GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

注射用重组人干扰素βser17的分离纯化及鉴定

重组人干扰素βser17(rhIFN βser17)工程菌经发酵培养后,采用高压匀浆破菌、离心分离包涵体、有机抽提、酸沉淀、SephacrylS -200、SephadexG -75等进行分离纯化,通过各种方法对终产品进行鉴定。结果显示,纯化的rhIFN- βser17比活性超过 2×107IU/mg,纯度超过 99%,其它各项质量指标均符合质量标准。通过研究建立了大规模制备rhIFN- βser17的纯化工艺,研制出了符合规程要求的注射用rhIFN -βser17纯品,为临床研究奠定了基础。     

Occurrence and Fate of Estrone, 17β-estradiol and 17α-ethynylestradiol in STPs for Domestic Wastewater

Estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) discharged from sewage treatment plants (STPs) into surface waters, are seen as a threat effecting aquatic life by its estrogenic character. Therefore, much research is conducted on the fate and removal of these compounds. Since these compounds are present in influents and effluents in the ng/l range, methods for detection deserve special attention. Most important processes that play a role in the removal of estrogens are: adsorption, aerobic degradation, anaerobic degradation, anoxic biodegradation and photolytic degradation. Halflifes tend to vary and are remarkably shorter when low initial concentrations are applied. In general anaerobic conditions result in longer halflifes then aerobic conditions. EE2 shows far most persistence of the compounds, thereby also the estrogenic effect in vitro is about 2–3-fold higher compared to E2. The three compounds show a higher affinity to sorb to sludge compared to other tested adsorption materials like sediment. Aerobic degradation is far the most efficient in removing these compounds, but adsorption seems to play a significant role in retaining the estrogens inside full-scale STPs. Removal rates in full scale plants depend on the HRT, SRT and loading rates, but lack of information on the exact dependency so far prevents an optimal design able to fully eliminate estrogens from wastewater.     

Structure and function of interleukin-17 family cytokines

The recently identified interleukin-17 (IL-17) cytokines family, which comprises six members in mammals (IL-17A-F), plays essential roles in the host immunity against infectious diseases and chronic inflammatory diseases. The three-dimensional structures containing IL-17A or IL-17F have become available and revealed the unique structural features of IL-17s as well as their receptors. Molecular modeling in this review shows that IL-17s may adopt a “cysteine knot” fold commonly seen in nerve growth factor (NGF) and other neurotrophins. Further modeling analysis unmasks a signature interaction feature of the IL-17F/IL-17RA complex, where a small loop of IL-17RA slots into the deep groove of the interface of IL-17F homodimer. This is quite different from the interaction between the best known four-helix cytokines and their cognate receptors. On the other hand, structure of IL-17A and its monoclonal antibody (CAT-2200) shows that, albeit that the antigenic epitope of IL-17A resides outside of the IL-17A homodimer interface, its physical proximity to the receptor binding groove may explain that antibody blockage would be achieved by interfering with the ligand-receptor interaction. This review is to summarize the advance in understanding the structure and function of IL-17 family cytokines, focusing mainly on IL-17A, IL-17F and IL-17E, in the hope of gaining better knowledge of immunotherapeutic strategies against various inflammatory diseases.     

Plasma cortisol and 17β-oestradiol levels in roach exposed to acute and chronic stress

Plasma cortisol levels were measured as an indicator of physiological stress in roach subjected to brief handling, or to a 14-day period of confinement, and in undisturbed control fish, during winter (water temperature 5° C) and summer (16° C), at which time plasma 17 β-oestradiol levels were also determined. Cortisol levels in undisturbed roach were low (mean 8·1 ng ml⁻¹ at 5° C; 1·4 ng ml⁻¹ at 16° C) and both handling and handling+confinement elevated blood cortisol levels significantly to 400 and 140 ng ml⁻¹, respectively (at 5° C) and 700 and 600 ng ml⁻¹, respectively (at 16) C). Blood cortisol levels had almost returned to baseline within 4 h following handling alone but in fish subjected to handling and prolonged confinement cortisol levels remained elevated for up to 168 h. Differences in baseline and poststress levels of cortisol, and in the rate of recovery from acute stress, were observed at the two different temperatures and the possible factors underlying these differences are discussed. Circulating levels of 17 β-oestradiol were reduced significantly within 24 h of exposure to either acute handling or chronic confinement indicating that the reproductive endocrine system in roach is sensitive to disruption by stressors.     

