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Biomedical subjects

W Qin

Publications and source records attributed to W Qin.

At least 55 records · Page 3Linked to original sources
PubMed1999

Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c.

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method. Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated. For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn). In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Algorithms↗
PubMed1999

Oxidative myocytes of heart and skeletal muscle express abundant sarcomeric mitochondrial creatine kinase.

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.

Animals↗
PubMed1999

Mammary gland expression of mouse mammary tumor virus is regulated by a novel element in the long terminal repeat.

Mouse mammary tumor virus (MMTV) infects both lymphoid tissue and lactating mammary gland during its infectious cycle, but some endogenous MMTVs are transcribed only in lymphoid cells. We found a lymphoid cell-specific endogenous MMTV that was converted to a milk-borne, infectious virus through recombination with an exogenously transmitted MMTV. The changed expression pattern correlated with the alteration of a single base pair in the long terminal repeat of the lymphoid cell-specific virus. Transgenic mice with the element from either the milk-borne or lymphoid cell-specific virus upstream of the chloramphenicol acetyltransferase reporter gene showed the same pattern of expression as the virus from which the regulatory sequences were derived. Electrophoretic mobility shift assays with mammary cell extracts showed that the site from the milk-borne virus was preferentially bound by a prolactin-inducible factor that poorly bound the altered site from the lymphoid cell-specific virus. The complex that formed on the milk-borne virus-specific oligonucleotide supershifted with anti-Stat5b antibody. Mice lacking either Stat5a or Stat5b had dramatically reduced levels of MMTV transcripts in mammary gland but not in lymphoid tissue. Thus, a member of the STAT family of transcription factors is involved in the tissue-specific expression of mouse mammary tumor virus in vivo. This is the first example of the involvement of a member of the STAT family of transcription factors in the control of tissue-specific expression.

Animals↗
PubMed1999

Insulin-induced vascular endothelial growth factor expression in retina.

PURPOSE: Clinical studies have demonstrated that intensive insulin therapy causes a transient worsening of retinopathy. The mechanisms underlying the initial insulin-induced deterioration of retinal status in patients with diabetes remain unknown. Vascular endothelial growth factor (VEGF) is known to be operative in the pathogenesis of diabetic retinopathy. The current study was conducted to characterize the effect of insulin on retinal VEGF gene expression in vitro and in vivo. METHODS: The effect of insulin on VEGF expression in vivo was examined by in situ hybridization studies of rat retinal VEGF transcripts. To examine the mechanisms by which insulin regulates VEGF expression, human retinal pigment epithelial (RPE) cells were exposed to insulin, and VEGF mRNA levels were quantified with RNase protection assays (RPAs). Conditioned media from insulin-treated RPE cells were assayed for VEGF protein and capillary endothelial cell proliferation. The capacity of insulin to stimulate the VEGF promoter linked to a luciferase reporter gene was characterized in transient transfection assays. RESULTS: Insulin increased VEGF mRNA levels in the ganglion, inner nuclear, and RPE cell layers. In vitro, insulin increased VEGF mRNA levels in human RPE cells and enhanced VEGF promoter activity without affecting transcript stability. Insulin treatment also increased VEGF protein levels in conditioned RPE cell media in a dose-dependent manner with a median effective concentration of 5 nM. The insulin-conditioned RPE cell media stimulated capillary endothelial cell proliferation, an effect that was completely blocked by anti-VEGF neutralizing antibody. CONCLUSIONS: Insulin increases VEGF mRNA and secreted protein levels in RPE cells through enhanced transcription of the VEGF gene. Intensive insulin therapy may cause a transient worsening of retinopathy in patients with diabetes through increased retinal VEGF gene expression.

Animals↗
PubMed1999

[Investigation of factors affecting the development of COPD].

