Mechanism of CCT2, a new downstream substrate of PDGFRα, on proliferation of tumor cells / 上海交通大学学报（医学版）

Objective: To study the molecular mechanism of chaperonin containing TCP1 subunit 2(CCT2), a new downstream substrate of platelet derived growth factor receptor α(PDGFRα), in tumorigenesis. Methods: Non-small cell lung cancer cell line H1703 was used. Western blotting was used to measure the phosphorylation of CCT2 upon PDGFRα inhibitor Gleevec treatment and PDGF stimulation. H1703 cells were divided into siCon group, siPDGFRα group and siCCT2 group; 48 h later, cell number counting was used to test the effect of CCT2 on cell growth after siRNA transfection. H1703 cells were divided into siCon group, siPDGFRα group, siAKT group and siCCT2 group; Western blotting was used to measure the protein level of PDGFRα and PARP. Cell fractionation was used to detect the cellular localization of CCT2 and co-immunoprecipitation was used to test the interaction between CCT2 and PDGFRα. Results: CCT2 phosphorylation was inhibited by Gleevec and induced by PDGF. Compared to the control group, the number of cells transfected by siCCT2 reduced by 30% (P=0.006). The protein level of PDGFRα was also decreased in siCCT2 transfected cells, whereas the cleavage of PARP was increased. CCT2 was localized in both cytoplasmic and membrane fractions and interacted with PDGFRα directly. Conclusion: CCT2 is a new downstream substrate of PDGFRα. CCT2 can promote tumor cells growth by interacting and stabilizing PDGFRα.

Detection of antibodies against immunodominant proteins of Chlamydia trachomatis in the sera of patients with urogenital Chlamydia trachomatis infection / 中华皮肤科杂志

Objective To detect antibodies against chlamydial plasmid-encoded protein 3 (Pgp3),outer membrane complex protein B C-terminal peptide (OmcBc),CT841 protein and heat shock protein 60 (HSP60) in the sera of patients with urogenital Chlamydia trachomatis infection.Methods Recombinant plasmids encoding the aforementioned four proteins and an empty plasmid were transformed into Escherichia coli separately followed by 2-hour isopropyl-1-thio-β-galactopyranoside (IPTG) induction and cell lysis.The expressed proteins were purified with glutathione magnetic beads and then used to coat 96-well enzyme-linked immunosorbent assay (ELISA) plates precoated by glutathione.Serum samples were collected from 20 patients with and 20 clients without urogenital C.trachomatis infection attending the sexually transmitted disease (STD) clinic of Tianjin Medical University General Hospital.ELISA with the expressed protein-coated plates was adopted to detect antibodies against these proteins in the serum samples.Results Of the 20 serum samples from C.trachomatis-infected patients,14 (70％)had anti-Pgp3 antibody,9 (45％) anti-OmcBc antibody,8 (40％) anti-CT841 antibody,and 5 (25％) anti-HSP60 antibody.Meanwhile,no antibody was detected in any of the serum samples from uninfected clients except for one with anti-HSP60 antibody.Conclusions Of the four studied C.trachomatis proteins,Pgp3 appears to have the strongest antigenicity with the highest antibody-detection rate,while HSP60 exhibits the weakest antigenicity with the lowest antibody-detection rate.

The Expression of Heat Shock Protein 60 kDa in Tissues and Cell Lines of Breast Cancer / 한국유방암학회지

