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Biomedical subjects

Ying Huang

Publications and source records attributed to Ying Huang.

At least 91 records · Page 5Linked to original sources
PubMed2005

Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance.

SLC7A11 (xCT), together with SLC3A2 (4F2hc), encodes the heterodimeric amino acid transport system x(c)-, which mediates cystine-glutamate exchange and thereby regulates intracellular glutathione levels. We used microarrays to analyze gene expression of transporters in 60 human cancer cell lines used by the National Cancer Institute for drug screening (NCI-60). The expression of SLC7A11 showed significant correlation with that of SLC3A2 (r = 0.66), which in turn correlated with SLC7A5 (r = 0.68), another known partner for SLC3A2, and with T1A-2 (r = 0.60; all P < 0.0001). Linking expression of SLC7A11 with potency of 1,400 candidate anticancer drugs identified 39 showing positive correlations, e.g., amino acid analogue, L-alanosine, and 296 with negative correlations, e.g., geldanamycin. However, no significant correlation was observed with the geldanamycin analogue 17-allylamino, 17-demethoxygeldanamycin (17-AAG). Inhibition of transport system x(c)- with glutamate or (S)-4-carboxyphenylglycine in lung A549 and HOP-62, and ovarian SK-OV-3 cells, reduced the potency of L-alanosine and lowered intracellular glutathione levels. This further resulted in increased potency of geldanamycin, with no effect on 17-AAG. Down-regulation of SLC7A11 by small interfering RNA affected drug potencies similarly to transport inhibitors. The inhibitor of gamma-glutamylcysteine synthetase, buthionine sulfoximine, also decreased intracellular glutathione levels and enhanced potency of geldanamycin, but did not affect L-alanosine. These results indicate that SLC7A11 mediates cellular uptake of L-alanosine but confers resistance to geldanamycin by supplying cystine for glutathione maintenance. SLC7A11 expression could serve as a predictor of cellular response to L-alanosine and glutathione-mediated resistance to geldanamycin, yielding a potential target for increasing chemosensitivity to multiple drugs.

Amino Acid Transport System y+↗
PubMed2005

Characterization of protein kinase C theta activation loop autophosphorylation and the kinase domain catalytic mechanism.

Protein kinase C theta (PKCtheta), a member of the Ca(2+)-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCtheta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCtheta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCtheta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCtheta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCtheta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKtheta(362)(-)(706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPgammaS on PKCtheta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCtheta. These results should aid the design and discovery of PKCtheta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.

Adenosine Diphosphate↗
PubMed2005

Oncogenic Ras-mediated downregulation of Gadd153/CHOP is required for Ras-induced cellular transformation.

Oncogenic Ras proteins transform cells via multiple downstream signaling cascades that are important for cell proliferation and survival. Gadd153, also known as CHOP, is a growth inhibitory and proapoptotic protein and its expression is upregulated by many agents that induce apoptosis. Here, we report our novel findings that oncogenic Ras downregulates Gadd153 expression at both protein and mRNA levels and that such downregulation occurs, at least in part, via decreases in GADD153 mRNA stability. Gadd153 downregulation is specific to oncogenic Ras since another oncogenic family member R-Ras2/TC21 does not downregulate Gadd153. We further demonstrate that the expression of exogenous Gadd153 interferes with Ras-induced oncogenic transformation, which suggests that downregulation of Gadd153 appears to be an important mechanism by which oncogenic Ras promotes cellular transformation. Thus, oncogenic Ras-mediated cellular transformation also involves downmodulation of important molecules such as Gadd153 that negatively regulate cell growth and survival.

3T3 Cells↗
PubMed2005

Compressed-CO2-assisted patterning of polymers.

We report a facile route to pattern polymer surfaces with the aid of compressed CO(2), termed the compressed-CO(2)-assisted imprint method. In this method, compressed CO(2) serves as a plasticizer for polymers (such as poly(methyl methylate) and polystyrene), which leads to a tremendous reduction in the glass transition temperature and viscosity of the polymers. Nylon fabrics and anode aluminum oxide porous membranes are used as molds, respectively, to pattern the softened polymers at relatively low temperatures, resulting in patterns at the scale of micrometers and nanometers on the surface of polymer films. The patterned structures can be tuned by changing CO(2) pressure and temperature in the imprinting process. This method is simple and environmentally benign. It also can be operated at low temperatures, for instance, ambient temperature.

