Biomedical subjects

Ming Yu

Publications and source records attributed to Ming Yu.

At least 37 records · Page 2Linked to original sources

PubMed2006

Preparation and characterization of a monoclonal antibody against human B lymphocyte stimulator.

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family. It is required for B cell development. Deregulation of BLyS was involved in the pathogenesis of B cell-related autoimmune diseases and multiple myeloma. To prepare monoclonal antibodies (MAbs) against BLyS, cDNA encoding soluble BLyS (sBLyS) was first amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers, and then inserted into a prokaryotic expression vector pET-30a. Right recombinant plasmid was expressed in Escherichia coli strain BL21(DE3), purified by nickel affinity chromatography. Isolated sBLys was used as an antigen to immunize mice. Splenocytes of one immunized mouse were fused with NS- 1. Hybridomas secreting antibodies against sBLyS were identified by ELISA. One positive clone was selected to produce antibody by injecting the hybridoma into the peritoneal cavity of mice. After collecting ascites, the antibody was purified by protein A affinity chromatography. Western blot and immunoflourescence demonstrated that the antibody could bind recombinant sBLyS and genuine membrane-bound BLyS (mBLyS) on U937. This MAb can be used as a detecting reagent to analyze the serum level of BLyS in patients with autoimmune diseases and the expression profile of mBLyS on multiple myeloma cells.

Animals↗

PubMed2006

[Construction and expression of chimeric anti-human CD20 monoclonal antibody].

AIM: To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody (mAb) and realize its expression. METHODS: The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of V(H) and V(L) were amplified from T vector and cloned into chimeric antibody expression vector (pCMV-V(H) and pCMV-V(L)), generating the expression vectors of chimeric anti-human CD20 mAb (C1-28) including light chain expression vector C1-28L and heavy chain expression vector C1-28H. The two plasmids were co-transfected into 293T cells with Lipofectamine 2000. RT-PCR was used to detect the transcription at mRNA level. C1-28 expression was detected by Sandwich ELISA and Western blot methods. RESULTS: mAb 1-28's genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG. CONCLUSION: These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗

PubMed2006

[Research on association between atherosclerotic brain infarction and human leukocyte antigen DR gene].

OBJECTIVE: To evaluate the relationship between atherosclerotic brain infarction (ABI) and human leukocyte antigen DR (HLA-DR) gene at molecular level. METHODS: By using polymerase chain reaction sequence specific probe (PCR-SSP), gene special position of the HLA-II DR alleles were determined in 31 patients and 30 healthy individuals as controls, all of them were inhabitants of Northeastern China. RESULTS: The HLA-DR B1*0301 gene frequency in the group of ABI was obviously higher than that of control group (RR=5.6842, P<0.05). CONCLUSION: The HLA-DR B1*0301 gene may be the susceptible gene of ABI in Northeastern China, or it may associate with other genes to produce the disease.

Adult↗

PubMed2006

[The research and development of new diagnostic gene chips for SARS coronavirus].

The loci of cDNA sequences for valid diagnosis have been identified through the selection of the genome of SARS coronaries. The gene chips for diagnosing such virus have been developed, based on our own-developed technology for manufacturing and application of gene chips. The diagnoses given by such gene chips were consistent well with the reports of clinical laboratories (94.29%) and the sensitivity reached 10(-2)/ml virus molecules. This method is well suited for the clinical use in SARS coronaries diagnosis.

Humans↗

PubMed2006

[Anti-CD28 antibody costimulation enhances anti-CD3 antibody activating T cells and lowering TGF-beta expression in vitro].

In order to study how to activate T cells and their immunological characteristics, the anti-CD3 and anti-CD28 McAbs were used to stimulate PBMNC, then their related immunological changes, such as lymphocyte transformation function, the percentage of CD8(+)CD25(+) cells and TGF-beta expression were deleted by lymphocyte transformation assay, flow cytometry and RT-PCR respectively. The results showed that in costimulation with anti-CD28 antibody stimulation, the activity of anti-CD3 antibody was significantly enhanced, the ratio of CD8(+)CD25(+) cells of T cells was obviously increased, while TGF-beta expression was down-regulated. It was concluded that the anti-CD28 antibody costimulation could provide stimulatory signal II, which make T cells more active, while the expression of TGF-beta significantly down-regulated.

