Ligand-receptor interactions as controlled by wild-type and mutant Thr(370)Lys alpha2B-adrenoceptor-Galpha15 fusion proteins

Fusion proteins were constructed between either a wild-type or mutant Thr370Lys alpha2B-adrenoceptor (alpha2B AR) and a mouse Galpha15 protein to analyze ligand-receptor interactions at a receptor/Galpha15 protein density ratio of 1. Activation of the wild-type alpha2B AR-Galpha15 fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50 = 7.37+/-0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pK(B) = 7.55+/-0.15). Whereas d-medetomidine and oxymetazoline were as efficacious agonists as (-)-adrenaline, the following ligands displayed partial agonist properties: BRL 44408 < atipamezole < clonidine < UK 14304 < BHT 920. A comparison with the mutant Thr370Lys alpha2B AR-Galpha15 fusion protein displayed similar Ca2+ kinetics and a ligand-mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands' intrinsic activities by coexpression of the mutant Thr370Lys alpha2B AR with either a Galpha15 or Galphao Cys351Ile protein. The Thr370Lys alpha2B AR-Galpha protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative alpha2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy.     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Photosynthetic characteristics and effect of ATP in transgenic rice with phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase and pyruvate orthophosphate dikinase genes

In the untransformed rice (WT) and transgenic rice with the PEPC and PPDK genes (CK) we determined activities of C₄ photosynthetic enzymes, photosynthetic response to irradiance and temperature, the metabolic index of active oxygen, and the yield component factors. The activities of C₄ photosynthetic enzymes in WT were very low, while those of corresponding enzymes in CK were highly observable. Moreover, after adenosine triphosphate (ATP) treatment, and under high irradiance and high temperature, the net photosynthetic rate of CK increased by 17 and 12 %, respectively, as compared to that achieved without ATP treatment. The resistance of CK against photo-oxidation was enhanced under these conditions, and CK yield increased by 15 %. ATP treatment enhanced the photosynthetic productivity of CK, thereby proving that ATP is the key factor in enhancing the photosynthetic capacity of transgenic rice with C₄ gene. Our new technical approach can be used in breeding rice with high photosynthetic efficiency and high grain yield.     

Responses to water stress of apoplastic water fraction and bulk modulus of elasticity in sunflower (Helianthus annuus L.) genotypes of contrasting capacity for osmotic adjustment

The responses to water stress of the bulk modulus of elasticity () and the apoplastic water fraction were examined using six sunflower cultivars of differing capacity for osmotic adjustment (OA). Water stress did not affect the partitioning of water between apoplastic (ca. 20%) and symplastic fractions in leaves which expanded during the exposure to stress in any genotype. Hence, no genotype-linked effects on either the buffering of cell water status during stress or on the estimates of bulk leaf osmotic potential could be expected. Genotypes differed in the degree of change in (estimated from pressure/volume [P/V] curves) and OA (estimated using both ln RWC/ ln o plots and P/V curves) induced by exposure to stress. In three genotypes increased significantly (p=0.05) as a consequence of stress, in another three change were small. OA was the only attribute of the three examined that could have contributed to turgor maintenance under stress. There was a strong negative association between leaf expansion and degree of OA across genotypes (r=–0.91) and a strong positive one between OA and (r=0.94). However all genotypes evidenced some degree of OA. These results are consistent with part of the genotype differences in OA being attributable to variations in leaf expansion during exposure to stress.     

Morphometric and phylogenetic analysis of theDaviesia ulicifolia complex (Fabaceae, Mirbelieae)

Daviesia ulicifolia is a widespread species that exhibits complex variation throughout its range. Using ordination and cluster analysis of morphometric characters we resolved ten terminal taxa for phylogenetic analysis. A data set including these and five closely related species was coded for a combination of morphometric and qualitative characters and analysed using parsimony. This revealed thatD. ulicifolia is paraphyletic by inclusion ofD. acicularis, D. arenaria andD. microcarpa. One terminal cluster is more similar to an outgroup species (D. arthropoda) than toD. ulicifolia and should be treated as a new species. Given recent theoretical and empirical studies showing paraphyly to be both expected and observed at species level, we propose that all existing species in this group continue to be recognised taxonomically. We suggest subdividingD. ulicifolia into several subspecies.     

