PubMed Health⌕ Search

Biomedical subjects

C Chang

Publications and source records attributed to C Chang.

At least 109 records · Page 6Linked to original sources
PubMed2000

Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Bacterial Proteins↗
PubMed2000

Nucleoside diphosphate kinase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray crystallographic analysis.

Nucleoside diphosphate (NDP) kinase is a key enzyme in maintaining cellular pools of all nucleoside triphosphates. NDP kinase from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 4000 as precipitant. The crystal is hexagonal, belonging to the space group P6(3), with unit-cell parameters a = b = 72.89, c = 100.87 A. The asymmetric unit contains two subunits of NDP kinase, with a corresponding crystal volume per protein mass (V(M)) of 2.38 A(3) Da(-1) and a solvent content of 48.3%. Native X-ray diffraction data to 2.30 A resolution have been collected using synchrotron X-rays.

Crystallization↗
PubMed2000

Lactate dehydrogenase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray analysis.

L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate. LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant. Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 2.9 A have been collected from a native crystal. The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A. The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7%. Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 1.9 A have been collected from a native crystal. The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A. The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8%. Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.

Cloning, Molecular↗
PubMed2000

Effects of genomic length on translocation of hepatitis B virus polymerase-linked oligomer.

Accurate translocation of the polymerase-linked oligomer to the acceptor site (DR1*) in reverse transcription is crucial for maintaining the correct size of the hepatitis B virus (HBV) genome. Various sizes of foreign sequences were inserted at different sites of the HBV genome, and their effects on accurate translocation of polymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in these insertion mutants: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicated that the minus strand of RC and type I DL form was elongated from DR1*, while the minus strand of the type II DL form was elongated from multiple internal acceptor sites (IAS), such as IAS2. These IASs were also found to be used by wild-type HBV but with a very low frequency. Mutation of IAS2 by base substitution abrogated polymerase-linked oligomer transferring to IAS2, demonstrating that base pairing also plays an important role in the function of IAS2 as a polymerase-linked oligomer acceptor site. Data obtained from our insertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site and the acceptor is important. The polymerase-linked oligomer prefers to translocate to an acceptor, DR1* or IAS2, which are ca. 3.2 kb apart. However, it will translocate to both DR1* and IAS2 if they are not located 3.2 kb apart. These results suggest that the polymerase-linked oligomer may be able to scan bidirectionally for appropriate acceptor sites at a distance of 3.2 kb. A model is proposed to discuss the possible mechanism of polymerase-linked oligomer translocation.

Animals↗
PubMed2000

Isolation and characterization of the androgen receptor mutants with divergent transcriptional activity in response to hydroxyflutamide.

A yeast genetic screening was developed to isolate androgen receptor (AR) mutants with divergent transactivation characteristics in response to hydroxyflutamide (HF), an active metabolite of flutamide used for prostate cancer treatment. Two mutants carrying the substitution C685Y or E708K were isolated and characterized. Substitution of C685Y for wild-type AR (wtAR) rendered the receptor supersensitive to androgenic activity from HF and female hormones such as 17beta-estradiol (E2) and progesterone (P). Similar effects were observed in the AR mutant, named T876AAR, isolated from LNCaP cells. Surprisingly, we found that C685YAR7, but not T876AAR7, could be activated by casodex (bicalutamide), a nonsteroidal pure antiandrogen, with an induction fold 3- to 5-fold times higher than that for wild type or T876AAR. By contrast, although replacement of E708K for wtAR showed little effect on dihydrotestosterone-mediated transactivation, E708KAR lost its transcriptional response from many other ligands. The effects of ligands on E708KAR could be controlled at the DNA-binding level owing to the finding of a significant decrease in the DNA-binding ability once E708KAR was bound to HF, E2, or P. Together, these results suggest that C685YAR can be a novel tool for assaying the androgenic activity from antiandrogens, and the mechanism revealed from E708KAR could provide a possible explanation for the partial androgen insensitivity syndrome in men with a natural E708KAR mutation.

Amino Acid Sequence↗
PubMed2000

Identification of an essential cis-acting element (TR2-PACE) in the 5' promoter of human TR2 orphan receptor gene.

The human TR2 orphan receptor (TR2) is a member of the steroid/thyroid hormone receptor superfamily. It has been shown to be expressed in a wide variety of tissues during development. Using deletion mutation analyses and transient transfection CAT assays, we demonstrated here that a DNA fragment of 103 bp, with a sequence from +65 to -38, containing an initiator is capable of serving as a core promoter to initiate basal level transcription; further extending of this core promoter sequence up to -441 maximizes the reporter gene expression. Within this positive regulatory region (-441/+65), we were able to narrow the regulation-responsible sequence down to a small 64-bp (-263/-201) DNA fragment named the TR2 promoter activating cis-element (TR2-PACE). Further deletion mutagenesis and shifting of the insert position followed by reporter assays demonstrated that this TR2-PACE is essential for high-level induction of a heterologous core promoter's activity in a position-dependent nature. In addition, orientation tests indicated that the sense, but not antisense orientation increased the TR2 core promoter activity. Moreover, electrophoresis mobility shift assays and Southwestern analyses suggested that TR2-PACE may interact with unknown specific nuclear proteins for its enhancer activity. Together, our data suggest that TR2-PACE is a position-dependent and, in the case of TR2 core promoter (TATA-less), an orientation-dependent cis-activating element required for maximal expression of the TR2 gene.