OBJECTIVE: To investigate the factors which affect the development of COPD. METHODS: 88 patients with mPAP less than 20 mm Hg were followed up by 5 - 20 years. Pulmonary ventilation, blood gas analysis, ECG, pulmonary hemodynamic parameters and oxygen saturation at night were performed one time every 5 years. RESULTS: The results showed that: 5 year, 10 year and 20 year's mortality is 6%, 25% and 43% respectively; MVV%, FEV(1)% and PaO(2) declined accompanied by the increase of PaCO(2); among 41 patients with mPAP 16 - 20 mm Hg, 20 patients' mPAP were more than 20 mm Hg and 15 patients died after 10 years' follow up. Among 16 patients with the declination of oxygen saturation, 8 patients' mPAP were more than 20 mm Hg and 6 patients died. 57 strains of infectious bacteria were found in 88 patients' sputum with 43 strains in the death group. CONCLUSIONS: These results demonstrated that COPD is a kind of chronic diseases with high death rate; infection, decline of oxygen saturation at night and mPAP more than 16 mm Hg were high risk factors in COPD.

Adult↗
PubMed1999

[Left ventricular function evaluation after mitral valve replacement with preservation the subvalvular apparatus].

The objective of this study was to evaluate the left ventricular function after mitral valve replacement (MVR) with preservation of all chordae. Fourteen patients with a diagnosis of mitral regurgitation were studied. These patients, 4 males and 10 females, age from 24 to 59 years, who underwent mitral valve replacement, in which all chordae tendineae were preserved. The mean follow-up interval was 6 months. Preoperation and postoperation multigated equilibrium radionuclide angiography was performed by Elscint Helix Apex SPECT. Each patient was injected with 740MBq 99mTc-HSA, rest ejection fraction (global EF) of LV (%), regional ejection fraction (EF) of LV (%), PER/S and PFR/S were measured. Regional EF was calculated by the method of dividing the left ventricle into five segments by radial axis. The results showed that the postoperative EF of the whole left ventricle of the chordae-preserved patients was significantly greater than their preoperative EF (40.0% +/- 17.8%, to 51.6% +/- 18.2%, P < 0.02), and also the regional EF at the lateral wall of the chordae-preserved MVR was significantly greater than the preoperative regional EF (an increase from 51.0% +/- 18.1% to 69.7% +/- 21.2%, P < 0.01). MVR with preservation of all chordae, the patient's left ventricular function remarkably improved.

Adult↗
PubMed1998

[Reference value of urinary arsenic for nonoccupational exposed subjects in Henan].

Urinary arsenic levels of 2065 nonoccupational exposed subjects in Henan were determined by spectrophotometric analysis. The results showed: (1) The difference was not significant among Zhengzhou, Luoyang, Anyang, Xinyang and Shangqiu (0.01 < P < 0.025). (2) The difference was not significant among different ages(0.01 < P < 0.025) and between sexes(P > 0.05). (3) The distribution was positive skewness. The median of urinary arsenic was 17.8 micrograms/L. The arithmetic mean was 20.7 micrograms/L. The standard deviation was 15.2 micrograms/L. The geometric mean was 17.3 micrograms/L. (4) It is suggested that the reference upper limit value of urinary arsenic for nonoccupational exposed subjects in Henan in 56 micrograms/L.

Adolescent↗
PubMed1998

Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues.

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

Amino Acids, Diamino↗
PubMed1998

Chemiluminescence flow-through sensor for copper based on an anodic stripping voltammetric flow cell and an ion-exchange column with immobilized reagents.