Journal Article↗
PubMed2005

ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma.

Enhanced drug efflux mediated by ABCB1 P-glycoprotein and related ATP-binding cassette transporters is one of several mechanisms of multidrug resistance thought to impair chemotherapeutic success in human cancers. In malignant melanoma, its potential contribution to chemoresistance is uncertain. Here, we show that ABCB5, which functions as a determinant of membrane potential and regulator of cell fusion in physiologic skin progenitor cells, is expressed in clinical malignant melanoma tumors and preferentially marks a subset of hyperpolarized, CD133+ stem cell phenotype-expressing tumor cells in malignant melanoma cultures and clinical melanomas. We found that ABCB5 blockade significantly reversed resistance of G3361 melanoma cells to doxorubicin, an agent to which clinical melanomas have been found refractory, resulting in a 43% reduction in the LD50 from 4 to 2.3 micromol/L doxorubicin (P < 0.05). Our results identified ABCB5-mediated doxorubicin efflux transport as the underlying mechanism of resistance, because ABCB5 blockade significantly enhanced intracellular drug accumulation. Consistent with this novel ABCB5 function and mechanism in doxorubicin resistance, gene expression levels of the transporter across a panel of human cancer cell lines used by the National Cancer Institute for drug screening correlated significantly with tumor resistance to doxorubicin (r = 0.44; P = 0.016). Our results identify ABCB5 as a novel drug transporter and chemoresistance mediator in human malignant melanoma. Moreover, our findings show that ABCB5 is a novel molecular marker for a distinct subset of chemoresistant, stem cell phenotype-expressing tumor cells among melanoma bulk populations and indicate that these chemoresistant cells can be specifically targeted via ABCB5 to enhance cytotoxic efficacy.

AC133 Antigen↗
PubMed2005

Chemoresistance to depsipeptide FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone] is mediated by reversible MDR1 induction in human cancer cell lines.

Histone acetylation status, an epigenetic determinant of gene transcription, is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The potent HDAC inhibitor FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone] is a substrate for multidrug resistance protein (MDR1) and multidrug resistance-associated protein 1 (MRP1), both of which mediate FK228 resistance. To determine the mechanisms underlying acquired FK228 resistance, we developed four FK228-resistant cell lines from HCT-15, IGROV1, MCF7, and K562 cells by stepwise increases in FK228 exposure. Parent and resistant cells were characterized using a 70-oligomer cDNA microarray, real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and cytotoxicity assays. At both mRNA and protein levels, MDR1, but not MRP1 or other potential resistance genes, was strongly up-regulated in all resistant cell lines. HAT or HDAC activities were unaffected in resistant cells, consistent with a lack of cross-resistance to HDAC inhibitors that are not MDR1 substrates. FK228 was found to reversibly induce MDR1 expression by HDAC inhibition and subsequent histone hyperacetylation at the MDR1 promoter, as shown by real-time RT-PCR, Western blot, and chromatin immunoprecipitation. This study reveals a significant role of histone acetylation in MDR1 transcription, which seems to mediate FK228 resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Chromic-P32 phosphate treatment of implanted pancreatic carcinoma: mechanism involved.