Antibodies, Monoclonal↗

PubMed2006

[Study on the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by tumor necrosis factor related apoptosis inducing ligand].

OBJECTIVE: To explore the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic. METHODS: Apoptosis, integration of mitochondria (including DeltaPsim, cardiolipin), activity of Caspase-9 and release of cytochrome c in colon carcinoma cells SW1116 treated with TRAIL, were detected by means of flowcytometry, fluorometer method and western-blot at the different time point. RESULTS: After treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with DeltaPsim, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was (48.12+/-2.21)micromol.L(-1).h(-1).mg(-1) protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk. CONCLUSION: Mitochondrial pathways involved in the apoptosis of colon carcinoma cell induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondrial.

Apoptosis↗

PubMed2006

[Study on the potential mechanisms of leukemia cell resistance to TRAIL-induced apoptosis].

OBJECTIVE: To explore the potential mechanisms of leukemia cell resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis. METHODS: Cells apoptosis, changes of mitochondrial membrane potential, activity of NF-kappaB, activity of caspase-8 and expressions of apoptosis-related proteins in TRAIL treated K562 and CEM cells, were detected by flowcytometry, ELISA and Western blotting methods, respectively. RESULTS: After treated with TRAIL, the apoptosis indexes were 29.98% and 14.1%, and mitochondrial membrane potential were decreased to 73.25% and 25.4% in K562 and CEM cells respectively. Constitutive level of caspase-8 expression in CEM was lower than that in K562 cells. Both cells became over-expressed Bcl-xL and down-regulated Bax. The ratio of Bcl-xL/Bax in CEM cells was higher than that in K562 cells. Compared with that in K562, the NF-kappaB activity increased significantly in CEM after treatment with TRAIL in early stage. CONCLUSION: CEM cells were more resistant to TRAIL-induced apoptosis than K562 cells did. The potential mechanisms associated with CEM drug resistance might be the lower expression of the constitutive level of caspase-8, lower sensitivity of mitochondrial inner membrane, early increase in NF-KB activity and altered expression of Bcl-2 proteins family.

Apoptosis↗

PubMed2005

Proteomics-based identification of human acute leukemia antigens that induce humoral immune response.

The identification of panels of tumor antigens that elicit an antibody response may have utility in cancer screening, diagnosis, and establishing prognosis. Until now, autoimmunity in cancer has been mainly revealed in solid tumors. The aim of this study was to apply the proteomic approach to the identification of proteins that commonly elicit a humoral response in acute leukemia (AL). Sera from 21 newly diagnosed patients with AL, 20 patients with solid tumors, and 22 noncancer controls were analyzed for antibody-based reactivity against AL proteins resolved by two-dimensional electrophoresis. As a result, autoantibody against a protein identified by mass spectrometry as Rho GDP dissociation inhibitor 2 was detected in sera from 15 of 21 patients with AL (71%). By contrast, such antibody was detected in sera from one of 20 patients with solid tumors (5%) and one of 22 noncancer controls (4.5%). Five other protein autoantibodies were also found in AL patients with a high frequency and constituted the major target antigens of the AL autoimmune response. The findings of autoantibodies against Rho GDP dissociation inhibitor 2 and other proteins in sera of patients with AL suggest that the proteomic approach we have implemented may have utility for the development of a serum-based assay for AL screening and diagnosis.

Adolescent↗

PubMed2005

A randomized treatment-placebo study of the effectiveness of acupuncture for benign vocal pathologies.

