青霉源抗菌肽类霉肽素的体外抑菌活性研究

目的 研究抗菌肽类霉肽素(AF)的体外抑菌活性.方法 通过抑菌圈法和二倍稀释法检测AF的抗菌谱和对金黄色葡萄球菌的MIC;通过检测在AF中传代菌的敏感性确定细菌是否容易对AF产生抗药菌;将AF和细菌在不同温度和pH环境作用后检测抑菌活性,明确AF发挥活性的最适温度和pH值.结果 AF对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的MIC为0.8 mg/ml,金黄色葡萄球菌在AF中传代200代后敏感性不变.在14℃到30℃之间AF的抑菌活性随温度升高而降低,在pH 3到pH 10之间AF的活性随pH值升高而降低.结论 AF是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性.     

青霉源抗菌肽类霉钛类的体外抑菌活性研究

 目的 研究抗菌肽类霉肽素（AF）的体外抑菌活性。 方法 通过抑菌圈法和二倍稀释法检测 AF 的抗菌谱和对金黄色葡萄球菌的 MIC;通过检测在 AF 中传代菌的敏感性确定细菌是否容易对 AF 产生抗药菌;将 AF 和细菌在不同温度和 pH 环境作用后检测抑菌活性,明确 AF 发挥活性的最适温度和 pH 值。 结果 AF 对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的 MIC 为 0.8 mg/ml,金黄色葡萄球菌在 AF 中传代 200 代后敏感性不变。在 14 ℃ 到 30 ℃ 之间 AF 的抑菌活性随温度升高而降低,在 pH 3 到 pH 10 之间 AF 的活性随 pH 值升高而降低。 结论 AF 是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性。     

丰肉结海绵相关真菌产黄青霉HLS111菌株活性代谢产物研究

目的对我国南海水域丰肉结海绵相关真菌产黄青霉菌HLS111的活性代谢产物进行研究。方法采用HPLC-DAD技术与活性筛选相结合的方法,通过硅胶柱、凝胶柱及HPLC等色谱方法对HLS111发酵产物进行分离纯化;通过核磁共振、质谱等波谱分析手段对分离得到的化合物进行结构鉴定;并对单体化合物进行抗肿瘤、抗HIV、抗炎活性测定。结果分离得到12个化合物。其中2个黑麦酮酸类化合物:黑麦酮酸F(1)和D(2);4个生物碱类化合物:环(L-色氨酸-L-苯丙氨酸)(3)、citreoindole(4)、meleagrin(5)、脑苷脂B(6);4个甾体类化合物:麦角甾醇(7)、(22E,24R)-5α,8α-过氧化麦角甾-6,22-烯-3β-醇(8)、globosterol(9)、β-谷甾醇(10);1个三萜类化合物:24-亚甲基环木菠萝烷醇-反式阿魏酸(11);1个蒽醌类化合物:大黄素(12)。对化合物4的氢谱、碳谱数据进行了归属。初步的药理研究表明,黑麦酮酸类化合物表现出较强的抗肿瘤活性,化合物citreoindole表现出一定的抗HIV及抗炎活性。结论海绵相关真菌产黄青霉HLS111菌株体现了次生代谢产物化学结构的多样性;黑麦酮酸类化合物为产黄青霉HLS111的主要抗肿瘤活性成分。     

内毒素结合肽模拟肽P11的体外活性研究

目的 对从噬菌体展示随机肽库筛选获得的内毒素结合肽模拟肽进行体外拮抗内毒素活性鉴定.方法 采用FMOC固相合成法化学合成内毒素结合肽模拟肽P11,并进行拮抗内毒素活性和细胞毒性测定.结果 亲和ELISA检测显示P11与LPS有较高的亲和力,通过生长曲线和流式细胞学分析细胞周期显示P11对人U937细胞生长无明显影响.流式细胞检测显示P11呈剂量依赖性抑制FITC-LPS与人外周血单核细胞(PBMC)结合.细胞因子生成抑制实验显示10 μg/ml P11可显著抑制LPS诱导PBMC和U937细胞TNF-α mRNA转录和蛋白表达.结论 体外活性鉴定结果表明化学合成的模拟肽P11可抑制LPS诱导的炎性反应.     