AIM: To study the effects of chromic-P32 phosphate (32P colloids) interstitial administration in Pc-3 implanted pancreatic carcinoma, and investigate its anticancer mechanism. METHODS: Ninety-eight tumor bearing nude mice were killed at different time points after the injection of 32P colloids to the tumor core with observed radioactivity. The light microscopy, transmission electron microscopy (TEM) and immuno-histochemistry and flow cytometry were used to study the rates of tumor cell necrosis, proliferating cell nuclear antigen index, the micro vessel density (MVD). The changes of the biological response to the lymphatic transported 32P colloids in the inguinal lymph node (ILN) were dynamically observed, and the percentage of tumor cell apoptosis, and Apo2.7, caspase-3, Bcl-2, Bax-related gene expression were observed too. RESULTS: The half-life of effective medication is 13 d after injection of 32P colloids to the tumor stroma, in 1-6 groups, the tumor cell necrosis rates were 20%, 45%, 65%, 70%, 95% and 4%, respectively (F = 4.14-105.36, P<0.01). MVD were 38.5+/-4.0, 28.0+/-2.9, 17.0+/-2.9, 8.8+/-1.5, 5.7+/-2.3 and 65.0+/-5.2 (t = 11.9-26.1, P<0.01), respectively. Under TEM fairly differentiated Pc-3 cells were found. Thirty days after medication, tumors were shrunk and dried with scabs detached, and those in control group increased in size prominently with plenty of hypodermic blood vessels. In all animals the ILN were enlarged but in medicated animals they appeared later and smaller than those in control group. The extent of irradiative injury in ILN was positively correlated to the dosage of medication. Typical tumor cell apoptosis could be found under TEM in animals with intra-tumoral injection of low dosed 32P colloids. The peak of apoptosis occurred in 2.96 MBq group and 24 h after irradiation. In the course of irradiation-induced apoptosis, the value of Bcl-2/Bax was down regulated; Apo2.7 and caspase-3 protein expression were prominently increased dose dependently. CONCLUSION: 32P colloids intra-tumor injection having prominent anticancer effectiveness may reveal the ability of promoting cell differentiation. The low dose 32P colloids may induce human pancreatic carcinoma Pc-3 implanted tumor cell apoptosis; Apo2.7, caspase-3, Bcl-2 and Bax protein participated in regulating the process of irradiation induced cell apoptosis.

Animals↗
PubMed2005

Induction of human T cell leukemia virus type I receptors on quiescent naive T lymphocytes by TGF-beta.

The retrovirus human T cell leukemia virus (HTLV) type I (HTLV-I) is primarily transmitted by breast-feeding or sexual contact, by cell-to-cell contact between T cells. TGF-beta, which has been shown to enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. In this study, the ability of TGF-beta to regulate expression of molecules involved in HTLV-I binding and entry was examined. Previous studies using a soluble form of the HTLV-I envelope protein SU have shown that quiescent human T cells do not express cell surface molecules that specifically bind SU. After T cell activation, HTLV SU binding proteins are rapidly induced. In this study, we report that TGF-beta induces expression of proteins that bind soluble HTLV SU and HTLV virions on naive CD4(+) T lymphocytes. The induction of these proteins occurred without cell cycle entry or expression of activation markers, involved TGF-beta-induced intracellular signaling, and required de novo transcription and translation. Treatment of naive CD4(+) T lymphocytes with TGF-beta induced expression of GLUT-1, which has recently been reported to function as a receptor for HTLV. Treatment of a TGF-beta-sensitive human myeloid cell line increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses. Although earlier studies suggested that HTLV SU binding proteins might be an early marker of T cell activation and/or cell proliferation, we report in this study that TGF-beta induces binding of HTLV virions and expression of glucose transporter type 1 in primary CD4(+) T lymphocytes that remain quiescent.

CD4-Positive T-Lymphocytes↗
PubMed2005

The synthesis of substituted bipiperidine amide compounds as CCR3 antagonists.

Bipiperidine amide 1 has been identified as a CC chemokine receptor 3 (CCR3) antagonist. Optimization of its structure-activity relationship has resulted in the identification of cis (R,R)-4-[(3,4-dichlorophenyl)methyl]-3-hydroxymethyl-1'(6-quinolinylcarbonyl)-1,4'-bipiperidine 14n, which exhibits potent receptor affinity and inhibition of both calcium flux and eosinophil chemotaxis.

Amides↗
PubMed2005

Grouping of streptomycetes using 16S-ITS RFLP fingerprinting.

