Biomedical subjects

Qin Wei

Publications and source records attributed to Qin Wei.

At least 19 recordsLinked to original sources

PubMed2006

Proteasome inhibition down-regulates endothelial nitric-oxide synthase phosphorylation and function.

Endothelial nitric-oxide synthase (eNOS) function is fundamentally modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (bovine)/1177 (human) by Akt has been shown to be the central mechanism of eNOS regulation. Here we revealed a novel role of proteasome in controlling eNOS serine 1179 phosphorylation and function. Rather than affecting eNOS turnover, proteasomal inhibition specifically dephosphorylated eNOS serine 1179, leading to decreased enzymatic activity. Blocking protein phosphatase 2A (PP2A) by okadaic acid or PP2A knockdown restored eNOS serine 1179 phosphorylation and activity in proteasome-inhibited cells. Although total PP2A expression and activity in cells were not affected by proteasome inhibitors, proteasomal inhibition induced PP2A ubiquitination and ubiquitinated PP2A translocated from cytosol to membrane. Further biochemical analyses demonstrated that eNOS associated with PP2A on cell membranes. Proteasomal inhibition markedly enhanced PP2A association to eNOS, and this increase of PP2A dephosphorylated eNOS and its upstream kinase Akt. Taken together, these studies identified a novel pathway in which proteasome modulates eNOS phosphorylation by inducing intracellular PP2A translocation.

Animals↗

PubMed2006

A highly sensitive and rapid assay for protein determination in human urine and penicillin using a Rayleigh light-scattering technique with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt.

A simple and sensitive method was conducted for the determination of trace amounts of proteins with benzeneazo-8-acetylamino-1-naphthol-3,6-disulfonic acid sodium salt (azophloxine, AP) using a Rayleigh light-scattering (RLS) technique. At pH 2.60 and in the presence of an emulsifier OP microemulsion, the RLS of AP can be greatly enhanced by proteins, owing to the interaction between AP and protein. The enhanced intensity is proportional to the concentration of proteins. Four proteins, including bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lys) and gamma globulin (gamma-G) have been tested. For example, the linear range of BSA was 0 - 0.06 microg mL(-1) with detection limits of 2.38 ng mL(-1). The method was applied to the analysis of protein in human urine and penicillin samples with satisfactory results. The relative standard deviation was in all instances less than 4.0%, and the recovery was in the range of 97.5 - 104%.

Azo Compounds↗

PubMed2006

[Microemulsion sensitized fluorophotometric determination of protein with probe of phenylfluorone-molybdenum (VI) complex].

The fluorescence spectral behavior of interaction of phenylfluorone(PF)-Mo(VI) and protein was investigated in Triton X-100 microemulsion medium at pH 2.0. A novel method for the determination of protein using phenylfluorone (PF)-Mo(VI) as a fluorescence spectrum probe was developed. Excitation and emission wavelengths are 465 and 525 nm, respectively. The effective factors and the optimum conditions have been studied, and the reducing value of fluorescence intensity is in proportion to the concentration of proteins in the range 0-6.00 microg x L(-1) for bovine serum albumin, 0-4.00 microg x L(-1) for human serum albumin, 0-5.00 microg x L(-1) for ovalbumin, and 0-4.00 microg x L(-1) for lysozyme. The Triton X-100 microemulsion was efficiently used to enhance the sensibility and stability of the system, and the limits of detection are 5.4, 5.2, 1.5 and 8.2 ng x L(-1), respectively. Most of foreign substances do not interfere with the determination, and this method has good selectivity and high sensitivity. It has been applied to the determination of proteins in the urine samples with satisfactory results.

Animals↗

PubMed2006

[Study on fluorescence spectral behavior of trimethoxyphenylflurone with gallium in microemulsion].

In the present paper, the fluorescence spectral behavior of gallium with trimethoxyphenylflurone (TM-PF) in microemulsion was studied. In the buffer medium of HAc-NaAc at pH 4.95, the fluorescence intensity is in proportion to the concentration of gallium (III) in the range of 0-0.045 microg x mL(-1). The CTMAB microemulsion was efficiently used to enhance the sensibility and stability of the system, and the limits of detection were 1.2 ng x mL(-1). The coexistent metal ions can be separated and gallium can be enriched by the extraction of butyl acetate, greatly improving the selectivity and sensibility of the system. The method possesses high sensitivity as well as high selectivity. It can be used to determine trace amount of gallium in ore samples successfully.

English Abstract↗

PubMed2006

[Cloning, prokaryotic expression of chicken interferon-alpha gene and study on antiviral effect of recombinant chicken interferon-alpha].

