The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Haplotype, which is the sequence of SNPs in a specific chromosome, plays an important role in disease association studies. However, current sequencing techniques can detect the presence of SNP sites, but they cannot tell which copy of a pair of chromosomes the alleles belong to. Moreover, sequencing errors that occurred in sequencing SNP fragments make it difficult to determine a pair of haplotypes from SNP fragments. To help overcome this difficulty, the haplotype assembly problem is defined from the viewpoint of computation, and several models are suggested to tackle this problem. However, there are no freely available web-based tools to overcome this problem as far as we are aware. In this paper, we present a web-based application based on the genetic algorithm, named HapAssembler, for assembling a pair of haplotypes from SNP fragments. Numerical results on real biological data show that the correct rate of the proposed application in this paper is greater than 95% in most cases. HapAssembler is freely available at http://alex.chonnam.ac.kr/~drminor/hapHome.htm. Users can choose any model among four models for their purpose and determine haplotypes from their input data.     

Analyses of soluble and membrane proteomes of Ralstonia eutropha H16 reveal major changes in the protein complement in adaptation to lithoautotrophy

The soil-dwelling lithoautotrophic bacterium Ralstonia eutropha H16 utilizes hydrogen as the key source of energy during aerobic growth on hydrogen and carbon dioxide. We examined the soluble and membrane protein complements of lithoautotrophically grown cells and compared them to the protein complements of cells grown organoheterotrophically on succinate. (14)N/(15)N-based inverse metabolic labeling in combination with GeLC-MS led to the identification of 1452 proteins, 1174 of which could be quantitated. Far more proteins were found to be more abundant in the lithoautotrophically than in the organoheterotrophically grown cells. In addition to the induction of the key enzymes of hydrogen oxidation and carbon dioxide fixation, we observed several characteristic alterations in the proteome correlated with lithoautotrophic growth. (I) Genes for three terminal oxidases were upregulated. (II) NAD(P) transhydrogenase and enzymes for the accumulation of poly(3-hydroxybutyrate) (PHB) showed increased protein abundance. (III) Lithoautotrophically grown cells were equipped with an enhanced inventory of transport systems. (IV) The expression of cell surface appendages involved in cell movement was markedly increased, while proteins involved in cell adhesion were decreased. Our data show that the hydrogen-based lifestyle of R. eutropha H16 relies on an extensive protein repertoire adapting the organism to the alternative energy and carbon sources.     

The early vertebrate Danio rerio Mr 46000 mannose-6-phosphate receptor: biochemical and functional characterisation

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, γ-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish. Electronic Supplementary Material Supplementary material is available for this article at . The zebrafish MPR 46 sequence data have been submitted to the GenBank database under accession no. DQ089037.     

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.     

Role of DptE and DptF in the lipidation reaction of daptomycin

Daptomycin and A21987C antibiotics are branched, cyclic, nonribosomally assembled acidic lipodepsipeptides produced by Streptomyces roseosporus. The antibacterial activity of daptomycin against gram-positive bacteria strongly depends on the nature of the N-terminal fatty acid moiety. Two genes, dptE and dptF, localized upstream of the daptomycin nonribosomal peptide synthetase genes, are thought to be involved in the lipidation of daptomycin. Here we describe the cloning, heterologous expression, purification and biochemical characterization of the enzymes encoded by these genes. DptE was proven to preferentially activate branched mid- to long-chain fatty acids under ATP consumption, and these fatty acids are subsequently transferred onto DptF, the cognate acyl carrier protein. Additionally, we demonstrate that lipidation of DptF by DptE in trans is based on specific protein-protein interactions, as DptF is favored over other acyl carrier proteins. Study of DptE and DptF may provide useful insights into the lipidation mechanism, and these enzymes may be used to generate novel daptomycin derivatives with altered fatty acids.     

The Siak, a tropical black water river in central Sumatra on the verge of anoxia

The Siak is a black water river in central Sumatra, Indonesia, which owes its brown color to dissolved organic matter (DOM) leached from surrounding, heavily disturbed peat soils. The dissolved organic carbon (DOC) concentrations measured during five expeditions in the Siak between 2004 and 2006 are among the highest reported world wide. The DOM decomposition appeared to be a main factor influencing the oxygen concentration in the Siak which showed values down to 12 μmol l⁻¹. Results derived from a box-diffusion model indicated that in addition to the DOC concentration and the associated DOM decomposition the water-depth also plays a crucial role in regulating the oxygen levels in the river because of its impact on the turbulence in the aquatic boundary layer and the surface/volume ratio of water in the river. Model results imply furthermore that a reduced water-depth could counteract an increased oxygen consumption caused by an enhanced DOM leaching during the transition from dry to wet periods. This buffer mechanism seems to be close to its limits as indicated by sensitivity studies which showed in line with measured data that an increase of the DOC concentrations by ~15% could already lead to anoxic conditions in the Siak. This emphasizes the sensitivity of the Siak against further peat soil degradation, which is assumed to increase DOC concentrations in the rivers.     

Impact of plasmid architecture on stability and yEGFP3 reporter gene expression in a set of isomeric multicopy vectors in yeast

Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.

     

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia

Background

The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis.