Biomedical subjects

Gang Wang

Publications and source records attributed to Gang Wang.

At least 109 records · Page 6Linked to original sources

PubMed2005

[Effect of tyrosine kinase inhibitors on airway remodeling in bronchial asthma].

OBJECTIVE: To investigate the effect of tyrosine kinase inhibitors (TKIs) on asthmatic rat airway remodeling. METHODS: The inhibitive effects of three TKIs (Genistein, jin-zhuan-ting and Tyrphostin AG1478) on proliferation of primary cultures of rat tracheal epithelial cells were assessed by MTT assay. Then, jin-zhuan-ting was adopted in the asthmatic rat model; immunohistofluorescene method was used to stain phosphorylated tyrosine (P-tyr) for disclosing the activation of EGFR; Sirius Red staining of submucosal collagen I and III was performed, and an analysis was made on the correlation between EGFR activation and collagen I and III deposition. RESULTS: All the three TKIs inhibited the growth of tracheal epithelial cells in a time and dose depending manner, and the inhibition rates among them showed no statistical differences; airway subepithelial collagen I and III deposition degrees were markedly elevated in asthmatic groups and jin-zhuan-ting reduced the deposition in a certain degree; EGFR activation (P-tyr) in airways epithelium of asthmatic groups was greatly increased in comparison with that of control groups, and it was evidently decreased in jin-zhuan-ting groups. Correlation analysis demonstrated that the amount of airway subepithelial collagen I and III was positively correlated to EGFR activation. CONCLUSION: TKIs may have preventive implications for asthmatic airway remodeling.

Animals↗

PubMed2005

[Recognition and study of susceptible gene to ankylosing spondylitis].

To study the potential correlations between variances of TNFalpha gene and onset of ankylosing spondylitis in Chinese population, We scanned and analyzed the promoters of TNFalpha genes in 75 AS patients from south of China and found -850 T mutation allele frequency rather high (39.3%).By case-control study, the distribution of TT genotype is significantly higher in AS patients than that in normal subjects (10.7% VS 2.1%,P=0.003); Mutation T allele has a remarkable difference between AS group and normal control (72.2% vs 21.4%,P=2.729 x 10(-9)). The difference in distribution of TX genotype and non -TX genotype is also significant statistically between different genders(male: P=3.42 x 10(-9);female: P=0.001). The result suggests that this variation has a strong association with AS in males and females. No similar reports about the association between AS and the T mutation allele have been acquired. Therefore, our hypothesis can be supported by our results on the whole and the -850 C-->T mutation allele in the region on promoter of TNFalpha gene is likely one of susceptible genes to AS.

Adolescent↗

PubMed2005

[Screening of human scFv against IL-8 from phage antibody library].

AIM: To screen human scFv against IL-8 from phage antibody library. METHODS: IL-8-His fusion protein was expressed in E.coli BL21 (DE3) transfected with prokaryotic expression vector pRSET-IL-8 and was purified by affinity chromatography. Specific antibody was screened by 3 rounds of panning of phage antibody library with the fusion protein. The antigen binding activity and DNA sequences of positive clones were determined and analyzed. RESULTS: After 3 rounds of panning, 2 positive clones were obtained which could bind IL-8 specifically. DNA sequence analysis showed both of the 2 VH genes belonged to human IgG VH3 subgroup, and the Vlambda gene belonged to human VlambdaDPL5 and VlambdaDPL2 subgroups,respectively. CONCLUSION: It is feasible to obtain human anti-IL-8 antibody by phage display technique, which provides the basis for further research on psoriasis and other related diseases.

Antibodies↗

PubMed2005

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein.

OBJECTIVE: To label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects. METHODS: GFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine. RESULTS: Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy. CONCLUSIONS: hIGF-I eukaryotic expression vector has been successfully labeled with GFP.

Cartilage, Articular↗

PubMed2005

[Construction and expression of scFv phage antibody from natural anti-keratin monoclonal antibody 3B4].

AIM: To construct and express the scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 in E.coli and then compare its antigenic specificity with that of the parental antibody 3B4. METHODS: The V(H) and V(L) of 3B4 were amplified by PCR from pMD-18T- mAb 3B4 V(H) and pMD-18T- mAb 3B4 V(L), and then cloned into the phagemid pscMH. E.coli was transfected by recombined pscMH and superinfected by the helper phage VCSM13. Then the antigenic specificity of the scFv concentrated from the supernatant of the transfected E.coli was detected by indirect ELISA. RESULTS: DNA sequence analysis showed that the scFv sequence was the same as the original sequence except that there was one base mutation in the FR1 of V(L). Indirect ELISA showed that the scFv phage antibody was polyreactive as mAb 3B4, but its antigenic specificity was not the same with that of mAb 3B4. CONCLUSION: The scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 was successfully constructed and expressed. Its antigenic specificity was different from that of its parental antibody 3B4.

Amino Acid Sequence↗

PubMed2005

[A specific human scFv against malignant melanoma selected from a large human phage antibody library].

