百日咳无细胞菌苗的生物学特征

作者介绍百日咳无细胞菌苗的简单制备方法及其生物学活性。将百日咳杆菌305株培养在液体培养基中,于稳定条件下培养5天,离心除菌体,上清液加酸沉淀,然后用缓冲液溶解,再经福尔马林脱毒处理。所分离的抗原复合物与鸡及绵羊红细胞具有明显的血凝活性。胆固醇可使血凝素活性稍有下降,可见该抗原复合物含有淋巴细胞增多促进因子-血凝素(LPF-HA)和丝状血凝素     

螠蛏共附枯草芽孢杆菌110次级代谢产物的抑菌抗肿瘤活性研究Δ

摘要：目的 检测螠蛏共附枯草芽孢杆菌110次级代谢产物的抑菌活性组分,并探讨抑菌活性组分的抗肿瘤作用。方法 用0.22 μm滤膜过滤将螠蛏共附枯草芽孢杆菌110的菌体和发酵液分开,用硫酸铵沉淀发酵液的蛋白组分并用SDS-PAGE检测,用滤纸片法分别检测菌株发酵液、菌体及胞外蛋白的抑菌活性,琼脂块法检测抑菌活性组分的抑真菌活性,用海虾毒性法和MTT法检测抑菌活性组分的抗肿瘤活性。结果 螠蛏共附枯草芽孢杆菌110的发酵液抑菌活性远远高于菌体的抑菌活性,发酵液的硫酸铵沉淀物具有很强的抑菌活性,硫酸铵沉淀的110活性粗蛋白主要由分子量约为60、45和25 kDa的3条带组成。110活性粗蛋白对8种真菌指示菌具有抑制作用,其中对辣椒青枯、番茄灰霉和瓜类枯萎的抑菌圈直径超过20 mm。该活性粗蛋白还对白血病细胞K562、肝癌细胞HepG-2和人乳腺癌细胞MDA-MB-231具有抑制作用,其中200 μg/mL的蛋白对人肝癌细胞HepG-2抑制率达74.01%。结论 螠蛏共附枯草芽孢杆菌110胞外蛋白有抗肿瘤活性和广谱抑菌活性,具有潜在的开发价值。     

红色诺卡氏菌菌体形态与细胞破碎关系的研究

本文为研究红色诺卡氏菌的培养条件对菌体形态的影响，探讨其与细胞破碎难易的关系。结果表明红色诺卡氏菌形态与糖类和酵母膏型号有密切关系，菌体呈菌丝或杆状，细胞易破碎。菌体呈球状，细胞不易破碎。而菌丝型菌体和球型菌体的细胞壁形态和成份均相同。     

粘液与非粘液型鲍曼不动杆菌诱导肺腺癌细胞产生IL-8的研究

目的 研究粘液与非粘液型的鲍曼不动杆菌活菌菌悬液、培养上清液及菌体裂解液诱导人肺腺癌细胞表达白介素-8(IL-8)的能力.方法 制备2×109 cfu/ml、2×108 cfu/ml、2×107 cfu/ml的活菌悬液、菌体裂解液以及培养上清液,分别与人肺腺癌细胞孵育,6、12、24h后提取腺癌细胞的总RNA,逆转录扩增(RT-PCR)IL-8的mRNA,扩增产物行1%的琼脂糖凝胶电泳.结果 两种鲍曼不动杆菌的活菌菌悬液、培养上清液、菌体裂解液在不同浓度、不同时间点与人肺腺癌细胞孵育后,均只在24h 时间点表达IL-8.粘液型的菌体裂解液只有 2×108 cfu/ml 时可以诱导人肺腺癌细胞表达IL-8;非粘液表型的菌体裂解液在各浓度则均不能诱导人肺腺癌细胞表达IL-8.结论 菌落差异的可以影响细菌诱导肺腺癌细胞IL-8的产生.细菌经过不同的方式处理后诱导细胞产生IL-8的能力发生变化.     

海绵共附生真菌抗菌活性次级代谢产物研究进展

海绵共附生真菌次级代谢产物具有独特的化学结构和显著的生物学活性,是创新药物研发的重要来源.这些次级代谢产物对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)等多种致病菌株具有潜在的抗菌活性.本文综述了2002-...     

