Biogenic methane production in formation waters from a large gas field in the North Sea

Methanogenesis was investigated in formation waters from a North Sea oil rimmed gas accumulation containing biodegraded oil, which has not been subject to seawater injection. Activity and growth of hydrogenotrophic methanogens was measured but acetoclastic methanogenesis was not detected. Hydrogenotrophic methanogens showed activity between 40 and 80°C with a temperature optimum (ca. 70°C) consistent with in situ reservoir temperatures. They were also active over a broad salinity range, up to and consistent with the high salinity of the waters (90 g l⁻¹). These findings suggest the methanogens are indigenous to the reservoir. The conversion of H₂ and CO₂ to CH₄ in methanogenic enrichments was enhanced by the addition of inorganic nutrients and was correlated with cell growth. Addition of yeast extract also stimulated methanogenesis. Archaeal 16S rRNA gene sequences recovered from enrichment cultures were closely related to Methanothermobacter spp. which have been identified in other high-temperature petroleum reservoirs. It has recently been suggested that methanogenic oil degradation may be a major factor in the development of the world’s heavy oils and represent a significant and ongoing process in conventional deposits. Although an oil-degrading methanogenic consortium was not enriched from these samples the presence and activity of communities of fermentative bacteria and methanogenic archaea was demonstrated. Stimulation of methanogenesis by addition of nutrients suggests that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from such petroleum systems.     

Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import

The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.The maintenance of peroxisome function depends on the formation of the peroxisomal membrane and the subsequent import of both membrane and matrix proteins. Without exception, peroxisomal matrix proteins are nucleus encoded, synthesized on free ribosomes, and subsequently imported in a posttranslational manner (40). The peroxisomal import apparatus can facilitate the transport of folded and oligomeric proteins over the peroxisomal membrane, with the basic principle of this translocation event still being unknown. Based on the concept of cycling receptors (9, 31), the receptor cycle is divided into four steps. In the first step, the cargo proteins are recognized in the cytosol by their cognate receptor protein Pex5 or Pex7. In general, this initial step depends on either one of the two well-characterized PTSs (peroxisomal targeting signals), PTS1 and PTS2, which are recognized and bound by the corresponding receptor proteins Pex5 and Pex7, respectively. In the second step, the cargo-loaded receptors dock with distinct proteins accessible at the surface of the peroxisomal membrane, namely, Pex13 and Pex14. These two proteins together with Pex17 are established components of the docking complex. A second complex of the peroxisomal protein import machinery acts downstream of the docking event and consists of the three peroxins Pex2, Pex10, and Pex12. A common feature of these proteins is a C-terminal RING (really interesting new gene) finger domain. The RING finger subcomplex and the docking subcomplex are both linked in a Pex8-dependent manner to form a larger assembly, the importomer (1). In the third step of the receptor cycle, the cargo is delivered to the peroxisomal matrix, and finally, the receptor is released from the peroxisomal membrane in an ATP-dependent manner and thus made available for proteasomal degradation or another round of import (for a review, see reference 27).With respect to the PTS1 receptor Pex5, recent reports demonstrated that this final ATP-dependent step in the receptor cycle is catalyzed by the AAA (ATPases associated with various cellular activities) peroxins Pex1 and Pex6 (33, 37). The signal for the export process is the attachment of a monoubiquitin moiety or, alternatively, the anchoring of a polyubiquitin chain (5, 35). This protein modification is in general facilitated by a three-step enzyme cascade (20). The ubiquitin (Ub)-activating enzyme (E1) activates the Ub and transfers it to the Ub conjugation enzyme (E2). In a final step, a protein-Ub ligase (E3) binds both E2 and substrate and thereby facilitates the conjugation of the Ub moiety onto the substrate protein. Saccharomyces cerevisiae harbors genes coding for one E1 enzyme, 11 E2 enzymes, and approximately 80 to 100 putative E3 enzymes (18, 29). It was demonstrated that the polyubiquitination of Pex5 primarily depends on the E2 protein Ubc4, which upon deletion can be partly replaced by Ubc5 or Ubc1 (22, 25, 36). Polyubiquitination of Pex5 is not a prerequisite for its function in peroxisomal protein import but might be a crucial step of a quality control system for the disposal of dysfunctional Pex5 (10, 22, 25, 36). Pex5 monoubiquitination is facilitated by the E2 protein Pex4 (Ubc10) in yeast or the Pex4-like UbcH5a/b/c in humans (14, 35, 47). The modification of Pex5 by a single Ub primes the receptor for its export back to the cytosol, where the Ub supposedly is removed prior to the initiation of a new receptor cycle (5, 14, 35). Although the functional relevance and the cognate E2 protein required for the different Ub modifications of Pex5 were identified, the factor(s) determining the substrate specificity, the protein-Ub ligase(s), remained unknown.Here we report on the discovery of the function of Pex2 and Pex12 as E3 proteins required for ubiquitination of the import receptor Pex5. These RING peroxins, defects of which cause the lethal peroxisome biogenesis disorders in humans, exhibit Ub-protein isopeptide ligase activity with Pex5 as the molecular target. Pex2 is shown to mediate the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.     

The mammalian and yeast translocon complexes comprise a characteristic Sec61 channel

In eukaryotes, protein translocation across and insertion into the membrane of the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex or translocon. In our previous electrophysiological studies, we characterized the mammalian Sec61 channel from Canis familiaris. Here we extended these initial results to the Sec61 channel from the yeast Saccharomyces cerevisiae and compared the basic electrophysiological properties of both channel preparations with respect to the gating behaviour, distribution of channel open states, ionic conductance, approximated pore dimensions, reversal potential and selectivity as well as voltage-dependent open probability. We found that the Sec61 complexes from both species displayed conformable characteristics of the highly dynamic channel in an intrinsically open state. In contrast, the bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, displayed under the same experimental conditions significantly different properties residing in an intrinsically closed state. We therefore propose that considerable differences between the respective eukaryote and prokaryote protein-conducting channel units and their regulation exist.     

