A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity.

Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome RNA (rRNA). As specific immunotoxins, emergence of inhibitors for RA may obtain access to antagonistics against ricin intoxication and contribute to ameliorate the concomitant side effects. Many experimental results showed that the engineered VHs, which possessed solubility, stability, small size and consequently easier to express, purify and manipulate in vitro, were self and long-lived molecules compared to synthetic peptides. In this study, based on the crystal structure of RA, a novel recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH(PT)) was obtained using computer-guided molecular design method. Theoretically, rVH(PT) could penetrate deeply into the active cleft of RA and act as a potent antagonist analogue to block the RA-rRNA interaction. Followed results showed that the recombinant VH(PT) was easily expressed of high-yield production and in a partially soluble fusion form in Escherichia coli. In vitro cytotoxicity experiments demonstrated that it possessed remarkable ability to block ricin-induced cytotoxicity. This study highlights the potential of human VH to display biostructure and biofunction of peptides designed on RA functional domain and could be useful in developing new antidotes with potential therapeutic uses to neutralize unintended exposure to ricin.

Expression of adhesion molecules in polymyositis / 医学研究生学报

Objective:To study the role of adhesion molecules in the pathogenesis of polymyositis. Methods:The abnormal expression of adhesion molecules on T cells in peripheral blood and muscle fibers from patients with myositis was analyzed by two colour immunofluoresence and RT PCR methods respectively. Results:The expression of adhesion molecules including lymphocyte function associated antigen 1(LFA 1 ),very late antigen 4(VLA 4) on T cells in peripheral blood and intercellular adhesion molecule l(ICAM 1) on muscle fibers from patients with myositis was markedly higher than that in the healthy control group. Conclusion: These findings suggested that adhesion molecules may be responsible for the migration of T cells and destraction of muscle fibers.

Soluble intracellular adhesion molecule-1 concentrations in the serum and peritoneal fluid from endometriosis patients / 医学研究生学报

Objectives:To investigate the level of soluble intracellular adhesion molecule 1 (sICAM 1) concentrations in the serum and peritoneal fluid from the patients with or without endometriosis.To discuss the relationship within sICAM 1 and pelvic endometriosis. Methods:35 serum and 30 peritoneal fluid of patients with endometriosis (test group) and 26 serum and 4 peritoneal fluid without endometriosis (control group) were studied.Soluble intracellular adhesion molecule 1 levels were detected by an enzyme linked immunoassay(ELISA).The results were analysed by T test. Results: The serum concentration of sICAM 1 was significantly higher in test group than controll group. However,There was no significant difference in between two groups. Conclusions: These results suggest that sICAM 1 could play some role in the persistence of endometriotic lesions.

Expression and purification of recombinant N-terminal peptide of human perforin / 医学研究生学报

Objectives: To express and purify N-terminal peptide of human perforin(hPFP-N). 　Methods: The recombinant expressive plasmid pGEX-KG/hPFP-N was constructed and then introduced into a strain of E. coli BL21(DE3). Upon the induction of IPTG, GST/hPFP-N fusion protein was expressed. The expressed fusion protein was localized in inclusion bodies which could be solubilized by sonication after detergent lauroylsarcosine was added. The fusion protein was purified by affinity chromatography with glutathione agarose. After being cleaved by thrombin, GST and uncleaved fusion protein were removed by glutathione agarose beads once more, then purified recombinant rhPFP-N protein was obtained.　 Results and Conclusions: GST/hPFP-N fusion protein can be effectively expressed in E. coli and the protein hPFP-N was obtained after the purification process.

Establishment and clinical application of quantitative RT-PCR method for detecting perforin mRNA / 临床检验杂志

Objective To establish the quantitative method for detecting perforin mRNA. Methods The competitive templates were prepared by restriction endonuclease method.The quantitative RT-PCR assay was established and used to detecting the perforin mRNA level in peripheral blood of tumor patients and healthy adults.Results Average perforin mRNA level of six healthy adults is 0.51±0.40 pmol/ml.The perforin mRNA level of patients with digestive tumors are significantly lower than that of healthy adults(P<0.01).But compared with healthy adults,no significant difference is showed in patients with breast cancer or leukemia.Conclusion The decreased perforin mRNA level probably attributes to the high incidence of cancers.Except for perforin-mediated cytolysis,additional effective mechanism against tumor cells maybe exist.