AIM: To obtain the specific human scFv against malignant melanoma (MM) using phage antibody library technology. METHODS: The library was panned with a certain kind of MM cells for 4 rounds. The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies, which were then examined by DNA sequence analysis and immunohistochemistry. RESULTS: In ELISA and immunohistochemical staining, the positive clone selected from the 80 random clones was able to bind one kind of MM cell specifically, while not able to bind other irrelavant cells or antigens, such as human's squamous carcinoma cells, keratinocytes, melanocytes, keratin, trypsinase, transferrin and mouse IgG, etc. The antibody's V(H) genes belonged to human IgG V(H)5 subgroup and the V(L) belonged to human V(kappa) subtype determined by DNA sequence analysis. CONCLUSION: One specific human phage antibody against MM cells was obtained by phage display technique, which may be useful for further development of tumor-targeted therapies.

Antibodies↗

PubMed2005

[Studies on subset, location and secretion of natural keratin-reactive B cells].

AIM: To clarify the subsets and location of B cells responsible for the production of natural anti-keratin autoantibody (AK auto Ab), and analyze the function of each subset. METHODS: Cells isolated from spleen and peritoneal cavity of C57BL/6 mice were cultured in vitro. FACS was employed to detect keratin reactive B cell subsets. The anti-keratin IgM level in supernatant was analyzed by ELISA, while ELISPOT was utilized to detect the B cells which can secrete AK auto Ab. RESULTS: Keratin reactive B cells were mainly marginal zone B cells in spleen, while they were mainly B-1a cells in peritoneal cavity. There were more B cells producing AK auto Ab in peritoneal cavity than in spleen. The anti-keratin IgM level in the supernatant of cultured peritoneal cells was much higher than that of splenocytes. CONCLUSION: There are three subsets of keratin-reactive B cells: B-1 B cells, follicular B cells and marginal zone B cells. B-1 B cells are active in secreting AK auto Ab.

Animals↗

PubMed2005

[Preparation of humanized anti-digoxin scFv and diabody].

AIM: To prepare humanized anti-digoxin(Dig) antibodies(single chain Fv, scFv) from a human phage antibody library and construct anti-Dig diabody vector. METHODS: A human phage antibody library was panned against immobilized Dig for four times, and the specificity of the selected scFv expression clones was identified by ELISA. Positive clones against Dig were analyzed by DNA fingerprint and sequencing. A clone which had high affinity to Dig was selected to construct a diabody vector. Diabody was secreted from E. coli by IPTG induction, and the specificity was also identified by ELISA. RESULTS: After four rounds of panning, four specific humanized anti-Dig antibodies(scFv) were obtained. DNA fingerprint and sequencing analysis proved that they had different Ab-encoding genes. The V(L) of the 4 expression clones belonged to lambda subgroup 1 and V(H) belonged to 3 and 4 subgroups, respectively. A scFv clone was picked out to construct diabody vector. The prepared humanized diabody reacted with Dig specifically. CONCLUSION: Humanized anti-Dig antibodies(scFv) have been got by using phage display. A diabody has been obtained from scFv. These humanized anti-Dig antibodies may be used for the diagnosis and therapy of Dig toxication.

Antibodies↗

PubMed2005

[Construction and expression of anti-keratin single-chain Fv antibody].

AIM: To construct an expression vector of human anti-keratin scFv and express functional scFv in E. coli. METHODS: The V(H) and V(L) genes were amplified by PCR and cloned into a scFv expression vector, which was expressed in E. coli. The expression of scFv was tested by SDS-PAGE; The antigen-binding characteristics of scFv were tested by ELISA. RESULTS: The V(H) and V(L) genes were cloned and expressed in E. coli successfully. The expressed scFv retained the antigen-binding characteristics of the parental antibody. CONCLUSION: A functional anti-keratin scFv was obtained.

Antibodies↗

PubMed2005

[Expression, purification and antiviral activities of a new recombinant human interferon-lambda2].

OBJECTIVE: To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities. METHODS: According to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells. RESULTS: The expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b. CONCLUSION: Recombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.

Animals↗

PubMed2004

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Animals↗

PubMed2004

Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6.

IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.

Animals↗

PubMed2004

Two-dimensional locally resonant phononic crystals with binary structures.

The lumped-mass method is applied to study the propagation of elastic waves in two-dimensional binary periodic systems, i.e., periodic soft rubber/epoxy and vacuum/epoxy composites, for which the conventional methods fail or converge very slowly. A comprehensive study is performed for the two-dimensional binary locally resonant phononic crystals, which are composed of periodic soft rubber cylinders immersed in epoxy host. Numerical simulations predict that subfrequency gaps also appear because of the high contrast of mass density and elastic constant of the soft rubber. The locally resonant mechanism in forming the subfrequency gaps is thoroughly analyzed by studying the two-dimensional model and its quasi-one-dimensional mechanical analog. The rule used to judge whether a resonant mode in the phononic crystals can result in a corresponding subfrequency gap or not is found.