The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

The Ca²⁺/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K_i value of 3.8 μm. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.     

Oral health care in long‐term care facilities for elderly people in southern Brazil: a conceptual framework

doi:10.1111/j.1741‐2358.2009.00280.x
Oral health care in long‐term care facilities for elderly people in southern Brazil: a conceptual framework Objective: To present a theoretical model for understanding oral health care for the elderly in the context of long‐term care institutions (LTCI). Methods: Open‐ended individual interviews were conducted with the elderly residing in LTCI, their carers, nursing technicians and nurses, directors of care, dental surgeons and managers of public health services. A grounded theory methodological approach was adopted for data collection and analysis. Results: The emerging core category revealed a basic social process: ‘Promoting oral health care for the elderly based on the context of LTCI’. This process was composed of two contradicting yet correlated aspects: the oral health care does not minimise the poor oral epidemiological condition, and at the same time, there was a continued improvement in the oral care expressed by better care practices. These aspects were related to the: attribution of meaning to oral health, social determination of oral health, the ageing process, interactions established in the oral health care practices, oral health care management in LTCI, inclusion of oral health care into the political–organisational dimension and possibility of conjecturing better oral health care practices. Conclusion: The core concept of ‘Promotion of oral health care for elderly people based on the context of LTCI’ is capable of explaining the variations in the structure and process of LTCI, as well as in helping to understand the meaning of the oral health care practices for the institutionalised elderly.     

Beta1-Adrenoceptor Polymorphism Predicts Flecainide Action in Patients with Atrial Fibrillation

Background

Antiarrhythmic action of flecainide is based on sodium channel blockade. Beta₁-adrenoceptor (β₁AR) activation induces sodium channel inhibition, too. The aim of the present study was to evaluate the impact of different β₁AR genotypes on antiarrhythmic action of flecainide in patients with structural heart disease and atrial fibrillation.

Methodology/Principal Findings

In 145 subjects, 87 with atrial fibrillation, genotyping was performed to identify the individual β₁AR Arg389Gly and Ser49Gly polymorphism. Resting heart rate during atrial fibrillation and success of flecainide-induced cardioversion were correlated with β₁AR genotype. The overall cardioversion rate with flecainide was 39%. The Arg389Arg genotype was associated with the highest cardioversion rate (55.5%; OR 3.30; 95% CI; 1.34–8.13; p = 0.003) compared to patients with Arg389Gly (29.5%; OR 0.44; 95% CI; 0.18–1.06; p = 0.066) and Gly389Gly (14%; OR 0.24; 95% CI 0.03–2.07; p = 0.17) variants. The single Ser49Gly polymorphism did not influence the conversion rate. In combination, patients with Arg389Gly-Ser49Gly genotype displayed the lowest conversion rate with 20.8% (OR 0.31; 95% CI; 0.10–0.93; p = 0.03). In patients with Arg389Arg variants the heart rate during atrial fibrillation was significantly higher (110±2.7 bpm; p = 0.03 vs. other variants) compared to Arg389Gly (104.8±2.4 bpm) and Gly389Gly (96.9±5.8 bpm) carriers. The Arg389Gly-Ser49Gly genotype was more common in patients with atrial fibrillation compared to patients without atrial fibrillation (27.6% vs. 5.2%; HR 6.98; 95% CI; 1.99–24.46; p<0.001).

Conclusions

The β₁AR Arg389Arg genotype is associated with increased flecainide potency and higher heart rate during atrial fibrillation. The Arg389Gly-Ser49Gly genotype might be of predictive value for atrial fibrillation.     

Differential surface expression and possible function of 9-O- and 7-O-acetylated GD3 (CD60 b and c) during activation and apoptosis of human tonsillar B and T lymphocytes

The disialoganglioside GD3 (CD60 a) and its O-acetylated variants have previously been described as surface molecules of human T lymphocytes of the peripheral blood system. Here we report the expression of the 9-O-, and 7-O-acetylated disialoglycans of GD3 (CD60 b and CD60 c respectively) on human tonsillar lymphocytes. CD60 b and c are surface-expressed on activated germinal centre B cells and colocalize in raft-like structures on the cell surface together with the cytoplasmic tyrosine kinase Lyn and Syk. Addition of CD60 b and c mAb together with anti-IgM/IL-4 to in vitro cultivated tonsillar B cells resulted in a costimulatory effect. During spontaneous and staurosporine-induced apoptosis a distinct population of activated annexin V+/CD60 b+/CD60 c- B cells was observed. CD60 b and c are also found on cells of the extrafollicular T cell area. On tonsillar T cells, CD60 b mAb had a costimulatory effect together with PHA while CD60 c mAb alone was sufficient to induce proliferation. In further contrast to B cells, during apoptosis a distinct CD60 b+ T cell subpopulation was not observed. Together, surface-expressed CD60 b and c are differently expressed on tonsillar B and T cells and may be involved in the regulation of activation and apoptosis of lymphocytes in secondary lymphatic tissue.     

The nucleotide sequence of the cytoplasmic 5S rRNA from the horsetail, Equisetum arvense

Using 3'- and 5'-end labelling sequencing techniques, the following sequence for the cytoplasmic 5S rRNA of the horsetail Equisetum arvense could be determined: (sequence in text). This sequence exhibits all features expected for higher plant cytoplasmic 5S rRNAs, and can be fitted to the secondary structure model for 5S rRNA proposed by De Wachter et al. (15).     

Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis.