Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.     

Individual and geographical variation in display behaviour of male harbour seals in Scotland

Studying variations in behaviour at the individual or population level enables insight into the reproductive strategies within a species. We examined individual and geographical variation in the vocal and dive behaviour of male harbour seals, Phoca vitulina, which is associated with aquatic mating. This display behaviour was recorded in the Moray Firth, Scotland, from July 1994 to 1997, and in Orkney, Scotland, during July 1998. One vocalization type was apparent in the Moray Firth and two in Orkney. Time parameters (total and pulse duration) varied between males in the population in the Moray Firth. We used both frequency and time parameters in a discriminant analysis, which showed that 73.2% of individual male vocalizations could be correctly classified; 94.6% of male vocalizations from the Moray Firth and Orkney could be correctly classified according to their geographical areas. Therefore, vocal variation was greater between geographical areas than between individuals. No individual variation was apparent between dive and surface interval durations. However, individuals varied significantly in the percentage of short surface intervals. Male harbour seals showed substantial variability in the parameters affecting their vocal and dive behaviour during the mating season. We suggest that these variations may be indicative of adaptations to varying environmental challenges influencing the reproductive strategies of discrete populations. Copyright 2000 The Association for the Study of Animal Behaviour.     

Pulmonary stromal-derived factor-1 expression and effect on neutrophil recruitment during acute lung injury

The severe and protracted inflammation that characterizes acute lung injury (ALI) is driven by the ongoing recruitment of neutrophils to the lung. Although much of the cytokine signaling responsible for the initial phase of ALI has been elaborated, relatively little is known about the mechanisms governing the recruitment of neutrophils from the bone marrow to the lung in the later period of this disease. Given its previously described chemoattractant effects on marrow neutrophils, we investigated whether stromal-derived factor-1 (SDF-1) (CXCL12) might participate in this later phase of recruitment. Using immunohistochemistry to examine both banked human lung specimens from patients with ALI and lungs from mice with LPS-induced pneumonitis, we found that pulmonary SDF-1 expression increases during ALI. We further determined that both lung SDF-1 protein expression and mRNA expression rise in a delayed but sustained pattern in this mouse model and that the major source of the increase in expression appears to be the lung epithelium. Lastly, we found that expression of the SDF-1 receptor CXCR4 rises in a similar temporal pattern on neutrophils in both the blood and airspace of LPS-injured mice and that Ab-mediated SDF-1 blockade significantly attenuates late but not early pulmonary neutrophilia in this model. These results implicate SDF-1 in neutrophil recruitment to the lung in the later period of acute lung injury and suggest a novel role for this cytokine in coordinating the transition from the inflammatory response to the initiation of tissue repair.     

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fcγ receptors (FcγR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil FcγRs were examined with a specific FcγR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of FcγRIIa and FcγRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative FcγR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.     

降解烤烟秸秆和烟碱菌株的筛选及其产酶特性

摘要:【目的】为获得能够降解烤烟秸秆和烟碱的菌株,并探索其降解烤烟秸秆的利用途径。【方法】以烤烟秸秆为唯一碳氮源,从烟田土壤中进行了菌株的筛选。采用形态学观察、生理生化特性鉴定、16S rRNA基因序列鉴定等方法对该菌株进行了鉴定,并对其以烤烟秸秆为底物进行液态发酵的产酶活性和木质纤维素降解效果进行了测定。【结果】结果表明:该菌株为巨大芽孢杆菌(Bacillus megaterium)。在以烤烟秸秆为主要营养物质液态发酵条件下该菌株具有较强的木质素降解能力,最大漆酶活力达到418.52 U/L,而木质素过氧化物酶和锰过氧化物酶的最大酶活分别为19.71 U/L 和64.71 U/L。此外,发酵20 d后该菌能够完全降解发酵液中的烟碱。【结论】本研究筛选到了1株能够较好降解烤烟秸秆和完全降解烟碱的巨大芽孢杆菌(Bacillus megaterium),且该菌株具有利用烤烟秸秆生产漆酶的应用价值。