On what makes the gamma subunit spin during ATP hydrolysis by F(1).
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Adenosine Triphosphate↗
Biomedical subjects
H Ren
Publications and source records attributed to H Ren.
Automated parallel DNA sequencing on multiple channel microchips.
We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.
Automation↗
Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides.
In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.
Adenosine Diphosphate↗
Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening.
Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.
Amino Acid Sequence↗
Migration time correction for the analysis of derivatized amino acids and oligosaccharides by micellar capillary electrochromatography.
Migration-time reproducibility is essential in the use of capillary electrophoresis to identify components in mixtures. Two methods based on the migration time of either one or two reference markers are proposed for improving migration time reproducibility. These methods were evaluated to determine the migration time reproducibility for phenylthiohydantoin-amino acids, fluorescein thiohydantoin-amino acids, and tetramethylrhodamine labeled oligosaccharides. In the best case, the relative standard deviation of the migration time was reduced from >3% without correction to <0.04% with the two-marker correction.
Amino Acids↗
Inhibitory Mg-ADP-fluoroaluminate complexes bound to catalytic sites of F(1)-ATPases: are they ground-state or transition-state analogs?
Schemes are proposed for coupling sequential opening and closing the three catalytic sites of F(1) to rotation of the gamma subunit during ATP synthesis and hydrolysis catalyzed by the F(o)F(1)-ATP synthase. A prominent feature of the proposed mechanisms is that the transition state during ATP synthesis is formed when a catalytic site is in the process of closing and that the transition state during ATP hydrolysis is formed when a catalytic site is in the process of opening. The unusual kinetics of formation of Mg-ADP-fluoroaluminate complexes in one or two catalytic sites of nucleotide-depleted MF(1) and wild-type and mutant alpha(3)beta(3)gamma subcomplexes of TF(1) are also reviewed. From these considerations, it is concluded that Mg-ADP-fluoroaluminate complexes formed at catalytic sites of isolated F(1)-ATPases or F(1) in membrane-bound F(o)F(1) are ground-state analogs.
Adenosine Diphosphate↗
Hepatitis B virus X protein activates expression of IGF-IR and VEGF in hepatocellular carcinoma cells.
OBJECTIVE: The expression of insulin-like growth factor I receptor (IGF-IR) and the vascular endothelial growth factor (VEGF) in HepG(2) cells transfected with a hepatitis B virus X (HBx) expression vector was investigated in an attempt to study their possible relationship to the growth of HBx-induced hepatocellular carcinoma (HCC). METHODS: The eucaryotic expression vector of HBx gene was constructed and introduced into HepG(2) cells. The modified cell HepaG(2)-X was synchronized in a quiescent state by culture of serum deprivation. The IGF-IR and VEGF were analyzed by immunohistochemical and Western blot technique. RESULTS: The positive rate of IGF-IR expression was 84%A3% in the transfected HBx gene cells, 26%A4% in X(0) control cells. The positive rate of VEGF expressed x cells was 83%A5%, X(0) cells was 28%A6% (P<0.001). The level of IGF-IR and VEGF in serum-starved x modified cells was 1.5 times higher than that of synchronized X(0) modified cells. CONCLUSION: Since the IGF-IR is a very important growth factor in sustaining the tumor abnormal growth and the VEGF has a crucial role in inducting tumor angiogenesis, our findings indicate that HBx may play an important role in the processes of HCC by activating IGF-IR and VEGF gene expression.
Carcinoma, Hepatocellular↗
[Effects of hepatitis B virus X gene on apoptosis and the activity of telomerase in HepG(2) cells].
OBJECTIVE: To observe the effects of hepatitis B virus X gene on hepatic carcinoma. METHODS: The expression vectors of HBs gene and HBc gene (pCEP4-S and pCEP4-C) were transfected into HepG(2) cells by electroporation technique, and the cells, HepG(2) cells which contained the expression vectors of HBs (pCEP4-X) were recovered and cultured stably. The activity index of telomerase and the apoptosis rates in three cell lines were determined by PCR-ELISA and flow cytometry. RESULTS: The 39.50% of cells expressed HBx gene came into S-phase of cell cycle, and the activity index of telomerase was higher in the cell line (3.95 +/- 0.07) than in other cell lines and control (F=20.85, P<0.01; d'=2.15, P<0.05) after synchronized culture. Induced by antisense oligonucleotides against telomerase the apoptosis of cell line expressed HBx gene reduced (apoptosis rate: 1.75%). The activity of cells also reduced with the dose increase of the antisense oligonucleotides and it showed a dose-response relationship. CONCLUSION: The activity of HepG(2) cell can be increased and the cell apoptosis can be reduced by HBx gene. It may be another malignant process of hepatocytes.
