Biomedical subjects

X Chen

Publications and source records attributed to X Chen.

At least 1,027 records · Page 57Linked to original sources

PubMed1998

[Cyclodextrin induced-room temperature phosphorescence for the determination of quinoline without deoxidization].

A cyclodextrininduced-room temperature phosphorescence (CD-RTP) system for the determination of quinoline without deoxidization procedure was developed based on the external heavy atom effect of dibromopropane (DBP), the RTP maximum wavelengths lambda(ex)/lambda(em) = 273/496nm. The difference of heavy atom effect between dibromopropane DBP and dibromoethane (DBE) was examined and the apparent formation constants of the ternary inclusion complexes for quinoline/beta-CD/DBP and quinoline/beta-CD/DBE systems were determined. As compared with DBE, the heavy atom effect of DBP was higher and the RTP intensity was stronger. The RTP intensity was linear for quinoline/beta-CD/DBP systems with quinoline concentration in the ranges of 5.0 x 10(-7)-5.0 x 10(-5)mol/L and 5.0 x 10(-5)-5.0 x 10(-4)mol/L, respectively. The detection limit was 7.8 x 10(-8)mol/L. The determination precision was satisfactory.

Calibration↗

PubMed1998

[Indirect sensitized upconversion in Tm3+ and Yb3+ codoped non-crystal pentaphosphate].

We report the blue upconversion luminescence of Tm3+ and Yb3+ codoped non-crystal pentaphosphate pumped by -798nm laser diode. The samples were directly excited to 3F4 level of Tm3+ ion. Two energy transfer processes between Tm3+ and Yb3+ contribute mainly to the population of 1G4 level of Tm3+. Phonon plays an important role in the upconversion process.

English Abstract↗

PubMed1998

Characterization of 5-oxo-L-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation of glutathione.

5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues. First, 5-OPase activity was measured in tissues of tumor-bearing rats, in the peripheral mononuclear cells of normal human subjects, and in surgically resected tumor and the adjacent normal tissues from patients. We found that the activity of 5-OPase in human kidney, liver, and lung is significantly lower than that found in rats. Second, we have raised a polyclonal IgG anti-5-OPase antibody by immunizing rabbits with purified 5-OPase from rat kidney. This antibody has very high affinity (shown by immunoprecipitation) and specificity (shown by Western blot) and cross-reacts with human 5-OPase (shown by Western blot and immunohistochemistry). It was then used to examine the distribution of 5-OPase in paired normal and neoplastic human specimens using Western blot and immunohistochemistry. Examination of paired normal and neoplastic tissues of stomach and lung revealed a significantly lower level of 5-OPase in tumor tissues than in the paired normal tissues. In colon tissues, there is no significant difference in 5-OPase level between the normal and tumor tissues. These findings could have implications for both carcinogenesis and therapy.

Animals↗

PubMed1998

[Gene diagnosis of 10 Chinese families with adult polycystic kidney disease from Guangxi region].

OBJECTIVE: To study presymptomatic gene diagnosis in adult polycystic kidney disease (APKD). METHODS: Linkage analysis and haplotype characterization were carried out in 10 unrelated Chinese APKD families from Guangxi region, including 97 individuals and 29 affected members. Members of these families were ultrasonographically examined. Linkage to PKD1 was investigated in all families with four microsatellite markers(SM7, CW2, AC2.5, and KG8). These flanking markers were amplified by PCR. RESULTS: The families were fully informative for gene diagnosis and no evidence of genetic heterogeneity was found. DNA analysis predicted 7 asymptomatic individuals(by 28 years old) as being affected, although they all presented negative ultrasonographs. CONCLUSION: Linkage analysis is still an important way in APKD presymptomatic gene diagnosis.

Adult↗

PubMed1998

Intravesical bacille Calmette-Guérin induces the antiangiogenic chemokine interferon-inducible protein 10.

