Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system.

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8-fold, while uncoupling protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type uncoupling protein 1. Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity. However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.     

Molecular cloning of rabbit CARP cDNA and its regulated expression in adriamycin-cardiomyopathy.

A full-length rabbit cDNA of cardiac adriamycin responsive protein (CARP) has been cloned. It shows high levels of identity at the amino acid sequence level (>86%) with the rat, mouse and human homologues. CARP mRNA levels are highly regulated in adriamycin-cardiomyopathy in rabbits.     

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFα is an apoptotic inducer in some cancer cells, it activates NFκB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFκB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFα, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTα, FasL, and TRAIL. Mechanistically, CCNs function through integrin α₆β₁ and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding α₆β₁-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an α₆β₁-HSPG-binding defective CCN1 are blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFα and related cytokines.     

Retention of low-fitness genotypes over six decades of admixture between native and introduced tiger salamanders

Background  

Introductions of non-native tiger salamanders into the range of California tiger salamanders have provided a rare opportunity to study the early stages of secondary contact and hybridization. We produced first- and second-generation hybrid salamanders in the lab and measured viability among these early-generation hybrid crosses to determine the strength of the initial barrier to gene exchange. We also created contemporary-generation hybrids in the lab and evaluated the extent to which selection has affected fitness over approximately 20 generations of admixture. Additionally, we examined the inheritance of quantitative phenotypic variation to better understand how evolution has progressed since secondary contact.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Two Amino-acid Carriers in Pea Chloroplasts

CHLOROPLASTS are more permeable to amino-acids^1,2 than are cell membranes³ and also discriminate between their optical isomers². This has led to the suggestion that the passage of amino-acids through the limiting membranes of chloroplasts is facilitated by carriers². Using the additional criteria of saturation and competition, we support this suggestion and show that at least two carriers are probably involved.     

The unpredictability of favourability: condition assessment and protected areas in England

The probability that protected areas will deliver their potential for maintaining or enhancing biodiversity is likely to be maximised if they are appropriately and effectively managed. As a result, governments and conservation agencies are devoting much attention to the management of protected areas. In the U.K., the demand for performance accountability has resulted in Public Service Agreements (PSA) that set out targets for government departments to deliver results in return for investments being made. One such target for England is to ensure that all nationally important wildlife sites are in favourable condition by 2010. Here, we tested the hypothesis, of potential strategic importance, that the ecological condition of these sites is predictable from relationships with a range of physical, environmental and demographic variables. We used binary logistic regression to investigate these relationships, using the results of English Nature’s 1997–2003 condition assessment exercise. Generally, sites in unfavourable condition tend to be larger in area, located at higher elevations, but with higher human population density and are more spatially isolated from units of the same habitat. However, despite the range of different parameters included in our models, the extent to which the condition of any given site could be predicted was low. Our results have implications for the delivery of PSA targets, funding allocation, and the location of new protected areas.