体外抑菌试验评价核糖体表达筛选的抗菌肽活性

目的 观察体外无细胞核糖体表达系统筛选得到的、能与细菌膜结合的多肽的体外抑菌活性变化,初步评价该筛选系统的可行性.方法 采用核糖体展示系统和细胞膜模型筛选可结合于细胞膜的多肽,对筛选的多肽进行结构预测分析.选择形成α-螺旋结构可能性大、溶解性尚可的多肽(C13)进行合成.利用改良的微量肉汤稀释法测定C13和硫酸庆大霉素对G+(金黄色葡萄球菌)和G-(大肠杆菌、伤寒杆菌)细菌的抗菌活性--最小抑菌浓度(MIC)和最小杀菌浓度(MBC).结果 C13对试验细菌的MIC均为400 mg/L,但在现有浓度范围对试验菌的MBC则未测得.结论 体外无细胞核糖体表达系统筛选的抗菌肽C13具有一定的抑菌活性,表明利用人工膜模型和核糖体表达进行抗菌肽筛选的方法是可行的,但有效抗菌肽的筛选需进一步的抑菌试验证实.     

体外抑菌试验评价核糖体表达筛选的抗菌肽活性

目的观察体外无细胞核糖体表达系统筛选得到的、能与细菌膜结合的多肽的体外抑菌活性变化,初步评价该筛选系统的可行性。方法采用核糖体展示系统和细胞膜模型筛选可结合于细胞膜的多肽,对筛选的多肽进行结构预测分析。选择形成α-螺旋结构可能性大、溶解性尚可的多肽(C13)进行合成。利用改良的微量肉汤稀释法测定C13和硫酸庆大霉素对G (金黄色葡萄球菌)和G-(大肠杆菌、伤寒杆菌)细菌的抗菌活性——最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果C13对试验细菌的MIC均为400mg/L,但在现有浓度范围对试验菌的MBC则未测得。结论体外无细胞核糖体表达系统筛选的抗菌肽C13具有一定的抑菌活性,表明利用人工膜模型和核糖体表达进行抗菌肽筛选的方法是可行的,但有效抗菌肽的筛选需进一步的抑菌试验证实。     

类胰高血糖素肽1—免疫活性细胞在胎儿胃肠道的分布

目的 研究类胰高血糖素肽－１（Ｇｌｕｃａｇｏｎ－ｌｉｋｅ ｐｒｐｔｉｄｅ,ＧＬＰ－１）在胎儿高肠道中的分布情况。方法 应用免疫组织化学法ＡＢＣ法,对５例约２３周左右胎儿胃肠道内ＧＬＰ－１免疫活性细胞进行定位研究。结果 胎儿胃肠道中ＧＬＰ－１免疫活性细胞密度最大是直肠,其次是结肠和回肠。结论 ＧＬＰ－１是免疫活性细胞主要分布在肠道远端。     

蝉拟青霉多糖免疫调节和抗肿瘤活性的实验研究

目的：研究蝉拟青霉总多糖(PcPSt)及其组分PcPSAc的免疫调节和抗肿瘤活性。方法：将实验动物分为5组：正常对照组、肿瘤对照组、PcPSt组、环磷酰胺（Cy）组和PcPSt + Cy组，除正常对照组外其余各组均复制荷瘤小鼠模型，每日分别向5组小鼠腹腔注射生理盐水（NS）、NS、PcPSt、Cy和PcPSt+Cy，10 d后，观察各组白细胞、脾指数、瘤重和抑瘤率等指标的变化。以cell counting kit 8（CCK-8）为指标，以脂多糖（LPS）为阳性对照，观察PcPSAc对体外培养的小鼠脾细胞增殖活性影响。以LPS为阳性对照，观察不同浓度PcPSAc刺激体外培养小鼠腹腔巨噬细胞（PMΦ）产生一氧化氮（NO）的作用。结果： PcPSt能增加荷瘤小鼠白细胞数量，缓解Cy所致荷瘤小鼠白细胞数的降低，提高荷瘤小鼠的脾指数，联用小剂量Cy时明显增强其抑制肿瘤作用。PcPSAc在600 mg/L和300 mg/L浓度时能提高体外培养小鼠脾细胞的增殖活性。PcPSAc在15-300 mg/L浓度范围内均能刺激体外培养小鼠PMΦ产生NO，其中以300 mg/L PcPSAc作用最强。结论：体内试验和体外试验表明蝉拟青霉多糖具有促进免疫功能和抗肿瘤的作用。     