A total of 463 Streptomyces and Kitasatospora type strains were screened using 16S-ITS RFLP fingerprinting (combined restriction digest using enzymes BstUI and HaeIII). In total, 59 clusters could be delineated, each comprising multiple strains with nearly identical patterns. Good correlation was found in general with phylogeny, as revealed by 16S rDNA sequencing. Most strains assigned to a particular 16S-ITS RFLP cluster were classified into the corresponding 16S sequencing cluster whether a 16S similarity cut-off value of 97 or 98% was used. We conclude that the taxonomic resolution of 16S-ITS RFLP fingerprinting is higher than that of 16S rDNA sequencing; this may provide a tool for reducing the number of laborious DNA-DNA hybridizations necessary for discovering potentially new species within Streptomyces.

Cluster Analysis↗
PubMed2005

Cyclooxygenase-2 interacts with p53 and interferes with p53-dependent transcription and apoptosis.

Cyclooxygenase-2 (COX-2) has been implicated in a variety of human malignancies and, accordingly, COX-2 selective inhibitors are being investigated as important chemopreventive and therapeutic agents. How COX-2 overexpression results in tumorigenesis and how COX-2 selective agents mediate their chemopreventive effects are issues that remain poorly understood. Here we report that the tumor suppressor p53 upregulates COX-2 expression and that COX-2 can in turn inhibit p53-dependent transcription. Additionally, a COX-2-selective inhibitor potentiates p53-induced apoptosis, which also supports the notion that COX-2 activity appears to interfere with p53 function. Expression of exogenous COX-2 in p53 wild-type cells does not affect the cytoplasmic or nuclear levels of p53, suggesting that COX-2 may not affect p53 turnover or subcellular localization. We further demonstrate that endogenous COX-2 interacts with p53 and that COX-2 and p53 interactions are a physiologically relevant event. Thus, p53 upregulates COX-2 and COX-2 in turn appears to negatively affect p53 activity via mechanisms that could involve physical interactions between COX-2 and p53. Based on our results, we propose that p53-dependent upregulation and activation of COX-2 appear to be yet another novel mechanism by which p53 could abate its own growth-inhibitory and apoptotic effects.

Apoptosis↗
PubMed2005

Facile synthesis of polyaniline nanofibers using chloroaurate acid as the oxidant.

This work demonstrated a facile route to the synthesis of polyaniline (PANI) nanofibers by polymerization of aniline using chloroaurate acid (HAuCl(4)) as the oxidant. The reduction of AuCl(4)(-) is accompanied by oxidative polymerization of aniline, leading to uniform PANI nanofibers with a diameter of 35 +/- 5 nm and aggregated gold nanoparticles which can precipitate from the liquid phase during the reaction. The resultant PANI nanofibers and gold particles were characterized by means of different techniques, such as UV-vis, FTIR spectroscopy, and scanning and transmission electron microscopy methods. It is found that the gold aggregates are capped with polyaniline, and the conductivity of the fibers is around 0.16 S/cm.

Journal Article↗
PubMed2005

Effects of the novel PDE4 inhibitors MEM1018 and MEM1091 on memory in the radial-arm maze and inhibitory avoidance tests in rats.

RATIONALE: Inhibition of cyclic AMP (cAMP)-specific phosphodiesterase (PDE4) enhances memory in rodents. MEM1018 and MEM1091 are newly developed PDE4 inhibitors that had not been evaluated as yet for their effects on working and reference memory. OBJECTIVE: Experiments were carried out to determine whether these two drugs alter memory and if these effects are associated with changes in intracellular cAMP in the brain. METHODS: The effects of MEM1018 and MEM1091 on memory deficits induced by the N-methyl-D-: aspartate (NMDA) receptor antagonist MK-801 were determined in the eight-arm radial maze and step-through inhibitory avoidance tasks in rats. Their effects on cAMP concentrations in primary cultures of rat cerebral cortical neurons and their potency for inhibiting recombinant PDE4 subtypes were examined. RESULTS: In the radial-arm maze, MEM1018 and MEM1091 (0.1-2.5 mg/kg, IP) enhanced working and reference memory impaired by MK-801 (0.1 mg/kg). In addition, both drugs antagonized the amnesic effect of MK-801 on passive avoidance behavior. Overall, the behavioral effects of MEM1018 and MEM1091 were similar to the prototypic PDE4 inhibitor rolipram (0.1 mg/kg). Consistent with this, and similar to the effects of rolipram, both MEM1018 (10-30 microM) and MEM1091 (10 microM) enhanced the ability of NMDA (30 microM) to increase cAMP concentrations in rat cerebral cortical neurons, in vitro. MEM1018 and MEM1091 showed greater relative selectivity for PDE4D than rolipram, although the general profiles of the three compounds were similar. CONCLUSIONS: The novel PDE4 inhibitors MEM1018 and MEM1091 enhance memory in a manner generally similar to rolipram. PDE4D may be the primary target for the PDE4 inhibitors in the mediation of memory.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

