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Biomedical subjects

W Qin

Publications and source records attributed to W Qin.

At least 37 records · Page 2Linked to original sources
PubMed2001

Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells.

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg(2+). At 0.5 mM Mg(2+), where only DNA ligase IV is expected to retain activity, low levels of end joining ( approximately 10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg(2+) in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Androstadienes↗
PubMed2001

Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II.

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Animals↗
PubMed2001

Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2.

Mutations in the BRCA1 or BRCA2 genes predispose to a wide spectrum of familial cancers. The functions of the proteins encoded by BRCA1 and BRCA2 remain to be elucidated, but their interaction and colocalization with hRAD51 suggest a role in homologous recombination and DNA double-strand break (DSB) repair. The role of BRCA1 and BRCA2 in the rejoining of ionizing radiation (IR)-induced DNA DSBs, which may represent a step in the overall process of repair, remains uncertain because recent reports provide conflicting results. Because elucidation of the role of these proteins in DNA DSB rejoining is important for their functional characterization, we reexamined this end point in cells with mutations in either BRCA1 or BRCA2. We show that two pancreatic carcinoma cell lines known to have either wild-type (BxPC3) or mutant forms (Capan-1) of BRCA2 rejoin IR-induced DNA DSBs to a similar extent following biphasic kinetics characterized by a fast and a slow component. Importantly, inactivation of DNA-dependent protein kinase (DNA-PK) by wortmannin generates similar shifts from the fast to the slow component of rejoining in BRCA2-proficient and BRCA2-deficient cells. This suggests that the functioning of either the fast, DNA-PK-dependent component or the slow, DNA-PK-independent component of rejoining is not affected by mutations in BRCA2. Also, a human breast cancer cell line with mutated BRCA1 shows normal rejoining of IR-induced DNA DSBs and levels of inhibition by wortmannin commensurate with the degree of DNA-PK inhibition. These observations fail to confirm a direct role for BRCA1 or BRCA2 in the rejoining of IR-induced DSBs in the genome of human tumor cells and, as a result, an involvement in nonhomologous end-joining. They are in line with similar observations with mutants deficient in genes implicated in homologous recombination and support the view that the radiosensitivity to killing of cells deficient in BRCA1 or BRCA2 derives from defects in this repair pathway.

Androstadienes↗
PubMed2001

Full-length cloning and 3'-terminal portion expression of human perforin cDNA.

BACKGROUND: Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. We report the full length of human perforin cDNA, which was cloned from liver tissue. RESULTS: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP. The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein. Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E. coli BL21 (DE3). The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes. CONCLUSION: These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perforin molecule. The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies.

Amino Acid Sequence↗
PubMed2001

Mutational analysis of the structure and functions of hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication. To further understand the structure and functions of NS5B, we introduced a series of 27 clustered and 19 point substitution mutations within and outside the well-known motifs conserved among RdRP by alanine scanning and investigated effects of these mutants on enzymatic activity of NS5B. Surprisingly, most of the mutations (22 of 27 clustered mutants) do not affect RdRP activity at all, indicating that the side chains of the corresponding amino acid residues are dispensable for the catalytic activity. On the other hand, 4 mutants, cm20t, cm194t, cm2t, and cm3t, are defective in RdRP activity. By further analysis with point mutations within these regions, E18, Y191, C274, Y276, and H502 were determined to be critical for the RdRP activity. Y276 was also shown to be critical for RNA template/primer association, although 3 amino acid sequences were identified to be important for RNA template binding by RNA-filter binding assays. Finally, 4 discontinuous sequences of NS5B (aa139-145, aa149-155, aa 365-371, and aa 382-388) were found to be essential for binding to NS5A as determined by glutathione S-transferase (GST)-pull down assays using GST-NS5A and FLAG-NS5B expressed in cotransfected cells, and GST-pull down assay in vitro. In light of the crystal structure models of NS5B recently reported, our results indicate that the RdRP activity of NS5B requires the longer loop and the helix located at the distal of the thumb, which are unique among RdRPs as well as reverse transcriptases.

Amino Acid Sequence↗
PubMed2001

A-6G variant of the angiotensinogen gene and essential hypertension in Han, Tibetan, and Yi populations.