Base Sequence↗
PubMed2000

Crystallization and preliminary X-ray crystallographic analysis of human nucleoside diphosphate kinase A.

Human nucleoside diphosphate kinase A catalyzes phosphoryl transfer and acts as a suppressor of metastasis. It has been crystallized using 2-methyl-2,4-pentanediol as a precipitant at 288 K. The crystal is monoclinic, belonging to the space group P2(1), with unit-cell parameters a = 74.21, b = 78.11, c = 82.29 A, beta = 101. 33 degrees. The asymmetric unit contains a homohexamer, with a corresponding crystal volume per protein mass (V(m)) of 2.27 A(3) Da(-1) and a solvent content of 46%. Native X-ray data to 2.15 A resolution have been collected using synchrotron X-rays.

Crystallization↗
PubMed2000

Donor origin of neuroendocrine carcinoma in 2 transplant patients determined by molecular cytogenetics.

Organ transplant recipients have an increased tumor incidence owing to their immunocompromised state. The origin of such tumors, whether donor or recipient, will have a clinical impact on decision-making concerning immunosuppressive therapy, retransplantation, and for recipients of other organs from the same donors. We report molecular cytogenetic determination of donor origin in 2 cases of small-cell neuroendocrine carcinoma developing in sex-mismatched transplant recipients (kidney and liver). Fluorescence in situ hybridization (FISH) analysis was performed on liver core needle biopsy material from the liver transplant patient and on liver fine needle aspiration cytopreparations from the kidney transplant patient. The results for the liver transplant patient were confirmed with microsatellite allelic analysis and with comparative genomic hybridization. In both cases, FISH showed the presence of only X chromosomes within the tumor cells, indicating the donor origin of the neoplasms. FISH is an excellent method to determine neoplastic origin in sex-mismatched transplant patients. HUM PATHOL 31:1425-1429.

Adult↗
PubMed2000

Uninsured under TennCare: a case study of public health clinic users.

BACKGROUND: We characterized public health clinic users 4 years after implementation of a major public health insurance reform and identified barriers to health care access. METHODS: We used face-to-face interviews and profile analysis of survey findings. RESULTS: The typical public health client household is larger, poorer, and more likely to be black than the wider population. The health status of participants is generally good; the level of insurance coverage is preponderantly TennCare; and 90% of respondents are willing to pay some premium. CONCLUSIONS: Lack of health insurance does not present a serious health care access barrier, nor do transportation or location; but taking time off from work with loss of wages is a problem for those employed. A cost-effective way to enroll uninsured children is to use the County Health Department clinics as a contact point.

Adult↗
PubMed2000

Functional analysis of androgen receptor N-terminal and ligand binding domain interacting coregulators in prostate cancer.

Several new androgen receptor (AR) coregulators, including ARA70, ARA55, ARA54, ARA160 and ARA24, associated with the N-terminal or the ligand-binding domain (LBD) of AR, have been identified by our group. We first identified the AR-LBD coregulators ARA70, ARA55, and ARA54. Our previous reports suggest that ARA70 can enhance the androgenic activity of 17 beta-estradiol (E2) and antiandrogens toward AR. It is of interest to compare and determine if the specificity of sex hormones and antiandrogens can be modulated by different coregulators. Our results indicate that, ARA70 is the best coregulator for increasing the androgenic activity of E2. Only ARA70 and ARA55 were able to significantly increase the androgenic activity of hydroxyflutamide, the active metabolite of a widely-used antiandrogen for the treatment of prostate cancer. Furthermore, our results suggest that among the LBD coregulators, ARA70 has a relatively high specificity for AR in the human prostate cancer cell line DU145. Together, our data suggest that the androgenic activity of some sex hormones and antiandrogens can be modulated by selective AR coactivators. In addition to the AR-LBD associated proteins, ARA24 and ARA160 have been identified as AR coregulators, interacting with the AR N-terminal instead of the LBD. Functional analysis revealed that the AR N-terminal coregulator ARA160 could cooperate with the AR LBD-associated coregulator ARA70. Our data indicate that ARA24 could also interact with AR, and that this binding is decreased by an expanding poly-glutamine (Q) length within AR. The length of the poly-Q stretch in the AR N-terminal domain is inversely correlated with the transcriptional activity of AR. Our data suggest that optimal AR transactivation may require interaction of AR with AR coregulators. The identification of factors or peptides that can interrupt androgen-mediated AR-ARA interactions may be useful in the development of better antiandrogens for treating androgen-related diseases, such as prostate cancer.

