Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.     

The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases

The Rhizobium leguminosarum bv. trifolii region encoding pssA and pssB genes was cloned. The pssB gene located upstream of the pssA encoded a 28.36-kDa protein which displayed 97.5% identity with the PssB of R. leguminosarum bv. viciae. Inactivation of the pssB gene by insertion of the lacZ-Gmr cassette resulted in the significant increased production of exopolysaccharide in comparison to the wild-type level. A mutant strain was also defective in nitrogen fixation suggesting a regulatory role of pssB in symbiosis with clover.     

Harmful singlet oxygen can be helpful

Highly reactive harmful singlet oxygen O2(1delta(g)) can be helpful while relaxing to its triplet ground state O2(3sigma(g)-). The energy emitted during this relaxation from the excited energy state is discernable at 634 nm. We report here on the effect of this energy as photon illumination and as energy transfer in air on the production of reactive oxygen species (ROS) by human monocytes, measured as isoluminol-enhanced chemiluminescence. We demonstrate up to 60% decrease in the secretion of ROS after 2-min illumination of the monocytes stimulated with phorbol myristate acetate (PMA). The results provide in vitro documentation of the utility of singlet oxygen energy in modifying cellular behaviour.     

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)

Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within atherosclerotic lesions. Reactive oxygen species produced by NADPH oxidase were found to trigger the cyclooxygenase-2 expression. The effects of preferential COX-2 inhibitors on ROS produced by Chlamydia-primed human monocytes (THP-1 cells) were evaluated by fluorescence, chemiluminescence, oxymetry, and EPR spin trapping. Fluorescence assays showed an increased production of ROS with Chlamydia versus cells primed by 10(-8)M PMA. COX-2 inhibitors inhibited in a dose-dependent manner the luminol-enhanced CL while ibuprofen and diclofenac increased the chemiluminescence response. By EPR spin trapping, COX-2 inhibitors, ibuprofen, and diclofenac, exhibited a dose-dependent inhibiting effect (10 and 100muM) on the EPR signal appearance. Our cell model combining EPR, chemiluminescence, and oxymetry appeared relevant to study the modulating effects of preferential COX-2 inhibitors on the cell oxidant activity and chronic inflammatory diseases.     

Homogeneous sandwich immunoassay based on the enzymatic complementation induced by single-chain Fv fragments

We describe a novel homogeneous sandwich immunoassay based on beta-galactosidase (beta-gal) complementation (the crab-claw sandwich enzymatic complementation immunoassay, CS-ECIA). We chose a high-molecular-weight antigen human serum albumin (HSA) as a model and constructed two chimeric proteins, in which a pair of single-chain Fvs (scFvs) recognizing distant epitopes of HSA was fused to either an N-terminal deletion mutant of beta-gal (deltaalpha) or a C-terminal deletion mutant of beta-gal (deltaomega). Upon simple mixing of the reagents with the sample, the two chimeric proteins became associated through binding separate epitopes on HSA that allowed reassociation of the two mutant enzymes. The resulting enzymatic complementation was measured as an increase in beta-gal activity using a luminescent substrate. With this CS-ECIA, a HSA concentration of 10-1000 pg/mL could be determined. In addition, the assay was easy to operate and required less time, handling, and sample volume than conventional sandwich enzyme-linked immunoassays. The assay will have general utility by substituting scFvs with other pairs of scFvs recognizing any polyvalent antigens.     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

人肝金属硫蛋白-I_A基因在鱼腥藻中的克隆与表达

将人工合成的人肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,简称ＭＴ）－ＩＡ基因插入至中间载体ｐＲＬ－４３９上强启动子ｐｓｂＡ后,再将其与穿梭载体ｐＫＴ－２１０相连,得到大肠杆菌－蓝藻穿梭表达载体ｐＫＴ－ＭＴ,用三亲接合转移法将ｐＫＴ－ＭＴ转入丝状体蓝藻－鱼腥藻７１２０,经链霉素筛选,得到了稳定的转人肝ＭＴ－ＩＡ基因鱼腥藻．纯化单藻落,液体扩大培养．从鱼腥藻中提取的质粒经Ｓｏｕｔｈｅｒｎ印迹分析,确定人肝ＭＴ－ＩＡ基因已转入鱼腥藻７１２０中,Ｗｅｓｔｅｒｎ印迹分析表明,金属硫蛋白在转人肝ＭＴ－ＩＡ基因鱼腥藻中得到了表达．经原子吸收光谱法测定表达量约为７００μｇＭＴ／ｇ鲜藻,重金属耐受性实验表明,得到了能耐受重金属－镉的转人肝ＭＴ－ＩＡ基因鱼腥藻,它将在清除水域中重金属污染和医药研究方面发挥重要作用．     

MDR1反义RNA降低细胞KB_(v200)的耐药性

由ＭＤＲ１基因过度表达所引起的肿瘤细胞对化疗药物的耐药性,是导致化疗失败的主要原因之一．针对ＭＤＲ１中一段包含转录启始位点、翻译启始位点和转录正调控区的序列,设计了反义ＲＮＡ并将其克隆到逆转录病毒载体ｐＬＸＳＮ上．用脂质体包裹载体导入ＭＤＲ１高表达的耐药细胞ＫＢｖ２００中,在反义ＲＮＡ转染的细胞中,ＭＤＲ１在ｍＲＮＡ和蛋白水平的表达都有下降,细胞内药物的浓度有所提高,对长春新碱、阿霉素的耐药性分别下降了６５％和４７％．实验结果表明,反义ＲＮＡ对ＭＤＲ１的表达有抑制作用,从而使肿瘤细胞内的药物浓度升高,其耐药程度下降．     

PKC对人mdr1基因转录调控的研究

研究了ＰＫＣα、β１和β２亚型对多药耐药基因ｍｄｒ１转录的调控作用．以ｍｄｒ１基因上游调控序列驱动的虫荧光素酶报告基因表达载体和ＰＫＣ基因表达载体共同转染ＣＯＳ７细胞后,测定虫荧光素酶报告基因表达水平．实验结果表明,ＰＫＣα、β１及β２对ｍｄｒ１基因的转录有下调作用,ＰＭＡ可进一步加强这一作用;在此转染体系中,ＰＫＣ抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ也可抑制ｍｄｒ１基因的转录,但在只转染ｍｄｒｌｕｃ的细胞中,ｓｔａｕｒｏｓｐｏｒｉｎｅ对ｍｄｒ１的转录则没有影响,提示ｓｔａｕｒｏｓｐｏｒｉｎｅ仅在ＰＫＣ过量表达的细胞中抑制ｍｄｒ１基因的转录．     

白首乌对超氧阴离子自由基清除作用的研究

为深入探讨耳叶牛皮消块根（白首乌）的药理作用,指导其保健食品的研究与开发,采用化学发光法测定了去皮白首乌及白首乌栓皮对超氧阴离子自由基（Ｏ－２）的清除作用。结果表明,两者均能直接清除Ｏ－２,其ＩＣ５０分别为６９６９μｇ／ｍｌ和２２９８μｇ／ｍｌ,而且后者对Ｏ－２的清除能力比前者高出３０多倍,并同时测定了赤何首乌、西洋参、灵芝、当归、黄芪、菊花和竹叶。结果表明,去皮白首乌清除Ｏ－２的能力显著低于赤何首乌和灵芝,与西洋参和当归相差不大,显著高于黄芪、菊花和竹叶。而白首乌栓皮清除Ｏ－２的能力却高于其中任何一种中药。