甲状腺激素对胸腺上皮细胞CK19蛋白表达的影响

目的探讨甲状腺激素对胸腺的发育的影响及可能的机制。方法将12只怀孕4d的大鼠随机分成A组和B组,A组正常饮水,B组孕鼠供以含有0．02％甲巯咪唑的饮水制备仔鼠甲状腺功能低下动物模型,将A组的仔鼠随机分成对照组和甲状腺素钠组,将B组的仔鼠随机分成甲低组和甲低＋甲状腺素钠组。甲状腺素钠组和甲低＋甲状腺素钠组于出生后15d给予腹腔注射甲状腺素钠（0．5mg／kg体重,1次／d）,连续给药25d。所有动物于出生后40d麻醉处死,测定仔鼠的胸腺重量及脏器指数;采用放射免疫技术测定仔鼠血清中三碘甲状腺原氨酸（triiodothyronine,T3）、四碘甲状腺原氨酸（tetraiodothyronine,T4）、促甲状腺激素（thyroid—stimulating hormone,TSH）水平,免疫组织化学技术检测胸腺上皮细胞细胞角蛋白19（cytokeratin 19,CK19）蛋白的表达量。结果与对照组比较,甲状腺素钠组仔鼠血清中T3、T4显著升高,TSH减少,胸腺重量增大;甲低组仔鼠血清中T3、T4明显降低,TSH显著增高,胸腺重量降低,胸腺上皮细胞CK19蛋白表达减少。与甲低组比较,甲低＋甲状腺素钠组仔鼠血清中T3、T4升高,TSH降低,胸腺指数增大,胸腺上皮细胞CK19蛋白的表达明显增多。结论甲状腺激素可以通过影响胸腺上皮细胞CK19的表达量,使胸腺发育或退化。     

斑马鱼心脏标志基因TNC多克隆抗体的制备及表达研究

TNC是心脏发育的标志基因,但该基因在斑马鱼中的表达尚未研究。斑马鱼TNC基因基因的开放阅读框含有5132bp,编码1710个氨基酸,采用生物信息学结合PCR的方法获得了斑马鱼TNC基因的片段。将所得的PCR片段插入原核表达载体pGEX-4T-1中,并将重组质粒（pGEX-4T-1-TNC）转化大肠杆菌BL21;通过IPTG诱导表达GST—TNC融合蛋白,通过尿素洗涤沉淀蛋白并切胶回收纯化融合蛋白,免疫新西兰大白兔制备多克隆抗体。Western blot和免疫组化分析表明,制备的抗体具有良好的高效价性和特异性。利用该抗体进行斑马鱼胚胎抗体染色分析表明,TNC蛋白在心脏组织中特异表达。     

Development of redox potential-controlled schemes for very-high-gravity ethanol fermentation

Fermentation redox potential reflects the momentary physiological status of organisms. Controlling redox potential can modulate the redistribution of intracellular metabolic flux to favor the formation of the desired metabolite. Accordingly, we have developed three redox potential-controlled schemes to maximize their effects on the very-high-gravity (VHG) ethanol fermentation. They are aeration-controlled scheme (ACS), glucose-controlled feeding scheme (GCFS), and combined chemostat and aeration-controlled scheme (CCACS). These schemes can maintain fermentation redox potential at a prescribed level (i.e., -50, -100, and -150 mV) by supplementing sterile air, fresh glucose media, or a combination of sterile air and fresh glucose media into a fermenter to counteract the decline of redox potential due to yeast growth. When ACS was employed, the fermentation efficiency at -150 mV is superior to the other two redox potential levels especially when the initial glucose concentration is higher than 250 g/l. The redox potential-controlled period for ACS, GCFS, and CCACS at -150 mV under the same 200 g glucose/l condition was 2.5, 21.7 and 64.6h and the corresponding fermentation efficiency was 85.9,89.3 and 92.7%, respectively.     

Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b

Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.     