Androgen Antagonists↗
PubMed2000

[Determination of chromium in geological samples with slurry sampling and probe atomization in GFAAS].

A method of loading a slurry prepared from a solid sample for analysis of chromium in geological samples using graphite furnace atomic absorption spectrometry had been approached by combining slurry sampling with probe atomization. The factors that influence the performances of chromium including ashing and atomization temperature, atomization surfaces were investigated. The results showed that the determination of chromium in geological samples could be performed by direct slurry sampling with the use of a calibration established with aqueous standards. The relative standard deviations(RSD) of the method were 3.0%-6.5%. The detection limit was 5.1 x 10(-12) g Cr. The Analytical results of the geological reference samples were consistent with the certified values.

English Abstract↗
PubMed1999

Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: a unique signaling pathway in the steroid receptor superfamily.

The androgen receptor (AR) binds to androgen response elements and regulates target genes via a mechanism involving coregulators. Here we demonstrate that the AR can interact with the testicular orphan receptor-4 (TR4) and function as a repressor to down-regulate the TR4 target genes by preventing the TR4 binding to its target DNA. Interestingly, the heterodimerization of AR and TR4 also allows TR4 to repress AR target gene expression. Simultaneous exposure to both receptors therefore could result in bidirectional suppression of their target genes. Together, these data demonstrate that the coupling of two different receptors, through the heterodimerization of AR and TR4, is a unique signaling pathway in the steroid receptor superfamily, which may facilitate further understanding of the complicated androgen action in prostate cancer or libido.

Dimerization↗
PubMed1999

Inhibition of CYP1A1 enzyme activity in mouse hepatoma cell culture by soybean isoflavones.

The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent Ki values of 325 microM and 140 microM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent Ki values of 55 microM and 50 microM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.

Animals↗
PubMed1999

Expression of antioxidant proteins in human intestinal Caco-2 cells treated with dietary flavonoids.

Dietary flavonoids are known to scavenge free radicals but little information is available on their roles in antioxidant protein gene expression. The goal of this paper is to investigate the effect of flavonoid treatment on the antioxidant protein expression in human intestinal Caco-2 cells. The antioxidant proteins of interest were metallothionein (MT), catalase (CAT), and superoxide dismutase (SOD). Treatment of Caco-2 cells with 100 microM genistein, biochanin A, daidzein or kaempferol significantly increased MT mRNA up to 15 fold. On the contrary, CAT mRNA level was not affected by various flavonoids. We also developed gel activity assays to determine the specific activities of CAT and Cu/Zn SOD in flavonoid-treated Caco-2 cells. Compared to the conventional spectrophotometric assays, the gel assays allow a separation of antioxidant activities of the enzymes from that of the flavonoids. CAT and Cu/Zn SOD were found not to be affected by 48-h treatment of 100 microM dietary flavonoids (genistein, biochanin A, daidzein, flavone, quercetin, or kaempferol). In conclusion, the effects of flavonoids on antioxidant protein expression are structure- and gene-specific. When evaluating antioxidant capacity of flavonoids, their ability to modulate antioxidant protein expression should also be taken into consideration.

Caco-2 Cells↗
PubMed1999

The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans.

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

Amino Acid Sequence↗
PubMed1999

Cutting edge: KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties.

Transmembrane adapter proteins are a class of molecules that mediate signals from an extracellular receptor to the cytoplasm of the cell. We have cloned a novel transmembrane adapter protein called KAP10, a approximately 10-kDa protein that is encoded within 100 bp of the DAP12 locus on human chromosome 19. KAP10 is predominantly expressed in immune cells, including NK cells, T cells, and monocytes. We show that KAP10, unlike other transmembrane adapter proteins, binds phosphatidylinositol-3 kinase following phosphorylation of a cytoplasmic YINM motif, which results in activation of Akt. In addition, we identify KAP10 as being able to bind the adapter protein Grb2. Based on our data, we suggest that this molecule is involved in stimulation and costimulation in cells of both myeloid and lymphoid origin.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Functional complementation of the Schizosaccharomyces pombe wis1 mutant by Arabidopsis MEK1 and non-catalytic enhancement by CTR1.

Arabidopsis thaliana MEK1 encodes a MAPKK homolog whose role in plants is currently unknown. High (but not low) expression of MEK1 rescued the Deltawis1 (MAPKK) mutant of the Schizosaccharomyces pombe Win1/Wis4-Wis1-Sty1 stress-activated MAPK pathway. Rescue was dependent upon upstream and downstream components of the pathway, suggesting that MEK1 might function in a homologous MAPK pathway in plants. When MEK1 was expressed at a low level, rescue of Deltawis1 was achieved by co-expressing Arabidopsis CTR1 (a putative MAPKK kinase (MAPKKK)). CTR1 constructs alone did not rescue the pathway, indicating that CTR1 augmented MEK1 function. Further data indicated that this enhancement was not due to CTR1 kinase activity.

Arabidopsis↗