Localization of the receptor binding site of IFN-alpha 2b

The receptor binding site of IFN-alpha is not precisely known. To further characterize this site, mAb against IFN-alpha 2b were selected that block the binding of radiolabeled IFN-alpha 2b to its cell surface receptor. These antibodies also neutralized the anti-viral and anti-proliferative properties of IFN-alpha 2b. A subset of these antibodies (group 1) do not recognize IFN-alpha 2a, either in solid-phase immunoassays or functional assays, whereas a second subset (group 2), with no cross-reactivity with group 1, recognizes both IFN-alpha subtypes. Because IFN-alpha 2b and IFN-alpha 2a differ by only alpha Arg23-Lys23 substitution, group 1 antibodies must recognize an epitope within the receptor binding region of IFN-alpha 2b that includes Arg23. Group 2 antibodies recognize a separate and distinct epitope within the binding site that does not include Arg23.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

嗜盐耐盐微生物抗盐胁迫相关离子转运蛋白研究进展

离子转运蛋白在维持细胞内pH稳态、离子动态平衡等方面发挥着重要作用。钠离子转运体和钾离子转运体在嗜盐耐盐微生物中广泛存在,其"保钾排钠"机制是微生物抗盐胁迫的两大策略之一。近年来,嗜盐耐盐微生物中许多新型钠、钾离子转运体被陆续发现,如RDD蛋白、UPF0118蛋白、DUF蛋白和KimA蛋白等;Fe³⁺、Mg²⁺等其他金属离子的转运蛋白也被证实可通过影响微生物胞内相容性溶质的合成起到渗透调节的作用。本文综述了嗜盐耐盐微生物中抗盐胁迫相关的各类离子转运蛋白,分析其分子结构和工作机理,并对这些蛋白在农业方面的应用进行了展望。继续发现新的离子转运蛋白,探究抗盐胁迫相关离子转运蛋白的结构和机理,解析各转运系统的协同作用及分子调控机制,将进一步加深对嗜盐耐盐微生物抗盐胁迫调控的认识,并为盐碱地农作物的改良等提供新的思路。     

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.     

Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.     

苹果柠檬酸合酶3基因家族成员鉴定及表达分析

柠檬酸合酶(citrate synthase 3, CS3)是细胞代谢途径中的关键酶之一,其活性调节着生物体的物质和能量代谢过程。本研究旨在从苹果全基因组中鉴定CS3基因家族成员,并进行生物信息学和表达模式分析,为研究苹果CS3基因的潜在功能提供理论基础。利用BLASTp基于GDR数据库鉴定苹果CS3家族成员,通过Pfam、SMART、MEGA5.0、clustalx.exe、ExPASy Proteomics Server、MEGAX、SOPMA、MEME和WoLF PSORT等软件分析CS3蛋白序列基本信息、亚细胞定位情况、结构域组成、系统进化关系以及染色体定位情况。利用酸含量的测定和实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术检测苹果6个CS3的组织表达和诱导表达特性。苹果CS3基因家族包含6个成员,这些CS3蛋白包括473−608个不等的氨基酸残基,等电点分布在7.21−8.82。亚细胞定位结果显示CS3蛋白分别定位在线粒体和叶绿体。系统进化分析可将其分为3类,各亚家族基因数量分别为2个。染色体定位结果显示,CS3基因分布在苹果不同的染色体上。蛋白二级结构以a-螺旋为主,其次是无规则卷曲,b-转角所占比例最小。筛选的6个家族成员在不同苹果组织中均有表达,整体表达趋势从高到低依次为MdCS3.4相对表达含量最高,MdCS3.6次之,其他家族成员相对表达量依次为MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5。qRT-PCR结果显示,MdCS3.1和MdCS3.3基因在酸含量较低的‘成纪1号’果肉中相对表达量最高,酸含量较高的‘艾斯达’果肉中MdCS3.2和MdCS3.3基因相对表达量最高。因此,本研究对不同苹果品种中CS3基因相对表达量进行了检测,并分析了其在苹果果实酸合成过程中的作用。结果表明,CS3基因在不同苹果品种中的相对表达量存在差异,为后续研究苹果品质形成机制提供了参考。     

椰心叶甲啮小蜂复眼和触角在交配中的作用及其超微结构的扫描电镜观察

对寄生蜂交配行为的了解将有助于发展对其行为调控的技术,提高寄生蜂对害虫控制的效能。为探讨椰心叶甲啮小蜂Tetrastichus brontispae Ferrière的复眼和触角在交配中的作用,用水溶性黑色素和液体石蜡分别涂抹该蜂复眼和触角后观察其交配行为,并利用扫描电镜（SEM）观察其复眼和触角的超微结构,分析雌、雄蜂触角感器的分布与数量差异。结果表明：椰心叶甲啮小蜂雄蜂的复眼在交配过程中起重要作用,雌蜂复眼作用不显著。在椰心叶甲啮小蜂求偶识别和接受过程中,雄蜂触角柄节部位起主要作用,其次是棒节部位,再次是鞭节的索亚节部位,而雌蜂触角鞭节索亚节部位起主要作用,然后是棒节部位,最后是柄节部位。扫描电镜观察表明椰心叶甲啮小蜂触角上共有8种感器,其中毛形感器和板形感器是主要感器,雌、雄蜂触角有明显的性二型现象,表现为触角大小不同及触角感器类型、大小、数量、分布不同。     

利用同源重组构建蓝藻集胞藻6803ndhO基因突变株及其分子鉴定

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。迄今为止,人们在蓝藻细胞中已鉴定出15种NDH-1复合体亚基(NdhA-NdhO)。然而,人们对NdhO亚基的研究尚不够,至今未见有反向遗传学等方面的研究。在通过构建同源重组载体、自然转化和多次继代筛选后,对转化子进行了PCR和蛋白免疫印迹鉴定。结果表明,卡那霉素基因已成功地插入到ndhO基因的保守区域,并完全破坏了ndhO基因的蛋白表达,从而获得了ndhO基因缺失的突变株,为进一步研究NdhO亚基对NDH-1复合体的稳定性和生理功能等奠定了实验基础。