[Construction of GPI-CTLA4Ig chimeric molecule and its expression on CHO-dhfr(-) cells].

Aim: To develop a novel immunosuppressant GPI-CTLAIg modified by glycosyl-phosphatidylinositol(GPI).

Methods: GPI-modified CTLAIg was produced by linking-up of CTLA4Ig with GPI-modification signal sequences from decay-accelerating factor (DAF). Chimeric molecule GPI-CTLA4Ig gene was cloned into eukaryotic expression vector pCI-dhfr.

Comparison of IL-6-induced STAT1 activation in different responsive cells.

Aim: To investigate the activation status of signal transducer and activator of transcription 1 (STAT1) induced by interlerkin-6 (IL-6) in different responsive cells.

Methods: Immunoblot analysis was performed with antibodies against STAT1 and tyrosine-phosphorylated STAT1.

Results: It's found that IL-6 had growth-promoting effects on 7TD1 and TF1 cells but had little effect on the tyrosine-phosphorylation of STAT1.

The difference of binding epitopes on human CD16 (FcgammaRIII) interacted with hIgG1 and monoclonal antibody B88-9.

The 3-D complex structures of extracellular domain of human CD16 (FcgammaRIII) and hIgG1 or B88-9 were obtained by means of computer-guided molecular modeling, respectively. The binding epitopes were predicted and binding energy was calculated theoretically. The epitopes of human CD16 interacted with hIgG1 and B88-9 were different and the binding energy of B88-9 was stronger than hIgG1.

[Identification of HCV antigen epitopes from a random 15-peptide library by anti-HCV polycloral antibody].

Aim: To analyze epitope recognized by anti-HCV antibodies; patients suffered from hepatitis C.

Methods: Anti-HCV Abs were purified from the patients serum through an affinity chromatography column which was prepared with sepharose 4B coupled with protein A. These Abs were used for biopanning of a phage-displayed random 15-peptide library.

Screening of functional antidotes of RNA aptamers against bovine thrombin.

A specific RNA aptamer (T705) against bovine thrombin had been obtained after seven rounds of SELEX (systematic evolution of ligands by exponential enrichment) selection from a random RNA library previously. In order to further investigate the relationship between the structure and function of this aptamer, three truncated RNA aptamers, T705a, T705b and T705c, were designed according to the secondary structure of T705 RNA. Our results showed that T705c keeping the precise stem-loop structure but lacking most of the stem region sequence of T705 could inhibit clot formation in vitro in the same way as its parental form.

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides.

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP.

Crystallization and preliminary crystallographic studies of the C-terminal domain of human FKBP52.

FKBP52 is a high-molecular-weight immunophilin belonging to the FKBP (FK506-binding protein) family. FKBP52 is one of several chaperone proteins associated with untransformed steroid receptors in steroid receptor-hsp90 heterocomplexes. Here, the C-terminal domain (amino acids 145-459) has been cloned, overexpressed and purified.

Design and assembly of anti-CD16 ScFv antibody with two different linker peptides.

Several studies have focused on the effect of different lengths of linker peptides on the properties of single-chain Fv (ScFv). The expressing level and stability of anti-CD16 ScFv with common linker peptide (Gly(4)Ser)(3) were very poor. Considering 3-D structures of heavy and light chain variable region gene of anti-CD16 antibody, a novel linker peptide PT7 (i.

A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells.

Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms. The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments. In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced.

Design and structure-based study of new potential FKBP12 inhibitors.

Based on the structure of FKBP12 complexed with FK506 or rapamycin, with computer-aided design, two neurotrophic ligands, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester and (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-phenylalanine benzyl ester, were designed and synthesized. Fluorescence experiments were used to detect the binding affinity between FKBP12 and these two ligands. Complex structures of FKBP12 with these two ligands were obtained by x-ray crystallography.

[Enhanced apoptosis-inducing effect of etoposide on leukemic cell lines M-07e and TF-1 by the proteasome inhibitor Z-LLL-CHO].

Recent researches indicate that ubiquitin-protea some pathway plays an important role in apoptosis regulation. Proteasome inhibitors induce apoptosis in many kinds of neoplastic cells, thus provide a great opportunity for exploring synergy of proteasome inhibitors and other apoptosis-inducing agents. In this study, the effect of the proteasome inhibitor Z-LLL-CHO combined with etoposide (VP16) on leukemic cell lines M-07e and TF-1 was investigated by MTT assay, trypan blue exclusion, flow cytometry and Western blot.