Acupuncture is a widely accepted treatment option for many medical ailments in China. Some reports claim that acupuncture is effective for treating dysphonia associated with benign pathological tissue changes. However, many of these reports are based on anecdotal evidence that lacks a scientific experimental design. The objective of this study was to investigate the effectiveness of intensive acupuncture therapy for dysphonias associated with benign pathological changes with a randomized, control design. Twenty-four subjects aged between 19 and 51 years were randomly assigned to either an experimental group or a placebo group. The experimental group received acupuncture on acupoints Renyin (Stomach Channel 9), Lieque (Lung Channel 7), and Zhaohai (Kidney Channel 6), which are all related to improving throat problems and vocal function, whereas the placebo group received acupuncture on acupoints Houxi (Small Intestine Channel 3) and Kunlun (Bladder Channel 60), which are not related to voicing. All subjects received 10 intensive acupuncture sessions within a 20-day period. Acoustic analysis of voice range profile, perceptual analysis of voice quality, and self-perceptions of quality-of-life (QOL) measurement by patients were the outcome measures for determining treatment efficacy. Results revealed significant improvement in the treatment group in all three aspects when compared with the placebo group. The acupuncture effect was maintained into the second week after the completion of acupuncture treatment.

Acupuncture↗

PubMed2005

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585).

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.

Animals↗

PubMed2005

Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification.

Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.

Acute Disease↗

PubMed2005

In vivo identification of the interaction site of ErbB2 extracellular domain with its autoinhibitor.

Direct interference with the transforming potential of ErbB2 has become a subject of great interest. Disruption of critical ErbB2 ectodomain interactions may lead to novel therapeutic approaches for the treatment of various tumors. The ErbB receptor signaling can be inhibited by rationally designed peptide mimetics based on the subdomains of ErbB ectodomain. The mimetics can bind to the ErbB receptor specifically and block inter-receptor interactions, resulting in the growth inhibition of ErbB2-overexpressing cells in vitro. In this study, three-dimensional structure of herstatin, an autoinhibitor of ErbB2 and ErbB2 ectodomain complex was constructed by computer-aided molecular modeling. The binding site on ErbB2 ectodomain for herstatin was determined at S1 domain. The mutants of ErbB2 ectodomain were constructed. The interactions of ErbB2 ectodomain and its mutants with herstatin were analyzed for the first time in living cells that coexpressed herstatin and ErbB2 ectodomain or the mutants. The S1 domain in ErbB2 ectodomain was verified as the interaction site with herstatin by immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET). The binding region of herstatin on ErbB2 ectodomain might be a potential target region for the drug design.

Amino Acid Sequence↗

PubMed2005

Controlled growth of Chinese hamster ovary cells and high expression of antibody-IL-2 fusion proteins by temperature manipulation.

Growth and the expression of the anti-ErbB2 scFv-Fc-IL2 fusion protein in Chinese hamster ovary (CHO) cells were in association at 37 degrees C. The expression of the fusion protein was no more than 25 microg/ml. At 30 degrees C the cell growth was arrested but the cells continued to produce the fusion protein up to 60-80 microg/ml. About 50% of CHO cells were rapidly blocked in G2/M phase after the temperature was shifted from 37 to 30 degrees C. Lowering temperature resulted in cell growth arrest, but maintained cell viability for a longer time and enhanced the production of the antibody-IL-2 fusion protein in CHO cells.

Animals↗

PubMed2005

Two new triterpenes from the husks of Xanthoceras sorbifolia.

Two new triterpenes, 22-angeloyl-21-epoxyangeloylbarringtogenol C (1) and 21,22-diangeloyl-24-hydroxy-R(1)-barrigenol (2), and the known 21,22-diangeloyl-R(1)-barrigenol (3) were isolated from the husks of Xanthoceras sorbifolia Bunge. Their structures were elucidated based on detailed NMR analysis. Compounds 1 and 2 inhibited significantly the growth of cancer cell lines (A375-S2, HeLa) with IC(50) values ranging from 2.9 to 76.3 micromol/L.

Antineoplastic Agents↗

PubMed2005

A novel neutralizing monoclonal antibody against cell-binding polypeptide of ricin.

One strain of neutralizing monoclonal antibody (MAb) against cell-binding polypeptide of ricin, named 3E1, was generated efficiently. The antibody recognized the linearity epitope of RTB located in a toxin structure domain characterized by Western blotting. The safe period of mice for intraperitoneal injection of 100 microg of antibody was 20 min after intraperitoneal injection of 2 microg of Ricin (10 times LD50). The neutralizing MAb we obtained could be developed into an immunotherapeutic agent to counteract the use of ricin as a terrorist or biological warfare weapon. It might be useful, as well, for antibody-based prophylaxis.

Animals↗

PubMed2005

[Construction and expression of anti-human CD3 chimeric antibody gene and preliminary study of expressed product].