The full length of chicken interferon alpha (ChIFN-alpha) gene was amplified by the polymerase chain reaction (PCR) from total liver genome of Sanhuang meat-chicken and sequenced. The amplified gene was about 582bp. The coding region for mature protein (489bp) was subcloned into pET-28a(+). The recombinant plasmid pET-28a(+)-IFNalpha was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE and Western-blot indicated that a 22kD fusion protein was expressed in the form of inclusion bodies with good immunity. The purity of inclusion bodies was above 70% and that of protein purified by nickel affinity chromatography was 95%. The recombinant protein could inhibit H9N2 avian influenza virus (H9N2 AIV) replication on chick embryo fibroblast. 2 microg of recombinant IFN-alpha could completely protect Chick embryo from H9N2 AIV infection. The recombinant IFN-alpha can also delay Newcastle disease virus (NDV) replication on chick embryo for 12 approximately 48h. Chicken administered recombinant IFN-alpha can resist the H9N2 AIV infection. The bioactivities of recombinant IFN-alpha purified by affinity chromatograph were 20 times higher than that of inclusion bodies.

Animals↗

PubMed2006

[Atomic emission spectrometry determination of Au, Pt and Pd after separation and enrichment by hyperbranched polymer].

The present paper shows that the trace amount of gold, platinum and palladium in hydrochloric acid solution can be concentrated by hyperbranched polymer. The new reagent has a rapid adsorption rate and big concentrating capacity. The determination of trace Au, Pt and Pd in sample using carbon powder and strontium carbonate as buffer was carried out by atomic emission spectrometry(AES). Zirconium was selected as internal standard line. The sample was directly loaded into ordinary electrode. The method is simple, rapid and accurate. The condition of determination, and factors of influence were studied. The analysis line of Au, Pt and Pd is 312.3, 306.5 and 311.4 nm respectively. The internal standard line of Zr is 310.7 nm. The linear range of the determination of Au, Pt and Pd is 0-0. 20%, 0-0. 40% and 0-0. 20% respectively. The detection limit of Au, Pt and Pd is 0.010%, 0.0030% and 0.0030% respectively. The method has been applied to the determination of Au, Pt and Pd with satisfactory results.

English Abstract↗

PubMed2006

[Effective constitutents contents in indigowood roots and leaves from different regions].

By the method of HPLC, and with indigo and indirubin as indicative constituents, this paper determined the effective constituents contents in indigowoad roots and leaves collected from different regions. The results showed that the total content of effective constituents in indigowoad root was the highest (16.27 mg x kg(-1)) from Hanyin of Shaanxi Province, followed by Longde of Ningxia Autonomous Region (15.67 mg x kg(-1)), Bozhou of Anhui Province (14.90 mg x kg(-1)), and Linquan of Anhui Province and Jiamusi of Heilongjiang Province (13.97 mg x kg(-1)), and that in indigowoad leaf had the same order, with the values being 698.32, 683.68, 680.11, 654.19 and 642.23 mg x kg(-1), respectively. The HPLC chromatograms of leaf samples collected from different regions were differed, not only in the test constituents, but also in the other constituents manifested as various peaks in the chromatogram. It was suggested that besides meteorological factors, the selection of geo-authentic region should be also involved in the cultivation of Isatis indigotica.

China↗

PubMed2006

[Study on the mechanism of interaction between TH-PF-Mo(VI) complex and bovine serum albumin by fluorimetric method].

The mechanism of interaction between bovine serum albumin (BSA) and trihydroxylphenylfluorone (TH-PF)-Mo(VI) complex in neutral solution was studied by fluorimetric method. The mechanism of fluorescence quenching of BSA caused by (TH-PF)-Mo(VI) complex probe was investigated and the binding constants under different temperature were measured. The binding constants of the reaction at 25 degrees C and 40 degrees C were calculated by fluorimetric method to be 4.78 x 10(4) L x mol(-1) and 3.72 x 10(4) L x mol(-1), respectively. According to the theory of Forster non-radiation energy transfer, the binding distance and transfer efficiency at 25 degrees C were calculated to be 2.89 nm and 0.314, respectively. Furthermore, the thermodynamic parameters were measured and the results indicated that electrostatic force played a major role in the interaction between TH-PF-Mo(VI) complex and BSA.

Animals↗

PubMed2005

Quenching of the intrinsic fluorescence of bovine serum albumin by phenylfluorone-Mo(VI) complex as a probe.

In this paper, the binding characteristics of bovine serum albumin (BSA) and phenylfluorone (PF)-molybdenum (Mo(VI)) complex have been studied by fluorophotometry. The binding constants are calculated at different temperatures. The binding distance and the energy transfer efficiency between PF-Mo(VI) complex and protein are obtained on the basis of the theory of Forster energy transfer. DeltaH and DeltaS are calculated to be -7.11 kJ mol-1 and 70.30 J mol-1 K-1, which indicate that electrostatic force plays major role in the interaction of PF-Mo(VI) complex and BSA. The experimental results show that BSA and PF-Mo(VI) complex have strong interactions and the mechanism of quenching belongs to static quenching.

Animals↗

PubMed2005

Interaction of m-nitrophenylfluorone-Mo(VI) complex as a probe with human serum albumin: a fluorescence quenching study.