人脂联素基因在L-02细胞中表达及其功能检测

目的:在人正常肝细胞株L-02中表达外源人脂联素蛋白,并观察其生物学活性。方法:采用脂质体法将成功构建的人脂联素真核表达质粒pcDNA3.1/CT-GFP-TOPO-Adiponectin转染L-02细胞;免疫组织化学定位观察脂联素的表达;Western blot检测培养上清及细胞沉淀中脂联素水平;酶联免疫法测定培养基血糖。结果:构建的真核表达质粒能有效转染L-02细胞,并在培养上清及细胞沉淀中检测到脂联素蛋白,该重组蛋白能够降低高糖处理的L-02细胞的血糖水平。结论:重组人脂联素蛋白能在L-02中有效表达,该蛋白定位在细胞质,并且能分泌到胞外,表达产物经体外实验证实具有生物学活性。     

银杏叶提取物GBE50对HepG2细胞甘油三酯代谢的影响

目的观察银杏叶提取物GBE50对HepG2细胞内甘油三酯含量的影响以及甘油三酯代谢关键基因CPT1A表达的调节。方法检测GBE50对培养细胞HepG2甘油三酯含量的影响;测定GBE50处理HepG2细胞24 h后CPT1A酶活性,用定量PCR检测药物处理后CPT1A基因表达水平的改变,Western blot检测CPT1A蛋白表达。结果 GBE50可降低培养细胞HepG2内甘油三酯含量。同时可以促进细胞CPT1A基因的表达,且能增加CPT1A酶的活性。结论 GBE50可有效降低HepG2细胞内甘油三酯的含量,CPT1A基因表达量的改变可能是GBE50调节HepG2细胞内甘油三酯水平的机制之一。     

促细胞代谢素注射液中多肽的分析及其与稳定性的关系

采用高效液相色谱法分析了"促细胞代谢素注射液"中的活性成份多肽, 并研究其与稳定性关系."促细胞代谢素注射液"中的多肽由十几种成份组成,其肽谱图与进口" 爱维治"基本相同;高温和强光照射能明显改变本品的肽谱图;室温考察时,其多肽组成稳定.本研究不仅为分析"促细胞代谢素注射液"的活性成份多肽提供了一种可行的方法,而且为研究其稳定性提供了科学的依据.     

Vero细胞检测出血性大肠杆菌O157:H7毒素的方法研究

目的建立用Vero细胞检测大肠埃希菌毒素的方法。方法用两种不同的培养基培养10株大肠杆菌和1株痢疾志贺杆菌,将其培养物上清和菌体裂解液加入Vero细胞中,观察对Vero细胞的毒性作用。结果两种培养基培养大肠杆菌产生的上清对Vero细胞的毒性作用差异不大,大肠杆菌培养上清的毒性作用大于菌体,而痢疾志贺菌菌体的毒性作用大于上清。结论Vero细胞毒性检测方法可以用于检测出血性大肠杆菌细胞毒素。     

乳杆菌A-2代谢产物对人舌鳞癌CAL-27细胞增殖与凋亡的初步研究

目的：初步研究乳杆菌A-2代谢产物（LSPM1和LSPM2）对人舌鳞癌CAL-27细胞的增殖抑制及诱导细胞凋亡.方法：乳杆菌A-2通过降解制备相应的代谢产物LSPM1和LSPM2;采用MTT法检测不同浓度的LSPM1和LSPM2（1.875mg/mL、3.75mg/mL、7.5mg/mL、15mg/mL和30mg/mL）对CAL-27细胞的体外增殖抑制作用;荧光显微镜下观察CAL-27细胞形态变化;采用流式细胞仪和DNA琼脂凝胶电泳法检测细胞凋亡情况.结果：不同浓度的乳杆菌A-2代谢产物LSPM1和LSPM2可以抑制CAL-27细胞的增殖,呈现明显的浓度依赖性;LPSM1可明显诱导CAL-27细胞凋亡,呈时间依赖性.结论：乳杆菌A-2代谢产物可以在体外抑制人舌鳞癌CAL-27细胞生长增殖,并诱导CAL-27细胞凋亡.     

Investigations into the membrane associated IL-1 like activity on mouse macrophages

The present study was undertaken to investigate the expression of membrane IL-1 (mIL-1) in mouse macrophages. Membrane IL-1 activity was measured on paraformaldehyde fixed peritoneal macrophages using EL4 6.1 lymphocyte cell line. Acid washing prior to fixation did not remove the mIL-1 activity, indicating that mIL-1 is an integral part of the membrane. Adherence to tissue culture plastic plates was found to be a potent inducer of mIL-1 activity in both resident and C. parvum activated macrophages. Dexamethasone reduced mIL-1 activity induced in vivo by C. parvum and also that induced by adherence in vitro.     

