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Biomedical subjects

M Zhang

Publications and source records attributed to M Zhang.

At least 145 records · Page 8Linked to original sources
PubMed2001

Interdomain chaperoning between PSD-95, Dlg, and Zo-1 (PDZ) domains of glutamate receptor-interacting proteins.

The multiple PSD-95, Dlg, and Zo-1 (PDZ) domain protein, glutamate receptor-interacting protein (GRIP), is involved in the clustering and trafficking of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor by directly binding to the cytoplasmic tail of the receptor's GluR2 subunit. Both the forth and fifth PDZ domains (PDZ4 and PDZ5) of GRIP are required for effective binding to the receptor. Using NMR and circular dichroism spectroscopic techniques, we show that PDZ5 is completely unstructured in solution. Freshly prepared PDZ4 is largely folded, but the domain can spontaneously unfold. Neither PDZ4 nor PDZ5 binds to GluR2 in solution. Unexpectedly, when PDZ4 and PDZ5 are covalently connected (i.e. PDZ45), both PDZ domains become well folded and stable in solution. The covalent linkage of the two PDZ domains is essential for proper folding of the tandem PDZ domains and its effective binding to GluR2. The interdomain chaperoning effect observed in the PDZ domains of GRIP represents a previously uncharacterized function of PDZ domains.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression.

Sterol 12alpha-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5'-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from -57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7alpha-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4alpha- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4alpha-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4alpha. Mammalian two-hybrid assay confirmed that HNF4alpha interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4alpha in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4alpha gene transcription. In summary, our data revealed the critical roles HNF4alpha play on CYP8B1 transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4alpha through interaction of HNF4alpha with SHP and reduction of HNF4alpha expression in the liver.

Base Sequence↗
PubMed2001

Development of Dengue virus type 2 replicons capable of prolonged expression in host cells.

BACKGROUND: As part of a program to develop a Dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ADE) of infection mediated by antibodies to Dengue virus structural proteins, we have begun to investigate the possibility of designing Dengue vaccines based on non-structural proteins. RESULTS: Dengue constructs which lack major structural proteins replicate intracellularly in tissue culture. These replicons are capable of prolonged expression of Dengue virus non-structural proteins for at least seven days in culture. CONCLUSIONS: Dengue virus genomes lacking major structural proteins can, like other flaviviruses, replicate intracellularly and express virus non-structural proteins with minimal toxicity to host cells. These findings pave the way for the development of dengue virus replicons as a form of live, attenuated virus vaccine.

Animals↗
PubMed2001

Sar studies of piperidine-based analogues of cocaine. Part 3: oxadiazoles.

The synthesis of novel 4beta-aryl-1-methyl-3alpha-(3-substituted-1,2,4-oxadiazol-5-yl)piperidines, bioisosteres of ester (+)-1, is described. The synthesized oxadiazoles were evaluated for their affinity to the DAT and their ability to inhibit monoamine reuptake at the DAT, NET, and 5HTT. The results show that affinity to the DAT and ability to inhibit the reuptake at the DAT, NET, and 5HTT is a function of the size of the substituent in the oxadiazole ring. (+)-(3R,4S)-4beta-(4-Chlorophenyl)-1-methyl-3alpha-(3-methyl-1,2,4-oxadiazol-5-yl)piperidine [(+)-2a], which is structurally and pharmacologically most similar to the ester (+)-1 in this series, showed at least a 2-fold longer duration of action when compared to ester (+)-1.

Carrier Proteins↗
PubMed2001

A novel method for the preparation of amino-substituted hypocrellin B.

A series of amino-substituted hypocrellins derived from hypocrellin B (HB) were synthesized by a novel mild method, in which the peri-hydroxylated perylenequinone structure of hypocrellin was preserved by the reaction of HB with an amine. The red absorption of the resulting products was significantly enhanced relative to the parent hypocrellins, which will significantly improve its photodynamic therapy effectiveness.