A novel chemiluminescence (CL) flow-through sensor based on immobilizing all the ingredients involved in the analytical reaction for the determination of copper is proposed. The analytical reagents including luminol and cyanide were coimmobilized permanently on an anion-exchange column, while the analyte copper was retained temporarily by electrochemical preconcentration on a gold electrode placed in an anodic stripping voltammetric flow cell. By injection of a volume of sodium hydroxide through the column with immobilized reagents, luminol and cyanide were eluted from the resins in alkaline aqueous solution and then reacted with copper stripped from the gold electrode to produce a CL signal, by means of which copper could be sensed. The sensor was not susceptible to interference by other metal ions associated with the CL reaction. The response to the concentration of copper was linear in the range of 0.01-10 micrograms/L and an extremely low detection limit of 8.0 x 10(-3) micrograms/L was achieved. A complete analysis could be performed in 4 min with a relative standard deviation of less than 8%. The column with immobilized reagents was readily prepared and could be reused over 200 times. The sensor was applied successfully to the determination of copper in human serum and natural water samples.

Chromatography, Ion Exchange↗
PubMed1998

RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

Amino Acid Sequence↗
PubMed1998

The human trifunctional enzyme of de novo purine biosynthesis: heterologous expression, purification, and preliminary characterization.

The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes glycinamide ribonucleotide synthetase, aminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity. The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.

Carbon-Nitrogen Ligases↗
PubMed1998

Protein kinase C alpha modulates growth and differentiation in Caco-2 cells.

BACKGROUND & AIMS: Caco-2 cells have been used extensively to elucidate events involved in intestinal cell proliferation and differentiation. Because individual isoforms of protein kinase C (PKC) and p21waf1, a cyclin-dependent kinase inhibitor, may regulate these processes, their role(s) on the growth and differentiation of Caco-2 cells were assessed. METHODS: Protein abundance and subcellular distribution of several PKC isoforms, as well as the expression of p21waf1, were examined in preconfluent and postconfluent cells. RESULTS: In cells at confluence (approximately 7 days postplating) and during their postconfluent phase (up to 20 days postplating), both total protein expression of PKC-alpha and its particulate distribution increased compared with their 3-day postplated counterparts. These findings were in agreement with those obtained by immunocytochemistry of PKC-alpha. In contrast, neither the total expression nor the subcellular distribution of PKC-betaI, -betaII, -delta, or -zeta changed significantly during these time periods. In addition, the expression of p21waf1, which can be induced by PKC-alpha, increased in postconfluent cells. CONCLUSIONS: PKC-alpha, but not other isoforms of PKC, may modulate the proliferation and differentiation of Caco-2 cells. This regulation appears to be mediated, at least in part, via a mechanism involving p21waf1.

Caco-2 Cells↗
PubMed1998

[Changes of carbohydrate metabolism in normal pregnancy and its relationship with placental lactogen concentrations].

OBJECTIVE: To observe the changes of glucose metabolism and its relationship with human placental lactogen (HPL) in normal pregnant women. METHOD: 94 normal pregnant women had their serum HPL concentration tested and had undergone a 75 g oral glucose tolerance tests (OGTT). Both serum insulin and glucose levels at different time intervals were determined, and the areas under curve (IAUC, GAUC) were caculated, also insulin resistance (IAI) was evaluated. RESULTS: GAUC, IAUC, IAI and HPL are all significantly increased with advance of gestation (P < 0.05). Multiple correlation coefficient study demonstrated that HPL is significantly positively correlated with GAUC, IAUC and IAI(P < 0.001). But when IAUC and IAI were controlled, the relationship between HPL and GAUC was not found. CONCLUSION: There are hyperinsulinemia and insulin resistance during pregnancy, and they are related to HPL. HPL is one of the factors that contributing to carbohydrate metabolism changes during pregnancy.

Adult↗
PubMed1998

[Ileocolonic pouch with appendix in situ through umbilicus as a continent conduit].