Journal Article↗

PubMed2004

Dendritic cells strongly boost the antitumor activity of adoptively transferred T cells in vivo.

Dendritic cells (DCs) have been well characterized for their ability to initiate cell-mediated immune responses by stimulating naive T cells. However, the use of DCs to stimulate antigen-activated T cells in vivo has not been investigated. In this study, we determined whether DC vaccination could improve the efficacy of activated, adoptively transferred T cells to induce an enhanced antitumor immune response. Mice bearing B16 melanoma tumors expressing the gp100 tumor antigen were treated with cultured, activated T cells transgenic for a T-cell receptor specifically recognizing gp100, with or without concurrent peptide-pulsed DC vaccination. In this model, antigen-specific DC vaccination induced cytokine production, enhanced proliferation, and increased tumor infiltration of adoptively transferred T cells. Furthermore, the combination of DC vaccination and adoptive T-cell transfer led to a more robust antitumor response than the use of each treatment individually. Collectively, these findings illuminate a new potential application for DCs in the in vivo stimulation of adoptively transferred T cells and may be a useful approach for the immunotherapy of cancer.

Animals↗

PubMed2004

Exogenous NADPH increases cerebral blood flow through NADPH oxidase-dependent and -independent mechanisms.

OBJECTIVE: NADPH, a substrate for the superoxide-producing enzyme NADPH oxidase, produces vasodilation in the cerebral circulation. However, the mechanisms of the effect have not been fully elucidated. We used a peptide inhibitor of NADPH oxidase (gp91ds-tat) and null mice lacking the gp91phox subunit of NADPH oxidase to examine the mechanisms of the cerebrovascular effects of exogenous NADPH. METHODS AND RESULTS: Cerebral blood flow (CBF) was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. Superfusion with NADPH increased CBF (27% at 100 micromol/L) without affecting the EEG. The CBF increase was attenuated by the free-radical scavenger MnTBAP (-54%, P<0.05) but not by the H2O2 scavenger catalase. The response was also attenuated by gp91ds-tat (-64%, P<0.05) and by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (-44%, P<0.05). The increase in CBF produced by NADPH was attenuated in gp91-null mice (-41%, P<0.05). NADPH increased production of reactive oxygen species, assessed by hydroethidine microfluorography, an effect blocked by MnTBAP or gp91ds-tat and not observed in gp91-null mice. CONCLUSIONS: These data suggest that the mechanisms of the CBF increases produced by exogenous NADPH are multifactorial and include NADPH oxidase-dependent and -independent factors.

Animals↗

PubMed2004

Differential binding of Mg2+, Zn2+, and Cd2+ at two sites in a hammerhead ribozyme motif, determined by 15N NMR.

A decamer duplex model of Domain II of the hammerhead ribozyme was synthesized with [8-13C-1,7,NH2-15N3]-guanosine at the known metal binding site, G10.1 and, for comparison, [2-13C-1,7,NH2-15N3]-guanosine at G16.2. The 15N NMR chemical shifts of the labeled N7s monitored during addition of Mg2+, Cd2+, and Zn2+ showed the same preference for binding at G10.1 over G16.2 for each metal. These results demonstrate that 15N labeling can be used to evaluate the binding of different metals, including Mg2+, to a given nitrogen, as well as to compare the binding potential of different sites.

Binding Sites↗

PubMed2004

NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius.

Angiotensin II (AngII), acting through angiotensin type 1 (AT1) receptors, exerts powerful effects on central autonomic networks regulating cardiovascular homeostasis and fluid balance; however, the mechanisms of AngII signaling in functionally defined central autonomic neurons have not been fully elucidated. In vascular cells, reactive oxygen species (ROS) generated by the enzyme NADPH oxidase play a major role in AngII signaling. Thus, we sought to determine whether NADPH oxidase is present in central autonomic neurons and, if so, whether NADPH oxidase-derived ROS are involved in the effects of AngII on these neurons. The present studies focused on the intermediate dorsomedial nucleus of the solitary tract (dmNTS) because this region receives autonomic afferents via the vagus nerve and is an important site of AngII actions. Using double-label immunoelectron microscopy, we found that the essential NADPH oxidase subunit gp91phox is present in somatodendric and axonal profiles containing AT1 receptors. The gp91phox-labeled dendrites received inputs from large axon terminals resembling vagal afferents. In parallel experiments using patch clamp of dissociated NTS neurons anterogradely labeled via the vagus, we found that AngII potentiates the L-type Ca2+ currents, an effect mediated by AT1 receptors and abolished by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride. The NADPH oxidase assembly inhibitor apocynin and the peptide inhibitor gp91phox docking sequence, but not its scrambled version, also blocked the potentiation. The results provide evidence that NADPH oxidase-derived ROS are involved in the effects of AngII on Ca2+ influx in NTS neurons receiving vagal afferents and support the notion that ROS are important signaling molecules in central autonomic networks.

Angiotensin II↗

PubMed2004

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.

Artifacts↗