Apoptosis↗
[Resistance of Bcl-2 adenovirus vector to HepG(2) cell apoptosis induced by ethanol].
OBJECTIVE: To evaluate the role of Bcl-2 family proteins in hepatocytic apoptosis caused by ethanol. METHODS: We induced hepatocytic apoptosis in HepG(2) cells with ethanol and detected the apoptosis with TUNEL assay. Bcl-2, Bax, and Bak protein expression in the hepatocytes was determined by immunohistochemistry method. We also observed the expression of Bcl-2 protein in HepG(2) cells infected with Bcl-2 adenovirus vector and its protection against hepatocytic apoptosis caused by ethanol. RESULTS: No obvious cytotoxicity was noted in 0.2% and 1% ethanol treatment group, while it developed in 3% ethanol treatment group and accompanied by a marked expression of Bax, Bak proteins. CONCLUSION: The overexpression of Bax, Bak proteins may play a role in HepG(2) cell apoptosis induced by ethanol and can be blocked effectively by Bcl-2 adenovirus vector.
Adenoviridae↗
Fas ligand expression and apoptosis in primary rat hepatocytes induced by lipopolysaccharide.
OBJECTIVE: To study hepatocyte apoptosis induced by lipopolysaccharide (LPS) directly and indirectly, and to elucidate the mechanisms of liver damage in endotoxemia. METHODS: Rat hepatocytes were isolated using collagenase perfusion, and cultured in RPMI 1640 medium. After 24h or 48 h of LPS treatment at various concentrations (1, 5, 10 mug/ml), membrane-bound Fas ligand (mFasL) expression in hepatocytes was determined by immunocytochemistry, and apoptosis was detected by TUNEL. In another set of experiments it was examined whether LPS-treated hepatocytes and its supernatants can stimulate apoptosis in LPS-untreated hepatocytes. RESULTS: LPS markedly stimulated mFasL expression and apoptosis in hepatocytes in a dose and time (24-48 h) dependent manner. In the co-culture system LPS-treated hepatocytes significantly induced LPS-untreated hepatocyte apoptosis. In contrast, there was no apoptotic cells observed in the supernatant stimulation system. CONCLUSION: LPS not only directly causes hepatocyte apoptosis, but also indirectly induces apoptosis of LPS-untreated hepatocytes by way of stimulating mFasL expression in hepatocytes.
Animals↗
Construction of the recombinant retrovirus vector of HBV-S gene and it's expression in eukaryotic cells.
OBJECTIVE: To investigate the effectiveness of recombinant retrovirus vector in gene therapy. METHODS: The retroviral vector PLXSN-S was constructed and transferred into PA317 by means of electroporation, then HepG(2), P815, and EL4 cells were infected with the pseudovirus produced from PA317, which highly expressed HBsAg. HBsAg expression was tested by RT-PCR and ELISA. RESULTS: HBsAg was expressed variously in the eukaryotic cells mentioned above. HBsAg (A value) of the cell supernatants (48 h) were 0.92, 0.09, 0.47, respectively. CONCLUSION: The vector used in this study is an effective one to carry genes of interest to target cells and it may be useful in the test for gene therapy.
Cell Line↗
[Antigen presenting role of dendritic cell proliferated from peripheral blood monocytes in patients with chronic hepatitis B].
OBJECTIVE: To study the presenting effect of dendritic cell (DC) proliferated from peripheral blood monocytes in patients with chronic hepatitis B on HBsAg. METHODS: DC proliferated from patient's peripheral blood monocytes by adding GM-CSF and IL-4 were incubated with HBsAg for 1.5h at different concentrations, then mixed with autologous T cell and cocultured in 96-well flat-bottomed microtiter plates for 5 d at 37 degrees C and 5% CO(2). (3)H-TdR was added 13h before the culture completion, and finally the cells were gathered and the cpm was tested. RESULTS: The proliferation of T cell by DC treated by HBsAg is significantly higher than untreated. CONCLUSION: DC cultured from peripheral blood monocytes in patients with chronic hepatitis B plays a role in presenting HBsAg.
Antigen Presentation↗
A clinical study on treatment of diabetic peripheral neuropathy with tang zhi min capsules.
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Adult↗
[Response of wheat seedlings with different drought resistance to water deficiency and NaCl stresses].