OBJECTIVE: To try to use appendix in situ without any special treatment as a conduit of the continent cutaneous ileocolonic reservoir. METHODS: An isolated ileocolonic segment, about 20 cm of terminal ileum and 20 cm of ascending colon, was detubularized by antimensenteric splitting and a pouch was completed by side-to-side anastomosis of the colon and the ileum loop. Both ureters were implanted into two separate submucosal tunnels of 3 cm long into the colon wall of the pouch. The appendix after tail was amputated was passed through the umbilicus hole with which the base of the cecum was sutured. RESULTS: As a conduit of the pouch, the appendix had a good continent ability when 8 patients were followed up for 10 to 30 months after operation. It was easy and convenient for the patients to make self-catheterization. The mean capacities of the pouch were 500-700 ml and then the pressures were 20-45 cm H2O while it was filled. There were no definite complications such as urine leakage, stoma stenosis, ureter reflux, and hydroureteronephrosis. CONCLUSION: The operation of the ileocolonic pouch with the appendix as a continent umbilical stoma is simple and easy to perform and has such advantages as good continent, convenient self-catheterization and provides a quality life for the patients undergoing total cystectomy.

Adult↗
PubMed1998

Molecular characterization of the creatine kinases and some historical perspectives.

Over the last 15 years, molecular characterization of the creatine kinase (CK) gene family has paralleled the molecular revolution of understanding gene structure, function, and regulation. In this review, we present a summary of advances in molecular analysis of the CK gene family with a few vignettes of historical interest. We describe how the muscle CK gene provided an essential model system to examine myogenic regulatory mechanisms, leading to the discovery of the binding site for the MyoD family of basic helix-loop-helix transcription factors essential in skeletal myogenesis and the characterization of the MEF2 family of factors with an A/T rich consensus binding site essential in skeletal myogenesis and cardiogenesis. Cloning and characterization of the four mRNAs and nuclear genes encoding the cytosolic CKs, muscle and brain CKs, and the mitochondrial (Mt) CKs, sarcomeric MtCK and ubiquitous MtCK, has allowed intriguing study of tissue-specific and cell-specific expression of the different CKs and analysis of structural, functional, regulatory, and evolutionary relationships among both the four CK proteins and genes. Current and future studies focus on understanding both cellular energetics facilitated by the CK enzymes, especially energy channelling from the site of production, the mitochondrial matrix and inner membrane, to various cytosolic foci of utilization, and regulation of MtCK gene expression at the cell and tissue-specific level as models of regulation of energy producing genes.

Amino Acid Sequence↗
PubMed1997

Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium.

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9. PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha. A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta. Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLDgamma and -beta are most active at microM Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [3H]PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha. These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha. The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.

Amino Acid Sequence↗
PubMed1997

Elements regulating cardiomyocyte expression of the human sarcomeric mitochondrial creatine kinase gene in transgenic mice.

Sarcomeric mitochondrial creatine kinase (sMtCK) is one component of a multiprotein, high energy channeling complex consisting of porin, mitochondrial creatine kinase, and adenine nucleotide translocase. To study the transcriptional mechanisms specifying sMtCK gene expression to the heart, transgenic mice were created carrying the 5'-flanking sequences of the human sMtCK gene ligated upstream of the human growth hormone (hGH) reporter gene. RNA blot hybridization demonstrated that the human sMtCK sequence, -485 to +6 base pair (bp), did not activate reporter gene expression to a detectable level. However, the human sMtCK sequence, -921 to +6 bp, expressed the hGH reporter gene at a high level in heart and skeletal muscle and at a very low level in esophagus and kidney, and it did not express the hGH gene in other organs tested (brain, lung, liver, spleen, bladder, uterus, and stomach). In situ hybridization revealed that reporter gene transcription was specified to cardiac and skeletal myocytes, recapitulating precisely the expression pattern of the endogenous gene. Sequence analysis identified several consensus binding sites between -921 and -757 bp, including four GATT motifs, one E box, and one MEF2 site. Further analysis of a third transgenic mouse strain demonstrated that the human sMtCK sequence, -757 to +6 bp, did not direct detectable expression of the hGH reporter gene. We conclude that this 160-bp genomic sequence, from -921 to -757 bp, is necessary in specifying expression of the human sMtCK gene to the oxidative and highly metabolically active heart tissue.

Animals↗