGF-7 binding. These results suggest that a complex of Ig module II and heparan sulfate is the base common active core of the FGFR ectodomain and that flanking structural domains modify FGF affinity and determine specificity.

Amino Acid Sequence↗

Candidate EPA, NIOSH method for determining carbon disulfide in air with capillary gas chromatography by orthogonal design.

This paper describes a candidate NIOSH EPA method for the determination of carbon disulfide in the air of workplaces with capillary gas chromatography using an orthogonal design. This method is designed to replace the packed column of the NIOSH method with a capillary column. The first part of this work concerned the setup of the method, particularly the choice of chromatographic parameters and finding their main favorable working ranges. The second part, using the statistical method orthogonal design, focused on optimizing the GC conditions, which were: column temperature, T(c) = 90 degrees C; injector temperature, T(i) = 140 degrees C; U section detector temperature, FPDU = 160 degrees C; L section detector temperature, FPDL = 210 degrees C; flow rate of carrier gas, F(c) = 20 cm/s; split ratio = 1/70; and injection volume = 1 microL. The quality control test showed that the coefficient of intra-day variation (CV) was 2.21%. A good logarithm linear correlation between the standard solutions and their peak areas was obtained. In general, the method reported here seems a valid candidate for a NIOSH EPA method due to its high precision and accuracy.

Air Pollutants↗