OBJECTIVES: Intravesical bacille Calmette-Guérin (BCG) induces a variety of cytokines into the urine of patients with superficial transitional cell carcinoma (TCC) of the bladder. Recent data have shown that some cytokines have antiangiogenic activity. We sought to determine whether the potently antiangiogenic chemokine interferon-inducible protein 10 (IP-10) and its inducing antiangiogenic cytokines, interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), are increased during intravesical BCG immunotherapy of bladder TCC. METHODS: Voided urine samples were collected sequentially from 8 patients before and after each weekly intravesical BCG treatment and from 4 patients receiving maintenance BCG treatments. The urinary output of IP-10, IFN-gamma, and IL-12 over 12 post-treatment hours was assessed by enzyme-linked immunosorbent assay. In vitro BCG and cytokine stimulations of human TCC and primary endothelial cell lines were also performed, and their supernatants were studied for IP-10. RESULTS: In all cases after intravesical BCG, patient urine was found to contain significant elevations of IP-10. Urinary IFN-gamma and IL-12 levels also increased in similar patterns after intravesical BCG. The peak weekly cytokine response per patient usually occurred between the fourth and sixth treatment for IFN-gamma and IP-10, but was less predictable for IL-12. Human TCC and endothelial cell lines were able to secrete IP-10 in response to BCG or interferon stimulation in vitro. CONCLUSIONS: Our small series demonstrates that IP-10 and its inducing cytokines are elevated in response to intravesical BCG. These data suggest that, in addition to a cellular immune response, BCG may induce a cytokine-mediated antiangiogenic environment that aids in inhibiting future tumor growth and progression.

Administration, Intravesical↗

PubMed1998

Fibrilization in mouse senile amyloidosis is fibril conformation-dependent.

Amyloidosis refers to a group of diseases characterized by tissue deposition of amyloid fibrils. A single intravenous injection of a very small amount of the native mouse senile amyloid fibrils (AApoAII) induced severe systemic amyloid deposition in young mice having the amyloidogenic apoA-II gene (Apoa2c). After AApoAII injection, amyloid deposition occurred rapidly and advanced in an accelerated manner, as observed in spontaneous senile amyloidosis in mice. However, the injection of denatured AApoAII, native apoA-II in high-density lipoprotein (HDL), and denatured apoA-II monomer, which have the same primary structure but without a fibril conformation, did not induce amyloidosis. No amyloid deposition was observed in mice having an amyloid-resistant apoA-II gene (Apoa2b) even 3 months after AApoAII injection. Significantly less amyloid deposition was observed in mice having both types of apoA-II genes heterozygously (Apoa2b/c). These findings suggest that the nucleation-dependent polymerization found in vitro also occurs in vivo, and that the fibril conformation is required for the injected amyloid fibrils to act as seeds in vivo. Fibril conformation-dependent fibrillization is proposed as a general model of the pathogenesis of various kinds of amyloidosis occurring in vivo; it may be useful in both elucidating the pathogenesis of amyloidosis and developing effective therapeutic modalities to treat this disease.

Aging↗

PubMed1998

Intrapersonal and maternal correlates of aggression, conflict, and externalizing problems in toddlers.

Research has shown that 2-year-olds engage in peer-directed aggression and initiation of conflict. However, there has been little consideration of the factors associated with variability in toddlers' aggression. One hundred and four toddlers (52 females) were observed for 35 min of free play with a same-sex peer, with both mothers present. Experience in early out-of-home care was not related to aggression. Toddlers' observed and mother-rated dysregulated temperament, and mothers' use of warmth and negative dominance during interactions with their children, were used to predict toddlers' aggression and maternal ratings of externalizing difficulties. Boys were observed to be more aggressive than girls. Regression analyses showed that, after controlling for main effects, the interaction of child gender, temperament, and maternal negative dominance predicted both outcomes. Observed aggression and mother-reported externalizing problems were associated significantly with dysregulated temperament only for boys with mothers who demonstrated relatively high levels of negative dominance.

Adult↗

PubMed1997

Hsp110 protects heat-denatured proteins and confers cellular thermoresistance.

