Homologous expression of Phanerochaete chrysosporium manganese peroxidase,using bialaphos resistance as a dominant selectable marker

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2–3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.Communicated by U. Kück     

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level

Summary DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4–5 × 10⁷ bp.     

Tet-on慢病毒调控系统的构建及其调控作用

Objective To establish Tetracycline-induced gene expression system for gene therapy based on third generation lentivirus system.Methods The mutant of the reverse Tet transactivator (rtTA and M2rtTA) was subcloned into a lentiviral vector with neo selection marker named as pELNS-rtTA-IRES-Neo and pELNS-M2rtTA-IRES-Neo. The Tet-responsive element (TETO and TREpitt) and green fluorescence proteint (GFP) were subcloned into a lentiviral vector with blasticidin selection marker named as plenti6-TETO-GFP and plenti6-TREpitt-GFP.293 cells were contransfect with pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP, or with pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt -GFP. Cells were treated by Dox to induce GFP expression. After 48hours, GFP expression in the co-transfected cells was observed under a fluorescent microscope.Results The first generation of Tetracycline-induced gene expression system named pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP vectors were successfully set up. GFP expression in cotransfected cells induced with Dox was about 90% positive, while there was 30% positive GFP expression observed in no Dox inducing group. The second generation of Tetracycline-induced gene expression system named pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt-GFP vectors were successfully set up. GFP expression in cotransfected cells induced with Dox was about 95% positive, while there was no positive GFP expression observed in no Dox inducing group.Conclusion Tetracycline-induced gene expression system based on lentivirus was successfully set up, which can induce gene expression effectively and tightly without obvious side-effects on cells by using the second Tetracycline-induced elements.     

The nature of extra-chromosomal maintenance of transforming plasmids in the filamentous basidiomycete Phanerochaete chrysosporium

Summary The nature of extra-chromosomal maintenance of the transforming plasmid p12-6 in Phanerochaete chrysosporium was studied. Our results indicate that the plasmid is maintained in the fungal transformants extra-chromosomally as part of a larger endogenous plasmid (designated pME) of P. chrysosporium. Using the total DNA of p12-6 fungal transformants, p12-6, as well as a larger plasmid, p511, were recovered in recA ^– E. coli strains while only p12-6 was recovered in recA ⁺ E. coli strains. The results also showed that the cytosine methylation system has no apparent effect on the strain-dependent recovery of p12-6 and p511 in E. coli from the total DNA of fungal transformants.     

Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi

The Lyme disease spirochete Borrelia burgdorferi must repress expression of outer surface protein C (OspC) to effectively evade specific humoral immunity and to establish persistent infection. This ability largely relies upon a regulatory element, the only operator that has been reported in spirochetal bacteria. Immediately upstream of the ospC promoter, two sets of inverted repeats (IRs) constitute small and large palindromes, in which the right IR of the large palindrome contains the left IR of the small one, and may collectively function as the ospC operator. In the study, the large palindrome with or without the small IR was fused with an flaB promoter, which was used to drive expression of a promoterless ospC copy as a reporter gene, and introduced into OspC-deficient B. burgdorferi. The presence of the large palindrome alone significantly reduced ospC expression driven by the fused flaB promoter in the joint tissue of severe combined immunodeficiency (SCID) mice, and rescued spirochetes from elimination by passively transferred OspC antibody in infected SCID mice and specific immune responses elicited in immunocompetent mice, confirming a function of the IRs as an operator. Inclusion of the small IR further enhanced the ability of the large palindrome to reduce the activity of the fused flaB promoter, indicating that the small IR is a part of the operator. Taken together, the study led to successful verification and dissection of the ospC operator.     