PURPOSE: Breast cancer has been reported as the most common cancer of women in the United States, Western Europe and Korea and about 210,000 and 10,000 women in United States and Korea every year, respectively are diagnosed with it. Breast cancer is curable with an early diagnosis, and many researchers have made efforts to find a marker for this malady, heat shock protein (HSP) consists of 6 groups, it is highly preserved throughout both the prokaryotic and eukaryotic cells and it acts as a molecular chaperone that's involved in protein folding. HSPs have been recently reported to be related with breast cancer. In this study, we investigated the changes of expression of HSP60 in tissues and cell lines of breast cancer. METHODS: We obtained breast cancer tissues and normal tissues from breast cancer patients, and we purchased several cancer cell lines from American tissue culture correction. We treated the tissues and the cell lines of human breast cancer with heat shock protein. Proteins and mRNAs were isolated from the tissues and the cells and then we performed Western blotting, reverse transcriptase-Polymerase chain Reaction and fluorescence activated cell sorter analysis on them. RESULTS: On Western blot, HSP60 was more overexpressed in the tissues and the cell lines of breast cancer than in the normal breast tissues and cell lines. The expression of HSP60 showed 2 types of molecular weight differences in the tissues and cell lines of breast cancer, and specifically, low HSP60 was over-expressed in the cancer tissues. There was no difference between the breast cancer cell lines and the normal cell lines in the expressions of HSP60 mRNA, according to the treatment with heat shock. Also, there was no relationship between phosphorylation and the structural difference of HSP60 protein, according to HSP60 protein's molecular weight. The expression of HSP60 has been mostly reported at the mitochondria; however, in this study, it was more predominantly detected at the cytoplasm than at the mitochondria in the breast cancer cell lines. CONCLUSION: We conclude that HSP60 may be used as a diagnostic marker for breast cancer. Detailed investigation of the usefulness and significance of the HSP60 expression as a prognostic factor is required in further studies.

FURTHER EVIDENCE THAT GENOTYPE I AND GENOTYPE II OF CRYPTOSPORIDIUM PARVUM ARE DISTINCT

Three new genes of <I>Cryptosporidium parvum</I> were cloned, including a gene encoding methionine aminopeptidase, one encoding chaperonin containing T-complex protein 1 delta (TCP-1 delta) and one with unknown function. DNA sequence analysis indicated that these genes are quite conserved, but there were some base pair differences between genotype I and genotype II isolates. These differences were confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 3 genes from 41 isolates collected from different hosts and geographical origins. In brief, the band patterns generated by endonuclease Hind III or Hinf I restrictions of the gene of methionine aminopeptidase, Sac I restriction of the gene of chaperonin, or Ava II restriction of the unknown gene could differentiate the isolates of <I>C. parvum</I> into genotype I and genotype II. PCR primers based on these genes amplified only <I>C. parvum</I> genes. Even a single oocyst was detectable with these PCR primers. Thus the results provided further evidence that genotype I and genotype II are distinct, and our three new primers can be used to detect and characterize <I>C. parvum</I> isolates with high sensitivity.

Recent advances in heat shock protein 60 / 中国病理生理杂志

The family of HSP60 belongs to heat shock proteins with highly species conservatism and some important biologic functions. It can help other proteins for their assembling, folding and translocating, and plays a role in protecting cells against injuries and other types of stress. In addition, HSP60 is frequently recognized by the immune system as predominant antigens during infections and the progression of certain autoimmune diseases and might provide a novel strategy for the development of immunotherapeutics. This review focuses on distribution, molecular chaperone mechanism, function and gene expression regulation of HSP60. [

Cloning and Expression of Heat Shock Protein 60 Gene from Chlamydia trachomatis / 中华皮肤科杂志

90%inasinglestep,andtheproteinyieldwas2.5mg/L.ConclusionPurifiedChsp60kDantigenisobtained,andtheantigenmightbeappliedtodetectantibodyinpatientsinfectedwithChlamydialtrachomatis,andwillcontributetostudytheroleofChsp60inimmunopathogenesis.

Cloning of Chaperonin Gene from Acidianus tengchongrensis Using Inverse PCR Method / 微生物学通报

DNA fragments of ? subunit and ? subunit were amplified by PCR from the genome of Acidianus tengchongensis with the degenerated primers designed from the consensus amino acid sequence of Sulfolobus chaperonins. The two DNA fragments about 500bp were used as probe for southern hybridization to determine the suitable restriction endonuclease. Restriction endonuclease digested genomic DNA was circularized by self-ligation, and complete sequences of ? subunit and ? subunit were obtained by inverse PCR. The primers oriented in the reversed direction of the usual orientation to amplify the DNA sequence that flank the known region. The complete genes of ? subunit and ? subunit were PCR amplified from genomic DNA using two pairs primers.