柯萨奇病毒B组3型基因组剪切片段的序列分析

柯萨奇病毒B组(Coxsackievirus B,CVB)感染细胞时其基因组RNA存在不稳定现象,但产生机制尚不清楚。本研究将柯萨奇病毒B组3型(CVB3)感染细胞后,利用5′ cDNA末端快速扩增技术(5′ rapid amplification of cDNA ends,5′ RACE)扩增并克隆细胞内CVB3基因组片段,并对每条序列及其5′端的二级结构进行分析。结果获得的20条CVB3基因组片段,长度为 2 067～5 547 bp,片段断端主要分布于2Apro和2C编码区。RNAfold分析显示,这些片段多数在5′断点端形成二级茎-环结构。本研究显示,CVB在宿主细胞感染时可形成大量不完整基因组RNA片段,这些片段可在5′断点端形成局部双链结构,提示片段不是随机产生,可能是RNA酶剪切产物。此发现有助于理解CVB基因组不稳定的机制。     

Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) sequester in placenta by binding to the chondroitin 4-sulfate (C4S) chains of low sulfated chondroitin sulfate proteoglycan (CSPG). Placental CSPG, the natural receptor for IRBC adherence in the placenta, is the ideal molecule for studying structural interactions in IRBC adhesion to C4S, adhesion inhibitory antibody responses, and identification of parasite adhesive protein(s). However, because of difficulty involved in purifying placental CSPG, the commercially available bovine tracheal chondroitin sulfate A (bCSA), a copolymer having structural features of both C4S and C6S, has been widely used. To determine the validity of bCSA for C4S-IRBC interaction studies, we comparatively evaluated the characteristics of IRBC binding to placental CSPG and bCSA using three commonly used parasite strains. The results indicate that, in all three parasites studied, the characteristics of IRBC binding to placental CSPG and bCSA are qualitatively similar, but the binding capacity with respect to both the number of IRBCs bound per unit area of coated surface and binding strength is significantly higher for CSPG than bCSA regardless of whether parasites were selected on CSPG or bCSA. These results demonstrate that placental CSPG is best suited for studying interactions between parasite adhesive protein(s) and C4S, and have implications in understanding C4S-IRBC structural interactions.     

不同氮肥基追比例对高温胁迫下小麦籽粒产量和品质的影响

选用强筋小麦品种济麦20、烟农19、藁麦8901做试验材料,设置不同氮肥基追比例和籽粒灌浆中后期高温胁迫处理,研究了不同氮肥基追比例对高温胁迫条件下小麦籽粒产量和品质的影响.研究结果表明,追氮比例由50%增加到70%,3个品种的千粒重、籽粒产量、粗蛋白含量、湿面筋含量、醇溶蛋白含量、谷蛋白含量、HMW-GS含量、LMW-GS含量、HMW-GS/LMW-GS比值显著提高.济麦20和烟农19的谷蛋白大聚合体含量、谷蛋白大聚合体体积加权平均粒径和表面积加权平均粒径因追氮比例提高而升高, 藁麦8901则无显著变化.济麦20和烟农19的面团形成时间、面团稳定时间因追氮比例提高而延长, 藁麦8901基本不受影响.追氮比例由50%增加到70%,3个品种的籽粒支链淀粉/直链淀粉比值显著降低,淀粉糊化高峰黏度、低谷黏度、稀懈值、最终黏度和反弹值相应降低.总之,提高氮肥追施比例可在一定程度上缓解灌浆期高温胁迫对小麦粒重和蛋白质质量的不利影响,但对淀粉质量产生负面效应,且品种间存在差异.