The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.     

Synthesis and production of poly(3-hydroxyvaleric acid) homopolyester by Chromobacterium violaceum

Chromobacterium violaceum DSM 30191 accumulated a homopolyester of 3-hydroxyvaleric acid (3HV) up to 65% of the cellular dry matter during cultivation in fed-batch cultures with valeric acid as sole carbon source and during cell starvation of the nitrogen source. From fructose, gluconate, propionate or hexanoate a homopolyester of 3-hydroxybutyrate (3HB) was accumulated. Poly(3HV) homopolyster was also accumulated by two different strains of C. violaceum, whereas two other strains of C. violaceum and three strains of Janthinobacterium lividum accumulated poly(3HB-co-3HV) copolyesters from valerate. The composition of the biosynthetic poly(3HV) was confirmed by various nuclear magnetic resonance spectroscopic methods. Differential scanning calorimetry analysis of four poly(3HV) samples that were isolated from different batches of cells revealed glass transition temperatures between –10 and –12°C and melting points between 107 and 112°C. Viscosity measurements gave intrinsic viscosities between 62.5 and 124.8 × 10^–2 dl/g for these samples, indicating approximate relative molecular masses between 60 000 and 145 000 of the biosynthetic poly(3HV). Correspondence to: A. Steinbüchel     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.     

Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase

Thiosulphate is one of the products of the initial step of the elemental sulphur oxidation pathway in the thermoacidophilic archaeon Acidianus ambivalens. A novel thiosulphate:quinone oxidoreductase (TQO) activity was found in the membrane extracts of aerobically grown cells of this organism. The enzyme was purified 21-fold from the solubilized membrane fraction. The TQO oxidized thiosulphate with tetrathionate as product and ferricyanide or decyl ubiquinone (DQ) as electron acceptors. The maximum specific activity with ferricyanide was 73.4 U (mg protein)(-1) at 92 degrees C and pH 6, with DQ it was 397 mU (mg protein)(-1) at 80 degrees C. The Km values were 2.6 mM for thiosulphate (k(cat) = 167 s(-1)), 3.4 mM for ferricyanide and 5.87 micro M for DQ. The enzymic activity was inhibited by sulphite (Ki = 5 micro M), metabisulphite, dithionite and TritonX-100, but not by sulphate or tetrathionate. A mixture of caldariella quinone, sulfolobus quinone and menaquinone was non-covalently bound to the protein. No other cofactors were detected. Oxygen consumption was measured in membrane fractions upon thiosulphate addition, thus linking thiosulphate oxidation to dioxygen reduction, in what constitutes a novel activity among Archaea. The holoenzyme was composed of two subunits of apparent molecular masses of 28 and 16 kDa. The larger subunit appeared to be glycosylated and was identical to DoxA, and the smaller was identical to DoxD. Both subunits had been described previously as a part of the terminal quinol:oxygen oxidoreductase complex (cytochrome aa3).     

Predicting Survey Responses: How and Why Semantics Shape Survey Statistics on Organizational Behaviour

Some disciplines in the social sciences rely heavily on collecting survey responses to detect empirical relationships among variables. We explored whether these relationships were a priori predictable from the semantic properties of the survey items, using language processing algorithms which are now available as new research methods. Language processing algorithms were used to calculate the semantic similarity among all items in state-of-the-art surveys from Organisational Behaviour research. These surveys covered areas such as transformational leadership, work motivation and work outcomes. This information was used to explain and predict the response patterns from real subjects. Semantic algorithms explained 60–86% of the variance in the response patterns and allowed remarkably precise prediction of survey responses from humans, except in a personality test. Even the relationships between independent and their purported dependent variables were accurately predicted. This raises concern about the empirical nature of data collected through some surveys if results are already given a priori through the way subjects are being asked. Survey response patterns seem heavily determined by semantics. Language algorithms may suggest these prior to administering a survey. This study suggests that semantic algorithms are becoming new tools for the social sciences, opening perspectives on survey responses that prevalent psychometric theory cannot explain.     