Role of interferon, antibodies and macrophages in the protective effect of Corynebacterium parvum on encephalomyocarditis virus-induced disease in mice

Treatment of mice with Corynebacterium parvum enhances their resistance to encephalomyocarditis (EMC) virus infection. In EMC-virus-infected mice, pretreated with C. parvum, neither interferon production nor antibody responses were increased. A decrease of virus recovery was observed in cultures of macrophages taken from the peritoneum of mice early after injection of C. parvum and infected with EMC-virus in vitro. The data suggest that C. parvum acts by increasing intrinsic antiviral activity of macrophages.     

Mechanism of cell proliferation--cell cycle, oncogenes, and senescence

Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.     

Identification of isoflavone derivatives as effective anticryptosporidial agents in vitro and in vivo

We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein analogues (2-H isoflavones), 2-carboethoxy isoflavones, and the precursor deoxybenzoins were all evaluated. Excellent in vitro activity against Cryptosporidium parvum was observed for both classes of isoflavones in cell cultures, and the lead compound 19, RM6427, shows high in vivo efficacy against an experimental infection.     

Effect of OKY-046 and ONO-3708 on liver injury in mice

The effects of OKY-046, a selective thromboxane A2 (TXA2) synthetase inhibitor, and ONO-3708, a novel TXA2 receptor antagonist, on liver disease were investigated in mice. The liver injury was induced by either an injection of antibasic liver protein (BLP) antibody into DBA/2 mice that had been previously immunized with rabbit IgG or by an injection of bacterial lipopolysaccharide (LPS) into Corynebacterium parvum (C. parvum) pretreated DDY mice. 1) In both injury models, clear elevation of glutamate transaminase (GOT and GPT) activity due to extensive liver parenchymal cell damage was observed; this was confirmed by significant histopathological changes in the liver. 2) Typical histopathological changes in the liver were submassive hepatocellular necrosis in the anti-BLP antibody-induced injury model and focal necrosis in the LPS-induced model. Inflammation and increased cell infiltration in portal connective tissue were observed in both cases. 3) Administration of OKY-046 (50 mg/kg) and ONO-3708 (0.5, 1.0 and 2.0 mg/kg) suppressed the elevation of serum GOT and GPT levels and histopathological changes in both experimental liver injury models. 4) Indomethacin inhibited the development of liver disease caused by anti-BLP antibody but not by bacterial LPS. Prostaglandin I2 inhibited the elevation of serum GOT and GPT levels and histopathological changes of the liver in the mice treated with anti-BLP antibody and showed the tendency to inhibit the development of liver injury caused by bacterial LPS.     

Sequential induction of nitric oxide synthase by Corynebacterium parvum in different organs of the mouse.

1. The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2-/NO3- in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied. 2. C. parvum (100 mg kg-1, i.p.) induced a time-dependent expression of a Ca(2+)-independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta. 3. There was no significant induction of a Ca(2+)-independent NO synthase in the brain, nor was there any change in the Ca(2+)-dependent enzyme in this organ, during the study period. 4. C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 +/- 1 mmHg to 79 +/- 3 mmHg), which recovered gradually by day 28. 5. Plasma NO2-/NO3- was significantly elevated between days 8 and 24, with a maximum increase at day 12. 6. These results show that C. parvum induces a Ca(2+)-independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cyclosporin A on the production of interferon by human peripheral blood leukocytes in vitro

Cyclosporin A (CsA) was assessed for its effect on the production of antiviral activity by human peripheral blood leukocytes (PBL). CsA markedly reduced the production of interferon-gamma (IFN-gamma) in response to stimulation with lectin mitogens, bacterial products, alloantigens, or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). CsA-mediated suppression of IFN-gamma secretion was dose-dependent and did not result from a shift of kinetics of the production of antiviral activity. The production of IFN-alpha in response to stimulation with Corynebacterium parvum (CP), viruses, and synthetic polynucleotides was not affected by the addition of CsA. These findings confirm earlier observations that CsA predominantly acts on T lymphocyte function. CsA may prove a valuable agent to study the role of IFN-gamma in the pathogenesis of virus-associated malignant lymphoproliferative disease.     