Ubiquitin and Ubiquitin-like Modifiers in Plants

Posttranslational modifications of proteins by small polypeptides including ubiquitination, neddylation (related to ubiquitin (RUB) conjugation), and sumoylation are implicated in plant growth and development, and they regulate protein degradation, location, and interaction with other proteins. Ubiquitination mediates the selective degradation of proteins by the ubiquitin (Ub)/proteasome pathway. The ubiquitin-like protein RUB is conjugated to cullins, which are part of a ubiquitin E3 ligase complex that is involved in auxin hormonal signaling. Sumoylation, by contrast, is known for its involvement in guiding protein interactions related to abiotic and biotic stresses and in the regulation of flowering time. ATG8/ATG12-mediated autophagy influences degradation and recycling of cellular components. Other ubiquitin-like modifiers (ULPs) such as homology to Ub-1, ubiquitin-fold modifier 1, and membrane-anchored Ub-fold are also found in Arabidopsis. ULPs share similar three-dimensional structures and a conjugation system, including E1 activating enzymes, E2 conjugation enzymes, and E3 ligases, as well as proteases for deconjugation and recycling of the tags. However, each of the ULP posttranslational modifications possesses its own specific enzymes and modifies its specific targets selectively. This review discusses recent findings on ubiquitination and ubiquitin-like modifier processes and their roles in the posttranslational modification of proteins in Arabidopsis.     

过量表达番茄类囊体膜抗坏血酸过氧化物酶基因（StAPX）提高了烟草种苗的抗氧化能力

为了探讨叶绿体类囊体膜抗坏血酸过氧化物酶（tAPX）与其抗氧化性的关系,从番茄叶片中分离了叶绿体类囊体膜抗坏血酸过氧化物酶基因（StA跚并转入到烟草中。以野生型（WT）、转正义StAPX烟草株系T3-3和T3-6为试材,测定了外源过氧化氢诱导的氧化胁迫条件下APX酶活性、过氧化氢酶（CAT）活性、过氧化氢（H2O2）含量、叶绿素荧光参数及叶绿素含量等。Northern杂交显示StAPX因的表达受外源H2O2氧化胁迫的诱导。氧化胁迫下转基因烟草的APX酶活性和清除H2O2的能力都显著高于野生型,并且转基因烟草比野生型具有更高的PSII最大光化学效率及叶绿素含量。结果表明,．刚尸舶勺过量表达有助于提高外源H2O2诱导的转基因烟草的抗氧化能力。     

HRB2慢病毒表达质粒的构建核蛋白细胞内定位的研究

目的:构建重组HIV-1相关结合蛋白2(HIV-1 rev binding protein 2)基因的真核融合表达质粒plenti-OFP-HRB2,用慢病毒表达系统感染HEKTER细胞.对过表达GFP-HRB2基因的细胞在激光共聚焦显微镜下观察,研究HRB2蛋白在细胞中的分布规律.方法:Trizol法提取人睾丸组织总RNA进行RT-PCR,将纯化的扩增产物HRB2与克隆载体plp-GFP-Cl连接、转化感受态细菌E.coli XLblue.测序正确后将质粒plp-GFP-HRB2与真核表达质粒plenti-Cl分别进行双酶切,连接后转化.将构建正确的plenti-GFP-HRB2重组质粒、△8.91、pvsvg瞬时共转染293T细胞后,用荧光显微镜观察绿色荧光蛋白的表达.收集包装病毒后感染HEKTER细胞.细胞生长一周后,将细胞铺玻片上用激光共聚焦显微镜观察.结果:构建的plenti-GFP-HRB2真核表达质粒经PCR鉴定及测序均说明人源HRB2基因已与plenti-GFP载体正确重组.瞬时转染293T细胞后能观察到绿色荧光.稳定感染后的HEKTER细胞经激光共聚焦显微镜观察后发现,HRB2蛋白在核仁处富集,在细胞核的其它部位少量分布,在胞浆中几乎没有分布.结论:人源HRB2基因表达的相关蛋白具有一个KH结构域,属于KH结构域家族的成员.稳定表达GFP-HRB2融合蛋白的细胞系的成功构建,为深入研究HRB2的入核机制、HRB2蛋白的在细胞分裂、RNA剪切等生物活动中的作用奠定了重要的实验基础.     

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

多点接种方法计数土壤细胞生理类群的研究

多点接点计数方法是根据细菌分解固体培养基上不同的有机化合物的能力确定为不同的生理类群,由有机化合物的降解点数和根据MPN方法进行计数,对不同的耕作方法进行了测定表明,秸秆覆盖免肾够明显提高土壤中分解淀粉,木聚糖,纤维素,果胶,几丁质,卵磷质,脂类和蛋白质类群的细菌数量。