Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)—rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance

The aphid transmitted Turnip yellows virus (TuYV) has become a serious pathogen in many rapeseed (Brassica napus L.) growing areas. Three-years’ field trials were carried out to get detailed information on the genetics of TuYV resistance derived from the resynthesised B. napus line ‘R54’ and to develop closely linked markers. F₁ plants and segregating doubled-haploid (DH) populations derived from crosses to susceptible cultivars were analysed using artificial inoculation with virus-bearing aphids, followed by DAS-ELISA. Assuming a threshold of E ₄₀₅ = 0.1 in ELISA carried out in December, the results led to the conclusion that pre-winter inhibition of TuYV is inherited in a monogenic dominant manner. However, the virus titre in most resistant lines increased during the growing period, indicating that the resistance is incomplete and that the level of the virus titre is influenced by environmental factors. Bulked-segregant marker analysis for this resistance locus identified two closely linked SSR markers along with six closely linked and three co-segregating AFLP markers. Two AFLP markers were converted into co-dominant STS markers, facilitating efficient marker-based selection for TuYV resistance. Effective markers are particularly valuable with respect to breeding for TuYV resistance, because artificial inoculation procedures using virus-bearing aphids are extremely difficult to integrate into practical rapeseed breeding programs.     

Discovery of orally available integrin α5β1 antagonists

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin α5β1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin α5β1 inhibitor scaffolds for potential systemic treatment is described.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

p38 MAPK signaling pathway is involved in butyrate-induced vitamin D receptor expression

Previously, we have demonstrated that the butyrate-induced differentiation in the human colon cancer cell line Caco-2 occurs via upregulation of the vitamin D receptor (VDR). However, the downstream pathways involved are unknown. The mitogen-activated protein kinases (MAPKs) have been shown to play an important role in regulation of cell differentiation, and may therefore be a potential target of butyrate action. To assess their role in butyrate-mediated cell differentiation and VDR expression, we used the specific p38-MAPK inhibitor SB203580 and the ERK1/2 MAPK-inhibitor PD98059. The p38-MAPK inhibitor abolished the butyrate effect on VDR expression and cell differentiation, while the ERK1/2 inhibitor did not influence the butyrate-mediated induction of cell differentiation and VDR expression. The essential role of the p38 pathway in up-regulation of VDR expression was further confirmed by using the p38 stimulator arsenite. These results imply an important role of the p38-MAPK in regulation of cellular differentiation through upregulation of VDR expression by butyrate.     

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.     

Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.     

Fully human,HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.     

Cytochrome P460 Genes from the Methanotroph Methylococcus capsulatus Bath

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.