锰在免疫调节中的功能及其应用

锰是一种人体必需的微量元素,在发育、生殖、代谢、神经功能和抗氧化等方面发挥着重要的作用。近年来, 锰在机体免疫中的重要作用逐步被认识和重视。锰作为一种损伤相关分子模式,具有强劲的免疫激活能力。锰离子诱导细胞产生大量的包括Ⅰ型干扰素在内的细胞因子,调控机体的天然免疫和适应性免疫反应,在免疫佐剂和肿瘤免疫治疗方面展现出巨大的潜力。本文首先总结了锰在机体内的稳态调节及其在机体免疫中的作用,之后对锰及其衍生物在免疫佐剂和肿瘤免疫治疗方面的一系列应用进行了详细的讨论,并对其未来的发展进行了展望。     

A new CO2 inhalation system for studying regulation of breathing

This paper describes the development of a computer-controlled system that controls inspired CO2 or end-tidal PCO2 (PETCO2) to follow preprogrammed functions such as step, sinusoid, and pulse, under normoxic, hyperoxic, and hypoxic conditions. The system uses a proportional-integral (PI) controller that was optimized by adjusting the PI parameters so as to minimize the integral-time of absolute error (ITAE) performance parameter.     

Transgenic tools for analysis of the haematopoietic system: knock-in CD45 reporter and deletor mice

We have produced and characterised reporter knock-in CD45-YFP and CD45-Cre mice that drive expression of yellow fluorescent protein (YFP) and Cre-recombinase, respectively under control of the haematopoietic CD45 locus. CD45-YFP expression was characterised in various haematopoietic cells populations. The activity of CD45-Cre mice was assessed by crossing with silent GFP reporter mice. Flow cytometry analysis indicated that both CD45-YFP and CD45-Cre were strongly expressed in the CD45(+) compartment of peripheral blood. Expression of these markers in various populations of adult bone marrow, including primitive cell populations was also determined. These mouse models will be useful for the direct visualisation of haematopoietic cells, especially at the periphery, and for gene knockout studies, in the haematopoietic system.     

利用昆虫-杆状病毒表达系统表达人乳头瘤病毒16型L1蛋白

目的 获得人乳头瘤病毒16型（Human papillomavirus type 16,HPV16)L1蛋白。方法 以pFB1为载体构建HPV16L1杆状病毒表达质 ,并感染昆虫细胞Sf9结果 收集27℃,培养72h的感染重组病毒的Sf9,提取细胞蛋白。经SDS－PAGE蛋白电泳分析,发现有一相对分子质量为56000的蛋白表达,Western blot证实所表达的蛋白为HPV16L1。结论 昆虫一杆状病毒表达系统可有效地表达HPV16L1蛋白。     

锰对大鼠生精细胞Caspase-3 mRNA调控及支持细胞波形蛋白表达的影响

目的 探讨锰对大鼠生精细胞Caspase-3 mRNA调控及支持细胞波形蛋白（vimentin）表达的影响。 方法 将正常雄性SD大鼠48只,随机分为1个对照组和2个染锰组,给予各组大鼠腹腔注射生理盐水和15mg/kg、30mg/kg氯化锰。分别染锰4周和6周,随机处死各组大鼠8只。应用末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记技术（TUNEL） 、原位杂交法和免疫组织化学链酶亲和素生物素过氧化物酶复合物（SABC）法进行检测研究。 结果 1. 与空白对照组比较,15mg/kg、30mg/kg染锰组生精细胞凋亡指数（AI）均升高（ P<0.05, P<0.01）,Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞波形蛋白（vimentin）阳性细胞率均显著降低（ P<0.01）。2. 染锰剂量相同,染锰6周组与染锰4周组比较,生精细胞AI与Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞vimentin阳性细胞率均显著降低（ P<0.01）。3. 染锰时间相同,30mg/kg MnCl2组与15mg/kg MnCl2组比较,生精细胞AI与Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞vimentin阳性细胞率均显著降低（ P<0.01）。4. 各组大鼠生精细胞AI和Caspase-3 mRNA阳性细胞率呈正相关（ r =0.842, P<0.01）,与支持细胞vimentin阳性细胞率呈负相关（ r =-0.859, P<0.01）,各组大鼠生精细胞Caspase-3 mRNA阳性细胞率和支持细胞vimentin阳性细胞率呈负相关（ r =-0.975, P<0.01）。 结论 染锰大鼠生精细胞Caspase-3 mRNA表达增加导致生精细胞凋亡增加;支持细胞vimentin表达下降;这可能是锰生殖毒性的重要分子机制之一。