人工接种条件下植物病原细菌噬菌体来源的研究

对盆栽水稻采用针刺、喷雾、盆水等不同方法接种水稻细菌性条斑病病菌后 ,检测稻株发病和盆水中的噬菌体 ,结果表明 :未接种稻细条病菌的处理中 ,无稻株发病 ,盆水中也未测到噬菌体 ;3种接种处理均可引致稻株发病 ,并先后在其盆水中检测到噬菌体 ,说明噬菌体与致病菌有密切的伴随关系。在采用叶面喷雾和盆水接种稻白叶枯自然组合菌株B 1和单细胞系菌株Ⅳ 16试验中 ,B 1处理后能引起稻株发病 ,且在盆水中测到噬菌体 ;而Ⅳ 16处理后虽引起稻株发病 ,但盆水中无噬菌体 ,结果表明

Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8^＋ T cells from a HLA-A2^＋ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION： The procedure is simple and efficient for generating peptide-MHC tetramers.

Expression of a soluble form of CTLA4 on macrophage and its biological activity

Interaction between cytotoxic T lymphocyte-asso-ciated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1and B7-2) is of importance in the cellular events of lym-phocyte, including antigen-specific T-cell activation andinduction of autoreactive T-cell. We describe here the firstintroduction of a murine soluble CTLA4 gene, CTLA4Ig,to Mm1 cells, a macrophagic cell line. CTLA4Ig waJssuccessfully expressed on Mm1 cells and the expressedCTLA4Ig was found to be functionally active in their bind-ing to B7 molecules by flow cytometry and immunofluo-rescence studies- The biological activity of CTLA4Ig fromthe transfected Mm1 cells was studied and showed in-hibitory activity on mixed lymphocyte culture. A highCTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfec-tion and there had so far been no report on expression ofCTLA4Ig gene on Mm1 cells, these results suggested thatthe CTLA4Ig expressing Mm1 cells could be useful forExpression of CTLA4 on Mml and its biological activityanalysis of CTLA4 and B7 molecule interaction in bothmacrophage and T-cell.

Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis

AIM: To investigate whether serum levels of two soluble forms of extracellular cytokeratin 18 (M30-antigen and M65-antigen) may differentiate nonalcoholic steatohepatitis (NASH) from simple steatosis in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: A total of 83 patients with suspected NAFLD and 49 healthy volunteers were investigated. Patients with suspected NAFLD were classified according to their liver histology into four groups: definitive NASH (n = 45), borderline NASH (n = 24), simple fatty liver (n = 9), and normal tissue (n = 5). Serum levels of caspase-3 generated cytokeratin-18 fragments (M30-antigen) and total cytokeratin-18 (M65-antigen) were determined by ELISA. RESULTS: Levels of M30-antigen and M65-antigen were significantly higher in patients with definitive NASH compared to the other groups. An abnormal value (> 121.60 IU/L) of M30-antigen yielded a 60.0% sensitivity and a 97.4% specificity for the diagnosis of NASH. Sensitivity and specificity of an abnormal M65-antigen level (> 243.82 IU/L) for the diagnosis of NASH were 68.9% and 81.6%, respectively. Among patients with NAFLD, M30-antigen and M65-antigen levels distinguished between advanced fibrosis and early-stage fibrosis with a sensitivity of 64.7% and 70.6%, and a specificity of 77.3% and 71.2%, respectively. CONCLUSION: Serum levels of M30-antigen and M65-antigen may be of clinical usefulness to identify patients with NASH. Further studies are mandatory to better assess the role of these apoptonecrotic biomarkers in NAFLD pathophysiology.

THE INCREASE IN PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 EXPRESSION BY STIMULATION OF ACTIVATORS FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS IN HUMAN ENDOTHELIAL CELLS

Objective.To investigate the effect of peroxis ome proliferator-activated recept ors(PPARs )activators on plasminogen activator inhibitor ty pe-1(PAI-1)expression in human umbilical vein e ndothelial cells and the possi-ble mechanism.Methods.Human umbilical vein endothelial ce lls(HUVECs )were obtained from normal fetus,and cul-tured conventionally.Then the HUVECs were exposed to test agents(linolenic acid,linoleic acid,oleic acid,stearic acid and prostaglandin J 2 respectively)in varying concentrations with fresh media.RT -PCR and ELISA were applied to determine the expression of PPARs and PAI-1in HUVECs.Results.PPARα,PPARδand PPARγmRNA were detected by using RT-PCR in HUVECs.Treatment of HUVECs with PPARαand PPARγactivators---linolenic acid,linoleic acid,oleic acid and prostaglandin J 2 respectively,but not with stearic a cid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner.However,the mRNA expressions of 3subclasses of PPAR with their activators in HUVECs were not changed compared w ith controls.Conclusion.HUVECs express PPARs.PPARs activators may increase PAI-1expression in ECs,but the underlying mechanism remains uncle ar.Although PPARs expression was not enhanced after stimulated by their activators in ECs,the role of functionally active PPARs in regulating PA I-1expression in ECs needs to be further investigated by using transient gen e transfection assay.

EXPRESSION OF TISSUE－TYPE PLASMINOGENACTIVATOR IN SMOOTH MUSCLE CELLS OF INJUREDILIAC ARTERIES IN RABBITS

In this experiment, expression of tissue-type plasminogen activator (t-PA) in smooth muscle cells (SMCs) was measured at different intervals after the arterial injury. In the normal iliac arteries, only low levels of t-PA activity were estimated. t-PA activity in extracts of the iliac arteries increased significantlyat the 4th day after the injury, equivalent to the process that SMCs migrated from the media to the intima,and the t-PA activity was then decreased approximately to the normal level at the 7th day. Coexistent to the above data, results from in situ hybridization showed that the expression of t-PA mRNA in the intima as well as media increased also significantly at the 4th day after the arterial injury, and at the 7th day, tPA mRNA was detected only in those SMCs locating closely adjacent to the internal elastic lamina. These results suggest that t-PA might play an important role in SMC migration following endothelial injury, andantagonism of t-PA expression and/or activity within the vessel wall might be helpful in intervening the de velopment of restenosis following angioplasty.

Expression of Plasminogen Activator Inhibitor-2 is Negatively Associated with Invasive Potential in Hepatocellular Carcinoma Cells

Objective To investigate the association between plasminogen activator inhibitor(PAI)-2 expression and invasive potential in hepatocellular carcinoma(HCC) cells.Methods The HCC cell lines with high,low,and non-metastatic potentials,namely MHCC97-H,MHCC97-L,and SMMC-7721 respectively,were cultured in vitro.Matrigel invasion assay and Western blot of PAI-2 protein expression were conducted.Results The number of invaded cells in MHCC97-L was significantly higher than that in SMMC-7721(P=0.005),whereas that in MHCC97-H was higher than in MHCC97-L(P=0.017) and SMMC-7721(P=0.001).Contrarily,PAI-2 protein expression was gradually reducing from SMMC-7721,MHCC97-L,to MHCC97-H(MHCC97-H vs.MHCC97-L,P<0.001;MHCC97-H vs.SMMC-7721,P=0.001;MHCC97-L vs.SMMC-7721,P=0.001).The Pearson's correlation analysis revealed a significant negative association between invaded cell number and PAI-2 expression(r= 0.892,P=0.001).Conclusion PAI-2 expression may be negatively associated with the invasive potential of HCC.