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S Qian

Publications and source records attributed to S Qian.

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

Cloning and expression of L-asparaginase gene in Escherichia coli.

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72 h of cultivation and 5 h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.

Asparaginase↗
PubMed2001

Liver-derived dendritic cells induce donor-specific hyporesponsiveness: use of sponge implant as a cell transplant model.

Spontaneously accepted mouse liver allografts are capable of protecting subsequently transplanted donor organs from rejection; however, the underlying mechanisms are unclear. Dendritic cells (DC) residing in liver grafts are likely important in tolerance induction. DC propagated from mouse liver with GM-CSF are phenotypically and functionally immature. They are poor allostimulators in MLR and prolong survival of pancreatic islet allografts. It has been problematic to perform mechanistic studies in an islet transplant model because of difficulties in obtaining sufficient graft infiltrating cells. In this study, we used a sponge allograft model [i.e.. a subcutaneously implanted sponge matrix loaded with B10 (H2b) spleen cells]. To investigate the influence of administration of donor (B10) liver-derived DC on alloimmune reactivity of C3H (H2k) hosts, sponge graft infiltrating cells (SGIC) and recipient spleen cells were isolated, and their immunophenotype and donor-specific cytotoxic T lymphocyte (CTL) activity were examined. The results illustrate that donor-specific CTL activity of T cells are lower in recipients that had received systemic treatment with liver-derived immature DC, associated with a decrease in CD8+ cell population and an increase in Gr-1+ cells in SGIC, compared with recipients treated with mature bone marrow (BM)-derived DC. Interestingly, administration of liver DC directly into the sponge did not inhibit T cell responses. These data suggest that systemic administration of donor liver DC induces donor-specific hyporesponsiveness, probably not by direct inhibition of graft infiltrating T cells. The increased Gr-1+ cells may play immune regulatory roles in induction of host donor-specific hyporesponsiveness.

Animals↗
PubMed2001

Percutaneous transsplenic embolization of esophageal and gastrio-fundal varices in 18 patients.

AIM: Clinical application and potential complication of percutaneous transsplenic varices embolization (PTSVE) of esophageal or gastrio-fundal varices in patients with hepatocellular carcinoma (HCC) complicated with portal vein cancerous thrombosis (PVCT). METHODS: 18 patients with HCC complicated with PVCT and esophageal or gastrio-fundal varices who underwent PTSVE were collected. The rate of success, complication, mortality of the procedure and postoperative complication were recorded and analyzed. RESULTS: PTSVE were successfully performed in 16 of 18 cases, and the rate of success was 89%. After therapy erythrocyte counts decreased in all of the natunts. 5 of patients needed blood transfusion, 2 patients required surgical intervention because of and 11 patients with ascites were alleviated by diuresis. Among these 18 patients, the procedure-related mortality was 11% (2/18), one died of acute hepatic failure on the forth day after procedure, another died of acute renal failure on the fifth day. The patients were follow up for 1-12 mon exceptone. 13 of them died of their tumors but none of them experienced variceal bleeding. CONCLUSION: PTSVE is a relatively safe and effective method to treat esophageal or gastrio-fundal varices in HCC patients with PVCT when percutaneous transhepatic varices embolization (PTHVE) of varices is impossible.

Adult↗
PubMed2001

[Imaging evaluation and interventional therapy of hepatic metastases from small intestinal leiomyosarcoma].

OBJECTIVE: To evaluate the imaging appearances and the short-term curative effect of hepatic metastases from small intestine leiomyosarcoma treated by interventional methods. METHODS: Seven cases with 46 lesions of hepatic metastases from small intestinal leiomyosarcoma were analyzed retrospectively. The diameters of the metastases were from 2 cm to 18 cm. CT triphasic scan and hepatoarterial angiography were performed in all cases. The interventional therapy methods included transcatheter arterial chemoembolization (TACE) (all 7 cases), percutaneous left subclavian artery port-catheter system implantation (PCS) (1 case). Three cases were also treated with percutaneous tumor necrosis drainage (PTND) because of obvious liquefaction necrosis within the tumor. RESULTS: The lesions were substantial occupation and some were mixed density with cystic component. The cystic component increased in the larger tumor. The hepatic metastases were enhanced remarkably during the hepatic arterial-dominant phase scan and had abundant tumor vascular and tumor stain on hepatoarterial angiography. The size of tumor in 4 cases was reduced after TACE. The tumor in the patient who was treated with PCS had no changed. The size of the tumor in 3 cases was slightly enlarged. The tumors in the 3 patients treated with PTND were reduced remarkably. CONCLUSIONS: The predominant blood-supply of the hepatic metastases from small intestinal leiomyosarcoma is the hepatic artery abundant in blood vessels. TACE, PCS and PTND are effective methods to treat hepatic metastases from small intestinal leiomyosarcoma.