AIM: To construct and express anti-human CD3 chimeric antibody. METHODS: The genes of variable regions of the light chain (V(L)) and heavy chain (V(H)) were cloned respectively into the expression vectors (V(L) Express, V(H) Express), and co-transfected into COS-7 cells. Expression level of the chimeric antibody in culture supernatant was detected by ELISA. The antibody was purified through protein A affinity chromatography and identified by Western blot. Binding activity of the chimeric antibody to the antigen was determined by FACS. Biological activity of the chimeric antibody was determined by mixed T-lymphocyte culture test. RESULTS: The expression vectors were constructed and the anti-human CD3 chimeric antibody was expressed and purified successfully. Western blot showed that the purified antibody was human-mouse chimeric antibody. FACS result showed that the chimeric antibody had antigen-binding activity. Mixed T-lymphocyte culture test showed that the chimeric antibody could suppress proliferation of T lymphocytes. CONCLUSION: The anti-human CD3 chimeric antibody has been constructed and expressed successfully, which lays the foundation for its further study.

Animals↗

PubMed2005

[Experimental study of adoptive immunotherapy using CD3AK cells].

AIM: To analyze the immunological properties and biological activity of a monoclonal antibody (mAb) against CD3 molecule(yCD3), and to observe the tumor-suppressive activity of CD3AK cells in vitro and in vivo. METHODS: FCM was used to test the specificity of yCD3 and the immunological phenotype and cytokine production of CD3AK. 3H-TdR assay was used to measure the transformation of lymphocytes activated by yCD3. LDH assay was used to analyze the cytotoxic activity of CD3AK in vitro. Mice bearing tumors were used to observe the anti-tumor effect of CD3AK cells. RESULTS: yCD3 could bind specifically to T cells. 5 microg yCD3 could competitively inhibit 70% of standard anti-CD3 antibody to bind with CD3 molecules on cell membrane. 8 microg/L of yCD3 stimulated the proliferation of peripheral blood lymphocytes, which could be further boosted by IL-2 or anti-CD28 antibodies. Among activated CD3AK cells, CD3+, CD8+, and CD25+ cells increased. IL-2 and IFN-gamma producing CD3+ cells were also increased, to 3.29- and 2.47- fold, respectively, under the co-stimulation of anti-CD28 antibody. When the ratio of effective cells and target cells was 80:1, 57.54% target cells were killed. As compared with control, the percent of tumor inhibition in CD3AK cells treated tumor-bearing mice was 33.17%, and the inhibition rate of lung metastasis was 39.70%. The CD3AK cells treatment was more effective when combined with LAK cells. CONCLUSION: yCD3 could activate T cells and significantly induce the tumor-suppressive activity of CD3AK cells in vitro and in vivo, which lays a foundation for adoptive immunotherapy against tumors in clinical medicine.

Animals↗

PubMed2004

Proteomic analysis of interleukin 6-induced differentiation in mouse myeloid leukemia cells.

Cytokine-induced differentiation of myeloid leukemia cells has important therapeutic implications, but the mechanism remains to be clarified. M1 cell, a mouse acute myeloid leukemia cell line, which underwent growth inhibition, terminal differentiation and apoptosis in response to IL-6, was selected as an experimental model to study on the molecular mechanisms of myeloid cell differentiation on a proteome-wide scale. Cell differentiation was evaluated by cell morphology and CD11b expression. With two-dimensional (2D) gel analyses, 17 protein spots showed obvious changes in quantity during the process of differentiation were found. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) or/and nano-electrospray ionization MS/MS (ESI-MS/MS) analysis, 15 protein spots were identified. The mRNA levels of these 15 proteins during differentiation were also examined using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Except two proteins, the mRNA levels demonstrated similar expression patterns to what the proteomic analysis revealed. The identified proteins were known to be involved in different cellular functions, including protein synthesis, transcription, signal transduction, cell cycle control, cell rescue and defense, cellular organization, and metabolism. Notably, seven proteins were not described before to be involved in differentiation. Our data provide novel information for a better understanding of the mechanisms by which terminal differentiation of acute myeloid leukemia cells induced by IL-6.

Animals↗