Association between pulmonary function and peak oxygen uptake in elderly: the Generation 100 study

Chronic obstructive lung disease (COPD) is a common cause of death in industrialized countries often induced by exposure to tobacco smoke. A substantial number of patients with COPD also suffer from pulmonary hypertension that may be caused by hypoxia or other hypoxia-independent stimuli - inducing pulmonary vascular remodeling. The Ca2+ binding protein, S100A4 is known to play a role in non-COPD-driven vascular remodeling of intrapulmonary arteries. Therefore, we have investigated the potential involvement of S100A4 in COPD induced vascular remodeling. Lung tissue was obtained from explanted lungs of five COPD patients and five non-transplanted donor lungs. Additionally, mice lungs of a tobacco-smoke-induced lung emphysema model (exposure for 3 and 8 month) and controls were investigated. Real-time RT-PCR analysis of S100A4 and RAGE mRNA was performed from laser-microdissected intrapulmonary arteries. S100A4 immunohistochemistry was semi-quantitatively evaluated. Mobility shift assay and siRNA knock-down were used to prove hypoxia responsive elements (HRE) and HIF binding within the S100A4 promoter. Laser-microdissection in combination with real-time PCR analysis revealed higher expression of S100A4 mRNA in intrapulmonary arteries of COPD patients compared to donors. These findings were mirrored by semi-quantitative analysis of S100A4 immunostaining. Analogous to human lungs, in mice with tobacco-smoke-induced emphysema an up-regulation of S100A4 mRNA and protein was observed in intrapulmonary arteries. Putative HREs could be identified in the promoter region of the human S100A4 gene and their functionality was confirmed by mobility shift assay. Knock-down of HIF1/2 by siRNA attenuated hypoxia-dependent increase in S100A4 mRNA levels in human primary pulmonary artery smooth muscle cells. Interestingly, RAGE mRNA expression was enhanced in pulmonary arteries of tobacco-smoke exposed mice but not in pulmonary arteries of COPD patients. As enhanced S100A4 expression was observed in remodeled intrapulmonary arteries of COPD patients, targeting S100A4 could serve as potential therapeutic option for prevention of vascular remodeling in COPD patients.     

Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers

The purpose of this study was to comparatively analyze the genetic diversity of sesame (Sesamum indicum L.) using agro-morphological and molecular markers. Twelve sesame populations collected from three regions in Cambodia and Vietnam were used in this study. A high genetic variation was revealed both by agro-morphological and RAPD markers within and among the 12 sesame populations. The range of agro-morphological trait based average taxonomic distance among populations (0.02 to 0.47) was wider than that of RAPD based genetic distance (0.06 to 0.27). The mean distance revealed by agro-morphological markers (0.23) and RAPD markers (0.22) was similar. RAPD based analysis revealed a relatively higher genetic diversity in populations from South Vietnam as compared to the other two regions. Interestingly, populations from this region also had higher values for yield related traits such as number of capsules per plant, number of seeds per capsule, and seed yield per plant suggesting positive correlation between the extent of genetic variation within population and yield related traits in sesame. A highly significant positive correlation (r = 0.88, P < 0.001) was found between agro-morphological and RAPD markers in estimating the genetic distance between populations. Both methods suggested the existence of a substantial amount of genetic diversity both in the Vietnamese and Cambodian populations. Although both agro-morphological and RAPD markers were found to be useful in genetic diversity analysis in sesame, their combined use would give superior results.     

Analysis of genetic diversity of Guizotia abyssinica from Ethiopia using inter simple sequence repeat markers

Within and among population genetic diversity of 37 Guizotia abyssinica populations from Ethiopia were analyzed using inter simple sequence repeats (ISSRs). Five primers amplified a total of 118 genomic DNA fragments across a total of 370 individuals of which 106 were polymorphic (89.83%). The average number of polymorphic bands per primer was 21.2. More bands were generated by primer UBC 888 (BDB(CA)(7.) The total genetic diversity (Ht) and the coefficient of genetic differentiation (Gst) were 0.4115 and 0.0918 respectively, while the within population genetic diversity (Hs) and the among population genetic diversity(Dst) were 0.3738 and 0.03776 respectively suggesting more variability within the populations than among them. The standard genetic distances between the G. abyssinica populations of the eight regions ranged from 0.0281 (between Wollo and Gojam) to 0.1148 (between Jimma and Hararghe). Generally, the standard genetic distances are smaller between populations of neighboring regions and highest between those of Jimma and the other regions, ranging from 0.0696 (between Jimma and Shewa) to 0.1148 (between Jimma and Hararghe). The ISSR based UPGMA clustering using the standardized genetic distances matrix also placed populations from neighboring regions closer than those from farther apart areas, while the UPGMA clustering by regions based on the standard genetic distances produced three clusters following the proximity and the contiguity of the regions. The mean Shannon Weaver diversity indices for the populations of the eight regions ranged from 0.8197 (Jimma) to 0.9176 (Hararghe), with a mean of 0.8841 for the whole material.