Angiography↗
PubMed2001

[The effect of side chain structure on the excitation transfer and charge transfer processes in PPV/C60 composite system].

In this paper, we investigated the effect of side chains on the optical properties of PPV derivatives, the excitation transfer and charge transfer process in the PPV/C60 composite films. The results demonstrate that the absorption and PL emission bands of PPV shift towards lower photon energy with increasing of the length of PPV side chain, which indicates that energy gap of PPV derivatives become narrow with their side chain length increasing. In the PPV/C60 composite films, the emission of PPV derivatives was greatly quenched due to the efficient excitation transfer process between PPV and C60. We observed the absorption band of C60- located at 920 nm in the MD-PPV/C60 composite films, which reveals the existence of charge transfer process in the ground state of the composite films. This results show that the side chains of PPV derivatives not only influence the properties of absorbance and photoluminescence but also influence the electronic affinity of PPV polymers, which means that both physical and chemical properties of PPV can be monitored by controlling its side chain structure.

Electrochemistry↗
PubMed2000

Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Animals↗
PubMed2000

Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.

In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme.

Asparaginase↗
PubMed2000

Characterization of l-asparaginase fused with a protective ScFv and the protection mechanism.

A fusion protein of the protective scFv linked to the C-terminus of ASNase via (Gly(4)Ser)(6) peptide was constructed. The ASNase-scFv fusion protein expressed in Escherichia coli exists mainly in the form of inclusion bodies, and a small amount of it was soluble. The soluble form was purified by four-step purification and it has been demonstrated that ASNase-scFv fusion exists as a dimer. By assay of the stability against proteolysis, the ASNase-scFv fusion was found to be more stable than native ASNase but less stable than scFv-ASNase fusion. The results of immunological assay indicated that the immunogenicity of the fusion proteins increased while their binding capacity with the anti-ASNase serum decreased by comparison to the native ASNase. Moreover, here the comparison of the basic physical and chemical properties of the ASNase-scFv fusion, scFv-ASNase fusion, and native ASNase is presented. Based on the structural evidence and the biochemical analysis described in this paper, the protection mechanism proposed in our previous study was further supported. The scFv moiety of the fusion protein may confer the ASNase moiety resistance to proteolysis as a result of both steric hindrance such as blocking the cleavage sites of trypsin and a change in the electrostatic potential surface of the enzyme.

Animals↗
PubMed2000

Microchimerism, donor dendritic cells, and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells: modulation by tacrolimus.

Flt3 ligand (FL) is a potent hemopoietic growth factor that strikingly enhances stem cells and dendritic cells (DC) in vivo. We examined the impact of infusing FL-mobilized bone marrow (BM) cells on microchimerism and anti-donor reactivity in normal and tacrolimus-immunosuppressed, noncytoablated allogeneic recipients. BM from B10 (H2b) mice given FL (10 microg/day; days 0-8; FL-BM) contained a 7-fold higher incidence of potentially tolerogenic immature CD11c+ DC (CD40low, CD80low, CD86low, MHC IIlow) that induced alloantigen-specific T cell hyporesponsiveness in vitro. C3H (H2k) mice received 50 x 106 normal or FL-BM cells (day 0) and tacrolimus (2 mg/kg/day; days 0-12). On day 15, enhanced numbers of donor (IAb+) cells were detected in the thymi and spleens of FL-BM recipients. Tacrolimus markedly enhanced microchimerism, which declined as a function of time. Ex vivo splenocyte proliferative and CTL responses and Th1 cytokine (IFN-gamma) production in response to donor alloantigens were augmented by FL-BM infusion, but reduced by tacrolimus. Systemic infusion of purified FL-BM immature DC, equivalent in number to that in corresponding whole BM, confirmed their capacity to sensitize, rather than tolerize, recipient T cells in vivo. In vitro, tacrolimus suppressed GM-CSF-stimulated growth of myeloid DC from normal BM much more effectively than from FL-BM without affecting MHC class II or costimulatory molecule expression. Infusion of normal B10 BM cells at the time of transplant prolonged C3H heart allograft survival, whereas FL-BM cells did not. A therapeutic effect of tacrolimus on graft survival was observed in combination with normal, but not FL-BM cells. These findings suggest the need for alternative immunosuppressive strategies to calcineurin inhibition to enable the engraftment, survival, and immunomodulatory function of FL-enhanced, immature donor DC.

Adjuvants, Immunologic↗
PubMed2000

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

Animals↗
PubMed2000

Nonmuscle myosin II localizes to the Z-lines and intercalated discs of cardiac muscle and to the Z-lines of skeletal muscle.

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh-frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with alpha-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.