In this paper, the binding characteristics of human serum albumin (HSA) and m-nitrophenylfluorone (m-NPF)-molybdenum (Mo(VI)) complex have been studied by fluorophotometry. The binding constants are measured at different temperature. Based on the theory of Forster energy transfer, the binding distance and the energy transfer efficiency between m-nitrophenylfluorone-Mo(VI) complex and protein are obtained. According to the thermodynamic parameters, the main sort of binding force can be judged. The results indicate that HSA and m-NPF-Mo(VI) complex have strong interactions. The mechanism of quenching belongs to static quenching and the main sort of binding force is electrostatic gravitation.

Energy Transfer↗

PubMed2005

Rapid and highly sensitive determination of trace proteins using dibromomethyl carboxyazo-Pb(II) complex as a spectroscopic reagent.

BACKGROUND: The concentration of protein in body fluids is an important measure in disease diagnosis and in analytical biochemistry. Searching for a sensitive and rapid method for proteins is an ongoing subject of investigation. We describe the reaction of dibromomethyl carboxyazo-Pb(II) with proteins found in urine. METHODS: In a buffer containing of HCl and KCl at pH 1.80, dibromomethyl carboxyazo-Pb(II) complex combined with proteins to form a colored compound. The reaction was completed in 2 min and remained stable for 80 min. Under optimum conditions, the product of reaction between protein and dibromomethyl carboxyazo-Pb(II) absorbed at 530 nm and was linear in the range of 0-50 microg/ml of BSA. Moreover, the maximum binding number was defined to express the binding ability of dibromomethyl carboxyazo-Pb(II) to protein under a given set of conditions. RESULTS: The method was applied to the determination of urine proteins. The recovery of protein from human urine was 97.6-103% and the precision was <4.8%. Results compared favorably to the Biuret and Bradford protein methods on 50 urine samples. CONCLUSIONS: This method had high sensitivity and specificity and may be applicable to clinical analysis.

Animals↗

PubMed2005

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90.

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2005

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

Animals↗

PubMed2005

[Spectrophotometric determination of trace amount of copper with trimethoxyphenylfluorone in microemulsion after preconcentration and separation by sulphydryl dextran gel].

In this paper, the color reaction of copper with trimethoxyphenylfluorone (TM-PF) in microemulsion has been studied. Copper (II) reacts with TM-PF to form a stable 1:2 complex at pH 10.40. Its apparent molar absorptivity is 1.46 x 10(5) L x mol(-1) x cm(-1) at 552 nm. Beer's law is obeyed in the range of 0-0.6 microg x mL(-1). Sulphydryl dextran gel (SDG), in which sulphydryl group has a big share, is a new type of metallic ion adsorbent. It presents the advantage of higher adsorption capacity, better reuse ability and higher mechanical stability. So the coexistent metal ions can be separated effectively and copper can be enriched by SDG, greatly improving the selectivity and sensibility of the system. The method was used to determine trace amount of copper in blood, urine, iron ore and water samples with satisfactory results, the relative standard deviation is less than 4.8%, and the recovery is 94.2%-106%.

Copper↗

PubMed2005

[Studies on the determination of protein with spectroscopic probe of o-chlorophenylfluorone-molybdenum (VI) complex in microemulsion medium].

The interaction and absorption spectral behavior of o-chlorophenylfluorone (o-CPF)-Mo(VI) and protein are studied in OP microemulsion medium. Under the optimum condition, the molar absorptivity of o-CPF-Mo(VI)-BSA system at 532 nm is 6.12 x 10(6) L x mol(-1) x cm(-1), and Beer's law is obeyed in the range of 0-14 microg x mL(-1) of BSA. Moreover, the binding number between the complex and BSA determined by molar ratio and slope ratio methods is 91. The mechanism of interaction of BSA and o-CPF-Mo (VI) is also discussed. OP microemulsion media introduced into the protein determination increased markedly the sensitivity of the system. The result of experiment indicates that the method possesses a high sensitivity as well as a high selectivity. It can be used to determine protein in human urine with satisfactory results.

Emulsions↗

PubMed2005

[Study on spectral behavior of trimethoxyphenylflurone with cadmium in microemulsion].

In the present paper, the spectral behavior of cadmium with trimethoxyphenylflurone (TM-PF) in microemulsion was studied. In the buffer medium of borax-NaOH at pH 10.40, cadmium (II) reacts with TM-PF to form a stable 1:2 complex. Its apparent molar absorptivity is 6.10 x 10(4) L x mol(-1) x cm(-1) at 560 nm. Beer's law is obeyed in the range of 0-0.60 microg x mL(-1). The coexistent metal ions can be separated, and cadmium can be enriched by sulfhydryl dextrane gel (SDG), greatly improving the selectivity and sensibility of the system. The method possesses high sensitivity as well as h igh selectivity, and can be used to determine trace amount of cadmium in environmental samples successfully.

English Abstract↗

PubMed2004

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb.

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.

Animals↗

PubMed2004

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival.

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 microg/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 microg/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB(2) and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 microg/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB(2) in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Adenosine Diphosphate↗