Production and characterisation of mucoadhesive nanosuspensions for the formulation of bupravaquone

Bupravaquone is a new naphthoquinone antibiotic against Cryptosporidium parvum and other parasites. It has attracted interest for the treatment of C. parvum infections, because of the lack of a drug in the treatment of mostly AIDS patients. The bioavailability of bupravaquone is limited when given orally. To overcome the problem of the high elimination rate caused by diarrhoea, typical for C. parvum infections, bupravaquone was formulated as a mucoadhesive nanosuspension, i.e. combining the properties of mucoadhesive drug delivery systems, in this case hydro gels, with nanosuspensions. In this study different polymers/hydro gels were employed to create a prolonged retention time for the drug in the infected gastrointestinal tract (GIT). The second step to improve the bioavailability of bupravaquone was the formulation as nanosuspension. Therefore various concentrations of bupravaquone with different surfactants were tested. The production of these nanosuspensions was carried out by high pressure homogenisation. In addition to the classical stepwise production, about a new one step production method is described.     

Detection of Cryptosporidium parvum in human feces by PCR

C. parvum has a high pathologic potential also for man, especially for immununosuppressed patients. The microscopic detection of cysts in feces is neither easy nor always reliable. During the recent years, considerable progress has been achieved in establishing PCR-based approaches for i) sensitive detection of C. parvum in a variety of specimen types [3, 9-16, 18, 21, 24, 25, 27, 29-32, 34], ii) identification of individual genotypes of C. parvum [2, 4-7, 17, 19, 20, 22, 23, 26, 28, 33] and iii) viability testing of C. parvum organisms [8, 9, 13, 29, 30]. The protocols published so far include nested PCR [3, 8, 18, 34], RT-PCR [13, 29], and use of the UNG carryover prevention system [10]. The aim of this work was to establish a PCR system for the detection of C. parvum oocysts in stool samples, applying the same specimen preparation procedure as applied for immunofluorescence. In addition, we combined the UNG carryover prevention system with the use of long, PCR-generated digoxigenin-labelled probes, thus achieving a sensitivity comparable to nested PCR und circumventing the contamination risks associated with nested PCR protocols. We developed a simplified sucrose-cushion-based protocol for preparation of clinical specimens (adopted from [1]), satisfying both the needs of immunofluorescence and PCR. When tested with stool samples spiked with C. parvum oocysts, the analytical sensitivity of PCR was 3,500 oocysts/ml stool (immunofluorescence: 3,000 oocysts/ml stool), demonstrating that both methods were equivalent with respect to analytical sensitivity. However, when PCR and immnunofluorescence were applied to clinical samples (n=5) with known positivity for C. parvum, only the specimen with the shortest duration of storage (5 weeks) could be correctly identified by PCR (clinical sensitivity: 20%). Our results demonstrate, that the PCR approach presented in this work is not suited for highly sensitive detection of C. parvum in faeces. This was mainly due to the fact that sucrose-gradient purified material was used, which relies on the presence of morphologically intact oocysts in the specimens. Desintegration of oocysts by excystation and/or storage may lower the parasite yield of the protocol drastically. As a consequence, a protocol extracting the entire DNA from faeces should be used for PCR for detection of C. parvum [34].     

The effect of gomisin A on immunologic liver injury in mice

The hepatoprotective effect of Gomisin A (TJN-101), which is a lignan compound isolated from Schizandra fruits, was studied on three immunologic liver injury models in mice. The first liver injury model was produced by the injection of anti-basic liver protein (BLP) antibody into DBA/2 mice which had been previously immunized with rabbit IgG (RGG). Other models were effected by injection of anti-liver specific protein (LSP) antibody into DBA/2 mice or by the injection of bacterial lipopolysaccharide (LPS) into ddY mice pretreated with Corynebacterium parvum (C. parvum). TJN-101 inhibited the elevation of transaminase (GOT and GPT) activities and showed the tendency to inhibit the histopathological changes of the liver in all models. Moreover, TJN-101 inhibited deoxycholic acid-induced release of transaminase from cultured rat hepatocytes in vitro, but did not affect the formation of hemolytic plaque forming cells in immunized mice spleens and hemolytic activity of guinea pig complement in immunohemolysis reaction. These results, therefore, suggested that the hepatoprotective effect of TJN-101 could be related to the protecting effect of hepatocyte plasma membrane rather than the inhibiting effects of the antibody formation and complement activity.