To investigate the relationship between the A-6G variant in the promoter of the angiotensinogen gene and essential hypertension in Han, Tibetan, and Yi populations. All patients with essential hypertension were selected by WHO criteria. And the polymorphism of the A-6G variant was determined by PCR/RFLP. The data were analyzed by t test and chi2 test. There was no significant difference in the genotype or allele frequencies between normotensives and hypertensives in the Han, Tibetan, and Yi populations, respectively. However, when the subjects were divided into male and female subgroups, the genotype distributions among hypertensives and normotensives of the Tibetan female group were as follows: AA, 37% vs. 48%; AG, 52% vs. 48%; GG, 11% vs. 4%, respectively and the frequency of the G allele was significantly higher in hypertensives than in normotensives in the Tibetan female group (0.37 vs. 0.28, chi2=4.25, p<0.05). In addition, we observed that there was a significant difference between the Han and Tibetan normotensive groups in the distributions of the allele and genotype frequencies of the A-6G variant. The frequency of the G allele was 0.29 and 0.17 in the Tibetan normotensive and Han groups, respectively (p<0.001). The G allele of the A-6G variant was associated with hypertension in the Tibetan females, but not in the Yi or Han females. And we confirmed that there was a significant difference in the prevalence of the allele frequencies of the A-6G variant between the Han and Tibetan normotensive groups.

Adult↗
PubMed2001

VEGF-initiated blood-retinal barrier breakdown in early diabetes.

PURPOSE: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels. METHODS: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres. RESULTS: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature. CONCLUSIONS: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.

Animals↗
PubMed2001

Vitamin D receptor gene polymorphism and bone mineral density in patients with type 2 diabetes mellitus.

OBJECTIVE: To explore the relationship between vitamin D receptor (VDR) gene polymorphisms and bone mineral density (BMD) in patients with type 2 diabetes mellitus (DM) and to better understand the pathogenesis of osteoporosis. METHODS: Ninety seven patients with type 2 DM were recruited for this study. BMD was measured by single photon absorptiometry at the lower one third of the nondominant radius and ulna. Polymorphisms of the VDR gene were analyzed by DNA amplification with polymerase chain reaction (PCR) and endonuclease digestion with Bsm I. RESULTS: The respective frequencies of VDR genotypes were BB 18.6%, Bb 27.8% and bb 53.6%. The Z scores of the three groups were -1.57 +/- -0.60, -1.45 +/- -0.67 and -1.41 +/- -0.81, respectively. Although the BMD of the Bb genotype DM patients was higher than that of BB genotype DM patients and lower than that of bb genotype DM patients, there were no significant differences. CONCLUSION: These findings suggest a small influence of VDR gene polymorphism on the BMD of patients with type 2 DM. Further study on the value of VDR genotypes in the pathogenesis of osteoporosis in diabetes mellitus is still needed.

Adult↗
PubMed2001

[Pretreatment of benzoic acid wastewater].

The benzoic acid wastewater produced from toluene by liquid-phase oxidation is one of the difficult wastewater to treat because of multi-component, high CODcr value (20,000-100,000 mg/L) and nonbiodegradability. A pretreatment way of this wastewater by extraction based on chemical complexation was carried out, and tributyl phosphate (TBP), trialkylamine (7301) and trialkyphosphine oxide (TRPO) were used as complexing agent with n-octanol, kerosene as cosolvent and diluent respectively. Results showed that benzoic acid was recovery successfully and the CODcr value of wastewater was reduced to 1/6 of the initial value through multi-stages cross-flow extraction. Back-extraction was quite easy and the solvent could be reused.

Benzoic Acid↗
PubMed2001

[Extraction and stripping of H acid and DSD acid wastewater].

H acid and DSD acid are two important substrate of dyes, which wastewater is treated more difficulty because of containing multi-component, with high chroma value and nonbiodegradability. A treatment way of this wastewater by extraction based on chemical association was carried out. Trialkylamine (Alamine 336) and chlorinated quaternary ammonium salt (Aliquat 336) were used as reacting agent with n-octanol, kerosene as modifier and diluent respectively. Results showed that the extraction efficiency depended on solution pH. Alamine 336 could be used to treat waste water with low pH while Aliquat 336 was high efficiency for waste water with pH > 6. Recovery yield of H acid and DSD acid was greater than 99% and the chroma value of wastewater was reduced effectively by simulating multi-stages cross-flow extraction. Back-extraction is quite easy and the solvent could be regenerated.