The 110-kDa heat shock protein (hsp110) has long been recognized as one of the primary heat shock proteins in mammalian cells. It belongs to a recently described protein family that is a significantly diverged subgroup of the hsp70 family and has been found in organisms as diverse as yeast and mammals. We describe here the first analysis of the ability of hsp110 to protect cellular and molecular targets from heat damage. It was observed that the overexpression in vivo of hsp110 conferred substantial heat resistance to both Rat-1 and HeLa cells. In vitro heat denaturation and refolding assays demonstrate that hsp110 is highly efficient in selectively recognizing denatured proteins and maintaining them in a soluble, folding-competent state and is significantly more efficient in performing this function than is hsc70. hsp110-bound proteins can then be refolded by the addition of rabbit reticulocyte lysate or hsc70 and Hdj-1, whereas Hdj-1 does not itself function as a co-chaperone in folding with hsp110. hsp110 is one of the principal molecular chaperones of mammalian cells and represents a newly identified component of the primary protection/repair pathway for denatured proteins and thermotolerance expression in vivo.

Animals↗

PubMed1997

A ribozyme-mediated, gene "knockdown" strategy for the identification of gene function in zebrafish.

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Animals↗

PubMed1997

Protein half-lives of two subunits of an NMDA receptor-like complex, the 71-kDa glutamate-binding and the 80-kDa CPP-binding protein.

We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997, Biochem. Biophys. Res. Commun. 237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (+/- 2.4) and 18 (+/- 1.2) hours for CBP and GBP, respectively.

Animals↗

PubMed1997

Differentiation-dependent expression of obese (ob) gene by preadipocytes and adipocytes in primary cultures of porcine stromal-vascular cells.

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 microg/ml, transferrin 5 microg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.

Adipocytes↗

PubMed1997

Variable efficiency of the thymidine kinase/ganciclovir system in human glioblastoma cell lines: implications for gene therapy.

The gene therapy strategy using the hsvl-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells, is needed. After proposing sensitivity criteria for the TK/GCV system and for the bystander effect, based on the levels of GCV that can be reached in vivo, we studied seven human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. Among six human glioblastoma cell lines stably transfected with the TK gene, five were sensitive to TK/GCV, and two had a good in vitro bystander effect. The in vitro transfectability of the cell lines tested was low (< or = 1%) compared to that of an established animal cell line, C6 rat glioma, in which 20-30% of the cells can be transfected routinely. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is an urgent need for agents to increase transfection efficiency.

Animals↗

PubMed1997

Fidelity of Escherichia coli DNA polymerase III holoenzyme. The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies.

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies finc = 5.6 x 10(-6), 4.2 x 10(-7), and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931-27935 (accompanying paper)).

Catalysis↗

PubMed1997

An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer's disease.

Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

Probing the structure of the cytoplasmic domain of the aspartate receptor by targeted disulfide cross-linking.

Applying the technique of targeted disulfide cross-linking to the cytoplasmic domain of the aspartate receptor of Salmonella typhimurium indicates a generally alpha-helical conformation of the linker region, and a close juxtaposition and a parallel alignment at the interface between the two subunits in the linker region. This conclusion is supported by the results from the Fourier transform of the hydrophobicity values of the amino acid sequences. Aspartate binding in the periplasmic domain causes a closer juxtaposition of the two subunits in the cytoplasmic domain, as indicated by the more rapid disulfide cross-linking on addition of aspartate.

Amino Acid Sequence↗

PubMed1997

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed1997

Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs.

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, (ACAG4TGTG4/TGTC4ACAC4) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we show by high resolution NMR that (C4TGTC4) forms a hairpin dimer with intercalated C+.C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+.C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C4TGTC4ACA4TGTC4) forms an intramolecularly folded hairpin with intercalated C+.C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C4TGTC4ACA)n (n>/=6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite.

Base Sequence↗

PubMed1997

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups.

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the approximately 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (approximately 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.

Alleles↗