The growth, photosynthesis, transpiration and antioxidative defence system of the seedlings of drought-tolerant wheat strain 8139 and drought-sensitive strain Ganmai No. 8 at 20% PEG 6000 and 1.2% NaCl stresses were compared. The results showed that strain 8139 had a strong drought resistance, but a weak salt resistance. The root growth of both wheat strains was inhibited significantly under salt stress, but stimulated slightly under drought stress. The net photosynthetic rate and water use efficiency of strain 8139 were significantly different from those of Ganmai No. 8 at every stage under both drought and salt stresses, and its transpiration rate was significantly different from that of Ganmai No. 8 only at 7th and 14th day after being stressed. The MDA content in strain 8139 after being stressed for 7 days was much lower than that in Ganmai No. 8 under drought stress, but there was no significant difference between the two strains under NaCl stress. Correspondingly, there was no significant difference in the fructan content and SOD and APX activities between strain 8139 and Ganmai No. 8, but a significant difference in GSH content was found under salt stress. Under drought stress, the contents of fructan and GSH and the activities of SOD and APX in strain 8139 were much higher than those in Ganmai No. 8 at different stage, and strain 8139 exhibited a strong antioxidative defence ability.
Disasters↗
[Effect of elevated CO2 concentration on photosynthesis and antioxidative enzyme activities of wheat plant grown under drought condition].
The photosynthesis and antioxidative enzyme activities of wheat plants grown in two open-top chambers with CO2 concentrations of 350 mumol.mol-1 and 700 mumol.mol-1 were examined under drought stress. The result showed that elevated CO2 concentration obviously enhanced the photosynthesis, stomatal resistance and water use efficiency, but decreased the transpiration of wheat. Doubled CO2 concentration significantly increased the activities of CAT, POD and SOD, which enhanced the abilities of antioxidative defence and drought tolerance.
Carbon Dioxide↗
[Concept of ecosystem management and its essential elements].
Ecosystem management originates from the tradition fields of natural resources management and utilization, and develops in the 1990's. Based on our best understanding of the ecosystem composition, structure and function, and in definite spatiotemporal scales, it integrates human values and social-economic principles into managing ecosystems to resotre and/or sustain ecosystem integrity and sustainability. Ecosystem management requires the collection of field data and the monitoring of ecosystem dynamics at multiple scales. The essential elements of ecosystem management include clear management goals, definite ecological boundaries and units, sound ecological understanding, appropriate scale and hierarchic structure, understanding of ecosystem uncertainty, adaptive management, cooperation between agency and individuals, and viewing human and its value as ecosystem components. The goal of ecosystem management focuses on ecosystem sustainbility.
Ecosystem↗
Relationship between serum or tissue ouabain and blood pressure in 1k1c hypertensive rats.
OBJECTIVE: To evaluate the role of endogenous ouabain (EO) in the development of hypertension and the characteristics of EO secretion in 1k1c (one kidney, one clipped) hypertensive rats. METHODS: EO content of serum and tissues in 1k1c hypertensive rats and normal control Sprague-Dawley (SD) rats was detected by the method of enzyme linked immunosorbent assay (ELISA). The relationship between serum or tissue ouabain and blood pressure was analyzed in 1k1c hypertensive rats. RESULTS: The ouabain content of serum, heart, kidney, adrenal gland, pituitary and hypothalamus was significantly higher in 1k1c hypertensive rats than that in normal control SD rats (2.25, 2.63, 3.35, 40.37, 3.34, 15.7 micrograms/kg tissue in 1k1c hypertensive rats vs 1.12, 1.79, 1.73, 27.54, 1.83, 10.10 micrograms/kg tissue in control SD rats, respectively. P < 0.05 for all of these comparisons). The ouabain content of the adrenal gland and the hypothalamus was higher than that of other tissues or serum, both in 1k1c rats and in control SD rats. The EO content of serum, kidney and hypothalamus was significantly correlated with blood pressure in 1k1c hypertensive rats (r = 0.59, 0.63, 0.52, respectively. P < 0.05). The ouabain content of heart, liver, adrenal gland and pituitary was not correlated with blood pressure. CONCLUSIONS: EO might play an important role in the development of hypertension in 1k1c hypertensive rats. The adrenal gland may be a major source of EO and the hypothalamus-pituitary-adrenal axis may be involved in the regulation of EO secretion.
Animals↗
[The mechanism of water retention in congestive heart failure].
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Animals↗