Expression of a novel recombinant dual human stem cell factor in insect cells.

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter.

[The effect of eIF-5A on the G1-S in cell cycle regulation].

Eukaryotic initiation factor 5A (eIF-5A) contains an unusual amino acid, hypusine, which is formed post-translationally. Although eIF-5A and its hypusine modification are essential for eukaryotic cell viability, the precise physiological function of it has remained elusive. The aim of the study is to investigate how hypusine formation modulate the proliferation, cell cycle and apoptosis in leukaemia cells.

Proteomic analysis of ubiquitin-proteasome effects: insight into the function of eukaryotic initiation factor 5A.

The global effect of ubiquitin-proteasome (UP) inhibitors on leukemic cell proteome was analysed. A total of 39 protein spots, affected by UP inhibitors, were identified, including 11 new apoptosis-associated proteins. They are involved in different cellular functions and four were associated with caspase-3 activation.

A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function.

Elevated erbB2 expression is detected in many in situ and invasive human ductal carcinomas. Anti-erbB2 antibody directed at the extracellular domain of erbB2 can result in an antitumor response in some patients with tumors overexpressing erbB2 oncoprotein. By combining interleukin 2 (IL-2) activities with a tumor specific antibody, immunotherapy of tumors might be more effective in the future.

Lineage-dependent NF-kappaB activation contributes to the resistance of human macrophages to apoptosis.

Granulocytes and mononuclear phagocytes develop from the same myeloid progenitor cells in the bone marrow via distinct differentiation pathways. Yet, it is known that mature macrophages are more resistant than granulocytes to spontaneous apoptosis in cultures without hematopoietic growth factors. This fact suggests that the development of resistance to apoptosis during myeloid differentiation is differentially regulated by a lineage-dependent mechanism.

[Expression and Characterization of Single-chain Fv-Fc Fusion Protein against Human P185(erbB2)].

In order to increase therapeutic effects and decrease immunogenicity of mouse McAb, the single-chain Fv (scFv) created by fusing the light and heavy chain variable region genes of anti-human P185(erbB2) McAb was conjugated to the Fc gene of human IgG1 to construct a scFv-Fc fusion gene. The scFv-Fc fusion gene was cloned into the expression vector pCIDN. The scFv-Fc fusion protein was synthesized as secreted two-chain molecule in CHO cells, and purified by affinity chromatography on recombinant protein A.

Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment.

In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells to eliminate those that recognize the common cellular components of both differentiated and undifferentiated PC12 cells. After six rounds of cell-based selection, both of individual aptamers and aptamers of the sixth round pool were found binding to differentiated PC12 cells, but not to the parental PC12 cells.

PI3-K/Akt pathway contributes to IL-6-dependent growth of 7TD1 cells.

BACKGROUND: Recently, growing evidence suggests the involvement of PI 3-K/Akt in IL-6-dependent survival and proliferative responses in several types of cells. However, whether PI 3-K/Akt plays the same role in IL-6-dependent growth of 7TD1 mouse-mouse B cell hybridoma is not known. METHODS: We investigated the activation status of Akt in 7TD1 cells induced by IL-6.

A novel erythroid differentiation related gene EDRF1 upregulating globin gene expression in HEL cells.

Objective: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression.

Methods: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2.

[Construction of Expression Vector for Human CD40-Ig Fusion Protein and Its Expression in COS-7 Cells].

CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection.

[The Comparison between the Killing Effects of Two Anti-T Immunotoxins on Target Cells].

The key to killing target cells by immunotoxin depends on the specific recognition of antibody to target cell and the cytotoxic effect of toxin. The comparative study of the killing effects of two anti-T immunotoxins, CD5:Ricin and CD5:rRA, on target cells was performed. The elimination rate of immunotoxins was analysed by flow cytometry and MLR.

[Induction of Apoptosis in Leukemic Cells by Inhibiting the Ubiquitin-Proteasome Pathway and Its Possible Mechanism].

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined.

RNA aptamers specific for bovine thrombin.

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported.

[Biological analysis of a novel gene related with interleukin-6].

Objective: To understand the action mechanism of interleukin (IL)-6 through investigation of its related genes.

Methods: Using electronic cloning with IL-6-related expressed sequence tags (ESTs), a novel gene at its full length of 924 bp was acquired. Reverse transcriptase-PCR method was subsequently employed to amplify the cDNA of this gene from the total RNA of human U937 cells, which were previously activated with 100 ng/ml IL-6 for 8 h.