Anti-HIV Agents↗
PubMed2001

An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p.

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.

Amino Acid Sequence↗
PubMed2001

A novel photosensitizer, 2-butylamino-2-demethoxy-hypocrellin B (2-BA-2-DMHB) - its photodynamic effects on HeLa cells: efficacy and apoptosis.

A novel hypocrellin congener, 2-butylamino-2-demethoxy-hypocrellin B (2-BA-2-DMHB) was found to be an effective photosensitizer. Compared with its parent compound hypocrellin B (HB), its absorption bands extended toward longer wavelength and the extinction coefficients raised to some degree (lambda(max) (nm) (log(epsilon)): 463 (4.06), 583 (4.09), 621 (4.10) for 2-BA-2-DMHB and 466 (4.06), 548 (3.70), 580 (3.52) for HB). And it also had a much higher photopotentiation factor than HB (i.e., more than 250 versus 10 at a dose of 24 J cm(-2) of red light on HeLa cells). This might be correlated to the higher ability of superoxide anion generation and the higher cellular uptake of 2-BA-2-DMHB. Meanwhile, the examinations of Hoechst 33342-labeled nuclei, DNA fragmentation on agarose gel and flow cytometry showed that 2-BA-2-DMHB induces apoptosis in photosensitized HeLa cells more quickly than HB, which might be correlated to the higher cellular uptake of 2-BA-2-DMHB and the difference of their cellular localization. The study suggested that 2-BA-2-DMHB was a well-suited candidate for a new generation of photodynamic therapy photosensitizer.

Apoptosis↗
PubMed2001

Cooperation of solid granule and solvent as porogenic agents. Novel porogenic mode of biporous media for protein chromatography.

A novel porogenic mode, cooperation of solid granule and solvent, has been introduced to prepare a biporous medium for protein chromatography. The matrix, a ternary copolymer of glycidyl methacrylate, triallylisocyanurate and divinylbenzene, was produced by a simple in situ polymerization with granules of sodium sulfate and cyclohexanol and dodecanol as porogenic agents. Functionalized with diethylamine, the resin (denoted as Resin C) was used as an anion exchanger. The pore structure, specific surface area and chromatographic properties of Resin C were determined and compared with those of the resin with only the solvents as porogen (Resin A) and the resin with only the salt granules as porogen (Resin B). The results indicated that Resin C contained regions of micropores with a maximum at approximately 55 nm and regions of macropores with a distinct maximum near 340 nm, which swelled to about 1 microm in aqueous solution. Compared with Resins A and B, the biporous medium Resin C simultaneously possessed a high specific surface area of 37.2 m2/g and a low back-pressure at mobile phase flow velocity up to 720 cm/h. The result of dynamic porosity showed that mobile phase was able to convectively flow through the macropores in Resin C. The dynamic adsorption capacity of Resin C for bovine serum albumin was as high as 57.0 mg/ml column volume (95.0 mg/g wet resin), basically identical to its static capacity, while that of Resin A was only 1.95 mg/ml column volume (3.12 mg/g wet resin), about 3% that of its static capacity. In addition, the column efficiency of Resin C was comparable to that of Resin B, but much higher than that of Resin A, indicating that the mass transfer behavior of proteins in the column was greatly improved by convective flow through the macropores.

Adsorption↗
PubMed2001

Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals.

Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others. Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase activity nor the related cholera toxin that ADP-ribosylates G(s) (but not G(i)) proteins blocked EAU induction or migration to chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it does so at least in part by disrupting signaling through G(i) protein-coupled receptors. Thus, the net effect of PTX on autoimmune disease would represent an integration of enhancing and inhibitory effects.

Adoptive Transfer↗
PubMed2001

Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering.