人源抗EV71病毒基因工程抗体的研究

目的 研制人源抗EV71病毒基因工程抗体.方法 采集多个患儿外周血淋巴细胞,构建人源抗EV71单链（scFv）噬菌体抗体基因文库.用纯化的EV71 VPI蛋白对抗体库进行筛选,将筛出抗体的轻链和重链通过序列测定确定抗体轻重链型别后分别克隆入全抗体表达载体pAC-LCH3R后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.结果 成功地获得了1株抗EV71病毒VPI蛋白的人源单克隆抗体,利用ELISA、IFA对所获人源单克隆抗体的功能特性进行鉴定,表达成分泌型IgG全抗体在体外与A型及C4亚型EV71病毒的中和反应结果 显示为阴性.结论 从免疫库中获得了1株人源非中和单抗,为EV71病毒引起的手足口病的鉴别诊断奠定了基础.     

类胰高血糖素肽—1免疫活性细胞在大鼠胃肠道的分布

目的 为了探讨类胰高血糖素肽—1(Glucagon—like peptide—1,GLP—1)在大鼠的胃肠道中的分布情况。方法 应用免疫组织化学ABC法,观察了6只Wistor大鼠胃肠道中GLP—1免疫活性细胞分布情况。结果 大鼠胃肠道中GLP—1免疫活性细胞密度最大的是回肠,其次是结肠和直肠。结论 GLP—1免疫活性细胞主要分布在小肠远端。     

Mixed bloodstream infection with Staphylococcus aureus and Penicillium chrysogenum in an immunocompromised patient: case report and review of the literature

We report a case of an immunocompromised patient who developed a mixed bacterial–fungal bloodstream infection involving Staphylococcus aureus and Penicillium chrysogenum . To date, no reports of such mixed bloodstream infection have been found.     

In vitro antibacterial activity of sucralfate

The effect of sucralfate (12.5 mg/ml) on the growth ofEscherichia coli (ATCC 25922),Enterococcus faecalis (ATCC 29212) and two isolates ofPseudomonas aeruginosa (ATCC 27853 and a multi-resistant clinical isolate) was studied in vitro at pH values of 3.0, 4.5, 6.0 and 7.4. A bacteriostatic effect of sucralfate was demonstrated forPseudomonas aeruginosa at a pH of 6.0 and 7.4 and forEscherichia coli andEnterococcus faecalis at a pH of 6.0. The bacteriostatic effect was most pronounced at high pH values. Sucralfate had no bactericidal effect on the bacteria tested at the concentration used.     

Effect of the Penicillium chrysogenum antifungal protein (PAF) on barley powdery mildew and wheat leaf rust pathogens

The small molecular mass antifungal protein of Penicillium chrysogenum (PAF) inhibited the growths of two obligate biotrophic fungal pathogens, Blumeria graminis f. sp. hordei and Puccinia recondita f.sp. tritici and, hence, mitigated the symptoms of barley powdery mildew and wheat leaf rust infections, respectively. PAF also affected adversely the germination of B. graminis conidia and P. recondita uredospores causing degenerative branching of germ tubes. Since powdery mildews and rusts cause serious economic losses the potential applicability of PAF to control these plant diseases is promising.     

Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4–6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus. Received: 5 January / 27 February 1998     

Allergic inflammation induced by a Penicillium chrysogenum conidia-associated allergen extract in a murine model

BACKGROUND: Recent evidence has shown that viable conidia from the fungus Penicillium chrysogenum induce allergic effects in mice. The present study was conducted to determine the specific allergic dose response of C57BL/6 mice to the protease extract, Pen ch, isolated from viable P. chrysogenum conidia. METHODS: Mice were treated with primary intraperitoneal (IP) injections of 10 or 100 microg of Pen ch adsorbed to alum, followed by weekly IP injections of 0.1, 1.0, or 10.0 microg Pen ch with alum for 4 weeks, and with 10.0 microg of Pen ch by intranasal (IN) inoculations the final 2 weeks before killing. RESULTS: Intraperitoneal injections of 10 and 100 microg of Pen ch for 5 weeks followed by 2 weeks of IN instillation of 10 microg induced significant increases of total serum immunoglobulin (Ig)E and IgG(1). Bronchoalveolar lavage cell counts revealed increased numbers of eosinophils and neutrophils. Histopathological examination of lungs detected perivascular inflammation by eosinophils and neutrophils and increased mucous production. CONCLUSIONS: The data presented in this study indicate that sensitization to protease allergens released by viable P. chrysogenum conidia in vivo induce a strong allergic inflammatory response in a murine model, which could have implications for people exposed to high levels of conidia of this organism.