Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

Tumor necrosis factor-related apop-tosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expres-sion vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant frag-ments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was trans-ferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently trans-formed lines were obtained by 100 mg/L spectino-mycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and culti-vation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%―0.67% of the total soluble proteins. These experimental results demonstrated the possi-bility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.

Cloning and identification of a novel binding protein of CD2 cytoplasmic domain

In order to understand why CD2 has a dual action of transduction of activation or apoptosis signals in T cells under different experimental conditions, we employed a yeast two-hybrid system to look for a binding protein of the cytoplasmic domain of CD2 which may be involved in this issue. A human T cell cDNA library was screened by a cDNA encoding the cytoplasmic domain of CD2 (Thr211-Gln336). The specificity of protein-protein interaction was verified by co-immunoprecipitation. The binding protein obtained, designated CD2cBP, was found to be homologous to v-fos transformation effector protein (Fte-1). As Fte-1 plays a role in cell transformation, growth, protein synthesis and protein-import into mitochondria, this result suggests that CD2cBP may be putatively involved in CD2-mediated signaling.

A highlight of recent advances in immunology

To celebrate the 60th anniversary of SCIENCE CHINA, six research groups of overseas and domestic Chinese immu- nologists published a series of review articles （SCIENCE CHINA Life Sciences, 2010, 53（2）： 157-158）, which high- lighted recent advances and their contributions to immu- nology.

Analysis of the ligand-binding domain of macrophage colony-stimulating receptor

RNAs have been extracted from human placenta. Extracellular regions of M-CSFR, D1-3, D2-3 and D3 motifs have been amplified with RT-PCR. The proteins have been expressed in E.coli. Enzyme-linked immusorbent assay (EIA) shows that recombinant D1-3 possesses binding ability 3 times that of D2-3. Kd, the dissociation constant of the former is 11 nmol/L, and that of latter is 39 nmol/L. D3 lacks binding ability. These data suggest that D2-3 is the main site for M-CSF binding, D1 is an assistant site and also contributes to the conformation of site for ligand binding.

PRMT5 suppresses DR4-mediated CCL20 release via NF-κB pathway

Construct expression vectors of pCMV-DR4-HA and pCMV-PRMT5-Flag,and transfect them into HEK293 cells to identify the interaction between TRAIL-R1 and PRMT5 and the molecular mechanism underlying DR4-mediated inhibition of chemokine CCL20 release via TRAIL receptor 1(DR4).Inflammatory cytokine was detected by RT-PCR and ELISA after TRAIL-R1 and/or PRMT5 transfection,respectively.NF-κB activity was detected by Dual Luciferase Reporter Gene Assay.ERK1/2 phosphorylation was analyzed by Western blot.PRMT5 could inhibit DR4-activated NF-κB activity and ERK1/2 phosphorylation.PRMT5 could inhibit NF-κB activition,ERK1/2 phosphorylation as well as CCL20 secretion via binding with DR4 in HEK293 cell,suggesting that PRMT5 may involve in DR4 dependent immune regulation.

Internalization and half-life of membrane-bound macrophage colony-stimulating factor

The binding of recombinant human macrophage colony-stimulating factor (M-CSF) soluble receptor (rh-M-CSF-sR) to membrane-bound macrophage colony stimulating factor (m-M-CSF) and the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes mediated by m-M-CSF were studied with a model of J6-1 cell line. The results indicated that m-M-CSF bound rh-M-CSF-sR with high affinity (Kd= 1.78×10 12 mol/L) and mediated a temperature- and energy-dependent internalization of rh-M-CSF-sR, and that internalized rh-M-CSF-sR could return to the cell surface in an m-M-CSF-bound state, suggesting that m-M-CSF may have a capability to mediate the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes. In addition, the half-lives of cell-associated M-CSF and its receptor of stimulated normal human cord blood mononuclear cells (CBMCs) and 4 leukemic cell lines were measured by indirect immunoflu-orescence and flow cytometry. The results showed that the half-lives of the various kinds of M-CSF isoforms and

Frequency of HLA-A*03 associates with HIV-1 infection in a Chinese cohort

During the early mid-1990s, a number of rural farmers across central China were employed to the unregulated plasma- selling-activity and many of them were infected by HIV-1. However, AIDS progression in the former blood donors （FBDs） is various. The aim of this study is to assess human leukocyte antigen （HLA） class I allele distribution in FBDs and evaluate its association with HIV-1 infection and disease progression. A total of 353 FBDs were enrolled in the cohort including 294 ART na＇fve HIV-1 seropositive and 59 HIV-1 seronegative age-matched subjects. The viral load and CD4/CD8 T cell counts were assessed in all subjects. Compared with HIV-seropositive group, the frequency of HLA-A＊03 in control was significantly higher. After classifying the HLA-B alleles of the subjects according to the presence of Bw4/Bw6 serological epitopes, detri- mental effect of HLA Bw6/Bw6 homozygosity was also confirmed in the HIV-seropositive subjects. This study provides nov- el evidence on HLA class I allele distribution and association of HLA-A＊03 frequency with HIV-1 infection and viremia in the HIV-1 infected FBDs, which may throw light on intervention strategy for the HIV-1 infection and our understanding how host immunity and genetic background affect HIV infection and AIDS progression.

Signal transduction pathways mediated by colony stimulating factor-1 receptor

Colony-stimulating factor-1 (CSF-1), which is necessary for cell proliferation and differentiation, regulates both immediate and delayed early responses throughout G1 phase. The binding of CSF-1 to its receptor (CSF-1R) triggers phosphorylation of the receptor and its intrinsic tyrosine kinase. The activated receptor binds directly to cytoplasmic effector proteins, which induce multiple-signal transduction pathways. CSF-1 can induce the c-myc gene expression via Ras and Ets-related proteins. The expression of c-fos/jun family genes is also targeted following the activation of Ras. CSF-1R activates STAT1 and STAT3 to participate in signaling, but JAKs do not appear to contribute to signaling by CSF-1R. CSF-1R activates PI3-kinase, and PI3-kinase can interact with downstream proteins by the MAPKK-related pathway independent of Ras/Raf. PC-PLC can enforce signaling in response to CSF-1. Furthermore, the turnover and dephosphorylation by the phosphatase SHPTP1 of CSF-1R are the major mechanism in the negative regulation of signaling by CSF-1R.