Chitosan scaffolds reinforced by beta-tricalcium phosphate (beta-TCP) and calcium phosphate invert glass were fabricated with a low-cost, bioclean freeze-drying technique via thermally induced phase separation. The microstructure, mechanical performance, biodegradation, and bioactivity of the scaffolds were studied. The composite scaffolds were macroporous, and the pore structures of the scaffolds with beta-TCP and the glass appeared very different. Both the compressive modulus and yield strength of the scaffolds were greatly improved, and reinforced microstructures were achieved. The bioactivity tests showed a continuous decrease in both Ca and P concentrations of a simulated body fluid (SBF) after the scaffolds with beta-TCP were immersed in the SBF for more than 20 h, which suggests that an apatite layer might be formed on the scaffolds. However, the same was not observed for the pure chitosan scaffolds or the scaffolds incorporated with the glass. This was further confirmed by micrographs from scanning electron microscopy. This study suggests that the desirable pore structure, biodegradation rate, and bioactivity of the composite scaffolds might be achieved through controlling the ratio of chitosan and calcium phosphates or beta-TCP and the glass.

Biocompatible Materials↗
PubMed2001

Structure-based design, synthesis and SAR of a novel series of thiopheneamidine urokinase plasminogen activator inhibitors.

The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.

Antimetabolites↗
PubMed2001

Induction of interleukin-6 and oncostatin M by radiation in Kaposi's sarcoma cell lines.

PURPOSE: To define the in vitro radiosensitivity of Kaposi's sarcoma (KS) and to explore the mechanism of its extreme clinical radiosensitivity. METHODS AND MATERIALS: The radiation survivals of the three KS cell cultures (KSY-1, KS-JD, KS6-3E) were determined by clonogenic assay and MTT assay. Supernatants from irradiated cells were collected at different time points for measurement of the interleukin 6 (IL-6), oncostatin M (OSM), and basic fibroblast growth factor (bFGF) by ELISA. Changes in the mRNA expression of these cytokines were examined by reverse transcription polymerase chain reaction and Southern blot hybridization. Fresh KS cells were preincubated with the irradiated supernatant before irradiation, and the change in survivals were assessed. RESULTS: The mean SF-2 (survival fraction after 2 Gy) for KS was 0.43. Preincubation with the irradiated supernatant reduced the SF-2 significantly from 0.43 to 0.33 (p < 0.05). Treatment with irradiated supernatant alone was not cytotoxic to the cells. Radiation induced IL-6 and OSM production by KS cells at the transcription level. A single dose of 2 Gy increased IL-6 and OSM mRNA expression of the KS Y-1 cells. This corresponded to an increase in the IL-6 and OSM levels in the culture medium. There was no significant change in the level of bFGF. Preincubation with recombinant human IL-6 or OSM sensitized KS in a dose dependent manner. CONCLUSION: The low SF-2 value for these KS cell lines correlates with the clinical radiosensitivity of KS. The high radiosensitivity may be due to radiation induction of cytokines such as IL-6 and OSM, which are radiosensitizers for KS cells.

Blotting, Southern↗
PubMed2001

minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel?

Inherited mutations and a polymorphism in minK-related peptide 1 (MiRP1) have been linked to congenital or acquired long-QT syndrome, pointing to the importance of MiRP1 in maintaining the cardiac electrical stability. We tested whether MiRP1 could affect the function of Kv4.x (x=2 and 3), the major pore-forming (alpha) subunits of transient outward (I(to)) channels in the heart. We used the Xenopus oocyte expression system to examine the effects of MiRP1 on Kv4.x channel gating kinetics and current amplitude and correlated these effects with MiRP1 expression level. MiRP1 slowed the rates of Kv4.2 activation and inactivation and shifted the voltage dependence of channel gating in the positive direction. These effects had a similar "dose" dependence: they plateaued at a cRNA ratio (MiRP1:Kv4.2) of 13:1, with half-maximum effects at estimated cRNA ratios of 2 to 4. On the other hand, MiRP1 had no significant effects on Kv4.2 current amplitude in the same range of expression level. When expressed at a comparable low level, MiRP1 had similar (although smaller) effects on Kv4.3 but could not modulate Kv1.4 (another alpha subunit of I(to) channels in the heart). Kv4.2 could be coimmunoprecipitated with epitope-tagged MiRP1, indicating that the 2 could form a stable complex. Our data suggest that MiRP1 may serve as a regulatory (beta) subunit of I(to) channels in the heart. This is supported by the observation that MiRP1 induced an "overshoot" of Kv4.2 current amplitude during channel recovery from inactivation, similar to the overshoot of I(to) described for human epicardial myocytes.