Actinin↗
PubMed2000

Prolongation of cardiac allograft survival using dendritic cells treated with NF-kB decoy oligodeoxyribonucleotides.

Dendritic cells (DC) classically promote immune responses but can be manipulated to induce antigen-specific hyporesponsiveness in vitro. The expression of costimulatory molecules (CD40, CD86, CD80) at the DC cell surface correlates with their capacity to induce or suppress immune responses. Expression of these molecules is associated with NF-kB-dependent transcription of their genes. DC tolerogenicity has been associated with impaired NF-kB-dependent transcription of costimulatory genes as well as NF-kB translocation to the nucleus. In this report, we demonstrate that double-stranded oligodeoxyribonucleotides containing binding sites for NF-kB (NF-kB ODN) are efficiently incorporated by bone marrow-derived DC and specifically inhibit NF-kB-dependent transcription of a reporter gene. Moreover, exposure of DC to the oligonucleotide decoys inhibited lipopolysaccharide (LPS)-induced nitric oxide production, a marker of DC maturation. Treatment of bone marrow-derived DC progenitors with NF-kB ODN selectively suppressed the cell-surface expression of costimulatory molecules without interfering with MHC class I or class II expression. Furthermore, NF-kB ODN DC induced allogeneic donor-specific hyporesponsiveness in mixed leukocyte cultures, and this was associated with inhibition of Th1-type cytokine production. Finally, infusion of NF-kB ODN-modified bone marrow-derived DC into allogeneic recipients prior to heart transplantation resulted in significant prolongation of allograft survival in the absence of immunosuppression. Specific interference with NF-kB and other transcriptional pathways involved in immune stimulation in DC using ODN decoy approaches could be one means to promote tolerance induction in organ transplantation.

Animals↗
PubMed2000

Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin.

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.

Asparaginase↗
PubMed2000

Organic vanadium chelators potentiate vanadium-evoked glucose metabolism in vitro and in vivo: establishing criteria for optimal chelators.

Several ligands, when complexed with vanadium, potentiate its insulinomimetic activity both in vivo and in vitro. We have recently found that L-Glu-gamma-monohydroxamate (HXM) and L-Asp(beta)HXM were especially potent in this regard. In the present study, we used vanadium-enriched adipose cells and cell-free experimental systems to determine the features of L-Glu(gamma)HXM and L-Asp(beta)HXM that turn these ligands into optimal-synergizing vanadium chelators. We found that L-Glu(gamma)HXM and L-Asp(beta)(HXM) possess the following characteristics: 1) They associate with vanadium(+5) at pH 7.2 within a narrow range of an apparent formation constant of 1.3 to 1.9 x 10(2) M(-1); 2) they have nearly the same binding affinity for the vanadyl(+4) cation and the vanadate(+5) anion at physiological pH values; and 3) they form intense ultraviolet absorbing complexes upon associating with vanadium(+4) at 1 and 3 M stoichiometry, respectively, at pH 3.0. Vanadium ligands lacking any of these three defined criteria synergize less effectively with vanadium to activate glucose metabolism.

Adipocytes↗
PubMed2000

[Effects of p16 and GM-CSF combined gene therapy on immune response of tumor-bearing mice].

OBJECTIVE: To investigate the effects of p16 and GM-CSF combined gene therapy on immune response of tumor-bearing mice (Renca). METHODS: The model of renal tumor-bearing mice was constructed by subcutaneous injection of Renca cells. The tumor-bearing mice were divided into 5 groups at random three days after injection of Renca cells. Different groups of mice were given different treatments. The antitumor immune responses and the survival time of mice were tested. Histopathologic analysis of tumor tissue was done. RESULTS: Enhancement of antitumor immune responses and prolongation of survival period were observed more obviously in tumor-bearing mice after combined therapy than in those of the other four groups. CONCLUSIONS: p16 and GM-CSF combined gene therapy can enhance the antitumor immune responses of mice and inhibit the growth of pre-established renal cell carcinoma.

Adenoviridae↗
PubMed2000

[Producing characterization of constitutive-form laccase by Basidiomycete].

A constitutive-laccase-producing fungus was gained from 20 strains of different sources with plate method. Laccase activity reached 0.58 IU/mL when the strain grew on DC medium with shaking cultivation, which was superior to stationary method by 4 times. The enzyme production paralleled with biomass in early 8 days, subsequently activities declined slightly though its biomass continued upward. Tyrosine and tryptone at the concentration of 4 g/L improved the enzyme production, reaching 2.49 and 2.0 IU/mL respectively. Some tested chemical agents seemed not to contribute to the increase of the enzyme production effectively except for syringaldazine and o-tolidine. Heatshock at 40 degrees C for 1 h made 1.3 times more enzyme expression.

Basidiomycota↗