益生菌对炎症性肠病幼鼠Th17细胞的调节作用

目的 观察鼠李糖乳杆菌（LGG）对炎症性肠病（IBD）幼鼠结肠白细胞介素-17A（IL-17A）水平的影响,探讨益生菌对Th17细胞的调节作用。方法 36只健康雄性SD幼鼠随机分4组：空白对照组、LGG对照组各8只,IBD组、IBD-LGG组各10只。利用2,4,6-三硝基苯磺酸（TNBS）诱导幼鼠IBD模型,观察一般状况、IBD疾病活动指数评分。第8天处死所有幼鼠,留取结肠标本,观察病理改变并采用免疫组织化学法测定结肠组织IL-17A的表达。结果 相比两对照组,IBD组、IBD-LGG组幼鼠一般状态差,IBD-LGG组便性状及隐血较IBD组缓解;IBD组、IBD-LGG组幼鼠结肠组织均见炎症改变,但IBD-LGG组较轻。IBD-LGG组DAI评分、IL-17A水平均低于IBD组,差异有统计学意义（P＜0.05）。结论 益生菌可减轻IBD幼鼠肠道炎症,其机制可能与益生菌调节Th17细胞进而调控IL-17A表达有关。     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

大气硝酸盐氧同位素异常及应用进展

近年来人为活动导致的大气硝酸盐不断增加,危害人体健康和生态环境。厘清大气硝酸盐的来源及形成机理至关重要。多氧稳定同位素技术是一种强有力的示踪手段,能够有效指示大气硝酸盐生成的氧化路径,在气溶胶、水体、土壤、森林、古气候研究中得到了广泛应用。本文总结了大气硝酸盐氧同位素异常(Δ17O)的测定方法(热裂解法、反硝化细菌法、化学法),探讨了Δ17O的产生原因,并围绕硝酸盐的形成过程阐明硝酸盐Δ17O的示踪机制,综述了Δ17O在大气化学反应机制研究中的应用。在此基础上,本文提出目前Δ17O研究的不足并对未来需要开展的研究进行了展望。     

干扰素阴道胶囊联合微波治疗宫颈炎合并高危型HPV感染对外周血Th17和Treg细胞及炎症因子的影响

目的探讨干扰素阴道胶囊联合微波治疗宫颈炎合并高危型人乳头瘤病毒(HPV)感染对外周血Th17和Treg细胞及炎症因子的影响。方法随机选择2015年1月-2018年1月在本院就诊的慢性宫颈炎合并高危型HPV感染患者90例,按随机数字表法分为观察组和对照组,每组45例。对照组给予重组人干扰素α2b栓,观察组给予重组人干扰素α2b栓联合微波治疗,治疗3个月后,观察两组患者的症状改善情况、HPV转阴率、血清炎性因子水平以及Th17和Treg细胞水平。结果观察组总有效为41例,占91.1%,对照组为73.3%,观察组显著高于对照组(P<0.05)。治疗后观察组的白带量改善率、白带脓性改善率、高危型HPV转阴率均显著高于对照组(P<0.05)。治疗前两组患者的白介素17(IL17)、肿瘤坏死因子β(TNFβ)、白介素23(IL23)水平比较,差异无统计学意义(P>0.05);治疗后两组患者的IL17、TNFβ、IL23水平均显著下降,且观察组下降更加显著(P<0.05)。治疗后观察组的Treg细胞、Th17细胞水平,Th17/Treg比值均较对照组低(P<0.05)。治疗前两组患者的滴虫、真菌、细菌性阴道炎、衣原体、解脲支原体感染人数比较差异无统计学意义(P>0.05),治疗后观察组的细菌性阴道炎、衣原体、解脲支原体感染显著降低,与对照组比较差异有统计学意义(P<0.05)。结论干扰素阴道胶囊联合微波治疗宫颈炎患者的治疗效果好,可显著改善患者症状,减轻体内炎症反应,增加HPV转阴率,调节Th17和Treg细胞,对预防宫颈癌有重要作用。