4-Aminopyridine↗
PubMed2001

Pharmacological and behavioral analysis of the effects of some bivalent ligand-based monoamine reuptake inhibitors.

Novel piperidine-based bivalent ligands were prepared in enantiomerically pure form and evaluated for their ability to inhibit reuptake of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) into rat brain nerve endings (synaptosomes). In this study, we have succeeded in using (1) the length of the linking chain connecting the two piperidine-based monomer units and (2) the absolute configuration of the piperidine monomer as a means to tailor activity and selectivity at the three monoamine transporters tested. In this series, the bivalent ligand 16, comprised of two (+)-trans-piperidine units linked by a pentamethylene spacer, exhibits a combination of high DA transporter (DAT) and 5-HT transporter (SERT) activity (K(i) = 39 nM and 7 nM, respectively). Piperidine 16 is capable of reducing cocaine's locomotor effects in mice while not having any effect on locomotion when tested alone. Additionally, compound 16 (1-10 mg/kg) does not substitute for cocaine in drug discrimination studies in rats. However, the analogous bivalent ligand 15 comprised of two (-)-trans-piperidine units, which is SERT selective, was less effective in antagonizing cocaine's locomotor stimulant activity. The piperidine-based bivalent inhibitors described herein constitute a new class of monoamine reuptake inhibitors that exhibit varying levels of monoamine transporter activity and selectivity, and these ligands may serve as lead candidates in the discovery of new therapeutics to treat a range of neurological disorders including cocaine addiction.

Animals↗
PubMed2001

Cellular localization and trafficking of the human ABCA1 transporter.

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.

ATP Binding Cassette Transporter 1↗
PubMed2001

Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic model system for the study of anionic phospholipid function in mitochondria.

Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.

Acridine Orange↗
PubMed2001

Enhancement of antigen-presenting ability of B lymphoma cells by immunostimulatory CpG-oligonucleotides and anti-CD40 antibody.

Immunostimulatory oligodeoxynucleotides containing the CpG motifs (CpG-ODN) can activate antigen-presenting cells including dendritic cells, macrophages, B cells, and enhance production of Thl cytokines. So, CpG-ODN has been regarded as a promising immune adjuvant. Using the A20 B lymphoma cell model, we investigated the effect of CpG-ODN on the immunogenicity of B lymphoma cells and whether CpG-ODN could enhance the antigen-presenting ability of B lymphoma cells. After incubation with CpG-ODN, proliferation of A20 cells remained unchanged. But CpG-ODN stimulation up-regulated the expression of MHC-I, MHC-II, CD40, ICAM-1 molecules in A20 cells, enhanced the antigen uptake ability of A20 cells, and promoted A20 cell production of IgM and IgG. More importantly, A20 cells activated by CpG-ODN could stimulate allogeneic T cells in MLR and antigen-primed T cells to proliferate more efficiently, suggesting the antigen-presenting ability of A20 B lymphoma cells could be enhanced by CpG-ODN stimulation and CpG-ODN-activated B lymphoma cells might be used as a potent cellular vaccine. Although anti-CD40 mAb was as effective as CpG-ODN at activating A20 cells and A20 cells expressed more CD40 molecules after CpG-ODN stimulation, a combination of CpG-ODN and anti-CD40 mAb had no synergistic effect on A20 cell activation. Our data expanded the potential application of CpG-ODN as an immunotherapeutic agent in cancer treatment.

Animals↗