Octyl-type monolithic columns of 530 microm i.d. for capillary liquid chromatography.

A novel monolithic capillary column (530 microm i.d.) was prepared for capillary liquid chromatography (CLC) by in situ copolymerization of octyl methacrylate (MAOE) and ethylene dimethacrylate (EDMA) in the presence of a porogen solvent containing 1-propanol, 1,4-butanediol, and water with azobisisobutyronitrile as the initiator. The influences of the contents of the porogen solvent, EDMA and the various concentration ratios of 1-propanol to 1,4-butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of monolithic capillary columns were characterized by SEM and mercury intrusion porosimetry, respectively. Chromatographic evaluations of the columns were performed under CLC mode. The results showed that good permeability and stability can be obtained under optimal experimental conditions. The separation results of some acid, neutral and basic analytes demonstrated the hydrophobicity and low affinity to basic analytes of the new column. Three metal ions, i.e. Mg(II), Zn(II) and Cd(II) were also separated under ion-pair mode on the new monolithic capillary column and the results were acceptable.

Chromatography, Liquid↗
PubMed2005

Efflux of depsipeptide FK228 (FR901228, NSC-630176) is mediated by P-glycoprotein and multidrug resistance-associated protein 1.

Depsipeptide FK228 [(E)-(1S,4S,10S,21R)-7[(Z)-ethylideno]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone], a novel histone deacetylase (HDAC) inhibitor, previously was reported to be a P-glycoprotein (Pgp) substrate. We now expand the investigation to demonstrate that FK228 is a substrate for Pgp and multidrug resistance-associated protein 1 (MRP1). Transport of FK228 across the Caco-2 cell monolayer in apical to basolateral (AP-->BL) and basolateral to apical (BL-->AP) directions in the absence and presence of Pgp and MRP inhibitors were investigated. An in vitro uptake study in human red blood cells (RBCs) and a cytotoxicity assay in MRP1(-) HL60 and MRP1(+) HL60Adr cells were conducted to show that FK228 is an MRP1 substrate. An FK228-resistant cell line (HCT15R) was developed from HCT15 colon carcinoma and characterized using a 70-oligomer cDNA microarray, reverse transcription-polymerase chain reaction, Western blot analysis, histone acetyltransferase (HAT) and HDAC activity assays, and cytotoxicity assays. FK228 showed a nearly unidirectional flux across the Caco-2 cell monolayer, with the BL-->AP apparent permeability coefficient (P(app)) 32 times that of AP-->BL without apparent saturation. Pgp inhibition decreased the BL-->AP P(app) and increased the AP-->BL P(app). RBC showed a concentration-dependent uptake and saturable efflux of FK228. HL60Adr cells were 4-fold more resistant to FK228 than HL60 cells, and the resistance was reversed by MRP inhibition. Up-regulation of Pgp, but not changes of MRPs or HAT/HDAC enzymatic activities, was the major mechanism for the acquired FK228 resistance. These studies demonstrate that FK228 is a substrate for Pgp and MRP1, and reversible Pgp up-regulation is predominantly involved in FK228 resistance in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection.

A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.

Adolescent↗
PubMed2005

Separation and determination of enkephalin-related peptides using capillary electrophoresis.

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.

Cerebrospinal Fluid↗