1-Octanol↗
PubMed2000

Improved detection limits and unbiased selectivity coefficients obtained by using ion-exchange resins in the inner reference solution of ion-selective polymeric membrane electrodes

By using a high concentration of an interfering ion and a low one of the primary ion in the inner reference solution of polymeric membrane ion-selective electrodes (ISEs), the lower detection limit may be improved and unbiased thermodynamic selectivity coefficients may be obtained. To this purpose, a cation-exchange resin is used here to keep the low concentration of the primary cation constant. Different compositions of the internal solution are required for obtaining optimal lower detection limits and unbiased selectivity coefficients. All ISEs studied here, i.e., for K+, Ca2+, and NH4+, based on valinomycin, ETH 5234, and nonactin/monactin, respectively, show improved lower detection limits in the range of 10(-7.6) (NH4+) to 10(-8.8) M (Ca2+). Nernstian responses and, therefore, unbiased selectivity coefficients are obtained with the K+-ISE for the discriminated ions, Na+, Mg2+, and Ca2+.

Journal Article↗
PubMed2000

Adenosine kinase of Arabidopsis. Kinetic properties and gene expression.

To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis. The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively. Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined. Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively. The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms. In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root. ADK1 transcript levels were generally higher than those of ADK2. ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds. The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis. They may also contribute to cytokinin interconversion.

Adenosine↗
PubMed2000

Low extracellular Ca(2+) activates a transient Cl(-) current in chicken ovarian granulosa cells.

The effects of low Ca(2+) on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca(2+) concentration (2. 5 mM) and with a holding potential of -80 mV, SOC was activated in all cells with command pulses more positive than -20 mV. In 2.5 mM Ca(2+), TOC appeared in 10% of cells at the command pulse of +80 mV and in 60-85% of cells at +100 to +120 mV. In low-Ca(2+) solution and command potential of +80 mV (holding potential of -80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca(2+), and TOC appeared in 43% of the cells that did not express it initially in normal Ca(2+). At both normal and low Ca(2+) levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl(-)-deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl(-) channel blocker. These findings suggest that chicken granulosa cells express a Ca(2+)-inactivated TOC carried by Cl(-). This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca(2+) deficiency.

Animals↗
PubMed2000

Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells.

We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-¿2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbon yl¿eth yl]phenyl]ethylamino-5'- ethylcarboxamidoadenosine ((125)I-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B(max)) of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K(d)) of 1. 17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC(50)): 5'-(N-ethylcarboxamido)adenosine (NECA) >/= CGS-21680 > 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A(2) receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A(2A) adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.

2-Chloroadenosine↗
PubMed2000

[Fluoride-oxide glass for high efficiency upconversion from IR to green].

In this paper, the fluoride-oxide glass doped with Er3+ for upconversion from IR to green was report. The component of sample in experiment is 60TeO2-8PbF2-10AlF3-10BaF2-10NaF-2ErO3/2, and the prepared method is obtained. The absorption spectrum was measured at room temperature. Intense green upconversion emissions from Er3+ doped the sample were observed under 808 nm and 970 nm excitation, and the upconversion emissions spectra under 808 nm and 970 nm LD excitation were measured at room temperature. The upconversion mechanics was discussed. The relationship between working currents of LD and intensity of upconversion emissions has been studied. The results was improved that needs of two photons for upconversion process under 808 nm and 970 nm excitation respectively.

English Abstract↗
PubMed1999

The source of heterogeneity in the heme vicinity of ferricytochrome c.

Heterogeneity in the heme vicinity of ferricytochrome c was reported to be detectable by a split of the NMR signal of the heme methyl 3 group [P.D. Burns and G.N. La Mar, J. Am. Chem. Soc. 101 (1979) 5844]. Using cytochrome c mutants and computer simulations of the native and mutated cytochromes, the source of this heterogeneity is found to originate from the His-33 residue motions. The H33F mutation abolished the NMR split and computer simulations of the H33F mutant revealed a narrower distribution of fluctuations of the radius of gyration, suggesting a more rigid structure due to the mutation. The stabilization of the mutant was further demonstrated by a reduction in the H33F mutant of 4 Kcal/mol in the calculated interaction energy between residue 33 and the rest of the cytochrome, in keeping with known experimental results.

Animals↗
PubMed1999

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

Aging↗