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.     

Topography strongly affects atmospheric deposition and canopy exchange processes in different types of wet lowland rainforest,Southwest Costa Rica

Bulk precipitation and throughfall were collected in a wet lowland rainforest in SW Costa Rica on an event basis to allow modelling the contributions of dry deposition and canopy exchange to nutrient inputs and internal cycling of nutrients. Estimates based on bulk precipitation underestimated total atmospheric deposition to tropical rainforests by up to 10-fold ignoring the contributions of dry deposition. Canopy exchange contributed most of the aboveground inputs to the forest soil of Na⁺, about half for K⁺, 10% for P and Mg²⁺ and negligible for N, C and other elements. Tree species composition did not account for the differences found in net throughfall between forest sites, and vegetation structure (plant area index) had only a small effect on net throughfall. Forest regrowth affected net throughfall through reduced soil fertility and differences in leaf traits. Topography most significantly affected net throughfall via increased dry deposition at sites of higher elevation and via soil fertility and increased canopy exchange at down slope sites.     

Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain.

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

Analytik Atypischer Fettsäuren in biologischen Matrizes

By combined application of chemical pretreatments, capillary gas-chromatography and mass spectrometry it was possible to enlighten the structure of atypical fatty acids with hydroxy groups and cyclopropane rings under the use of only a few of reference substances. The direct alkaline saponification of the sample with liberation of fatty acids and following methylation with boron trifluoride/methanol or diazomethane was proved to be the best method regarding to precision and speed of the sample cleanup.     

Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis. Additional detection of an exon potentially coding for a new peptide

From a genomic library of Xenopus laevis, two genes coding for different preprocaeruleins have been isolated and sequenced. These correspond to the type I and type III precursors analyzed previously at the cDNA level [Richter, K., Egger, R. and Kreil, G. (1986) J. Biol. Chem. 261, 3676-3680]. The type III gene comprises eight exons; the type I apparently contains eight exons as well, of which six have been sequenced. The genetic information for the dekapeptide caerulein is present on small exons of 45 base pairs. The two genes are highly homologous in their 5'-flanking region, the exon/intron boundaries, and long stretches of intron sequences. A possible scheme for the evolution of this small family of genes through exon and gene duplications is presented. In the type I gene, in place of one of the caerulein exons, a potential exon with conserved splice sites was discovered. If expressed in some frog cells, this exon would code for a new peptide 60% homologous to caerulein.     

A portable,adjustable finger printing stand

A lightweight finger printing stand is described which can be adjusted to the proper printing height. Based upon experience printing over 1,100 subjects, 12 advantages of using the stand are suggested.