Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008

Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR-008 antibiotic contains a 38-membered, poiyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

Extractive fermentation of lactic acid by immobilizedLactobacillus casei using ion—exchange resin

Summary A novel method of lactic acid fermentation byLactobacillus casei immobilized in Ca—alginate gels is described, in which an ion—exchange resin packed column is attached to a fermentor for separation of lactic acid from fermentative broth. The technique successfully alleviated the restriction imposed by lactic acid on bacterial growth and product formation. As compared to the conventional batch fermentation, the new fermentation technique enhanced the lactic acid productivity and sugar conversion rate from 0.328g/L·h and 88. 2% to 0.482g/L·h and 98.6%, respectively.     

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

基因工程链激酶(r—SK)纯化及其单克隆抗体制备和鉴定

采用Rotofor等电聚焦和分子筛技术纯化大肠杆菌表达的重组链激酶(r—SK),其纯度为97%。比活性1×10⁶IU／mg,回收率41％。分析所纯化的r—SK　N端氨基酸顺序证实其1—15个氨基酸顺序与SK基因的核苷酸顺序所示3联密码子一致。以纯化r—SK为抗原,通过杂交瘤技术构建了分泌抗r—SK单克隆抗体(McAb)杂交瘤细胞(4D11),该McAb属IgG1亚型．特异地作用于r-SK和C组口溶血性链球菌分泌的SK。用底物显色法测定该McAb可抑制SK对纤溶酶原的激活。Western blot法证实该McAb能识别分子量为16 250Da的r—SK的CNBr裂解片段。推测SK蛋白中第71残基至第237残基间的氨基酸顺序参与SK与纤溶酶原的结合。     

仲彬草属5种植物的核型研究

本文对我国西部高原仲彬草属Ｋｅｎｇｙｉｌｉａ５种植物的核型进行了分析。它们的染色体数目均为２ｎ＝４２,六倍体。核型是：糙毛鹅观草Ｋ．ｈｉｒｓｕｔａ,２ｎ＝６ｘ＝４２＝３６６＋６ｓｍ;青海鹅观草Ｋ．ｋｏｋｏｎｏｒｉｃａ,２ｎ＝６ｘ＝４２＝３６ｍ＋６ｓｍ：黑药鹅观草Ｋ．ｍｅｌａｎｔｈｅｒａ,２ｎ＝６ｘ＝４２＝３８ｍ＋４ｓｍ;硬秆鹅观草Ｋ．ｒｉｇｉｄｕｌａ,２ｎ＝６ｘ＝４２＝３８ｍ＋４ｓｍ;窄颖鹅观草Ｋ．ｓｔｅｎａｃｈｙｒａ,２ｎ＝６ｘ＝４２＝３８ｍ＋４ｓｍ。它们的核型属于１Ｂ或２Ｂ型。染色体中均未发现随体。     

麦蛾的求偶行为与区分等级的方法

求偶行为是麦蛾的本能,主要由自身生理状态决定,雌蛾全是自发行为,雄蛾多是对异性信息刺激的反应。两性求偶行为都有固定发生程序,有明显的阶段性。本文按其阶段程序特点把两性求偶行为各分为3等9级。非求偶行为概作0等0级。等级数值大小反映了性兴奋强度,统计分析级别数值,就能确定个体或蛾群求偶行为的动态。