Structure of the ATPase subunit CysA of the putative sulfate ATP-binding cassette (ABC) transporter from Alicyclobacillus acidocaldarius

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

Differential synthesis of two interleukin-1 receptor antagonist variants and interleukin-8 by peripheral blood neutrophils

With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.     

High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27(Kip) Cdk-complexes

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27(Kip1) levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27(Kip1) induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.     

Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.     

The Met-196 -> Arg variation of human tumor necrosis factor receptor 2 (TNFR2) affects TNF-alpha-induced apoptosis by impaired NF-kappaB signaling and target gene expression

Tumor necrosis factor-alpha (TNF-alpha)-induced signaling is pivotally involved in the pathogenesis of chronic inflammatory diseases. A polymorphism in the TNF receptor 2 (TNFR2) gene resulting in a juxtamembrane inversion from methionine (TNFR2(196MET)) to arginine (TNFR2(196ARG)) has been genetically associated with an increased risk for systemic lupus erythematosus and familial rheumatoid arthritis. Albeit the mutation does not affect the TNF binding kinetics of TNFR2, the present study provides evidence that the mutation results in a significantly lower capability to induce TNFR2-mediated NF-kappaB activation. Pretriggering of TNFR2 with a receptor-specific mutein leads to an enhancement of TNFR1-induced apoptosis, which is further increased in cells carrying the TNFR2(196ARG) variant. A diminished induction of NF-kappaB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, TRAF1, IL-6, or IL-8 is observed. The mutated form TNFR2(196ARG) shows a reduction of inducible TRAF2 recruitment upon TNF-alpha stimulation. The findings suggest a common molecular mechanism for the involvement of the TNFR2(196ARG) variant in the etiopathogenesis of different chronic inflammatory disorders.     

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.     

Analysis of xylem sap proteins from Brassica napus

Background  

Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs.     

Genome-wide single-nucleotide polymorphism map for Candida albicans

Single-nucleotide polymorphisms (SNPs) are essential tools for studying a variety of organismal properties and processes, such as recombination, chromosomal dynamics, and genome rearrangement. This paper describes the development of a genome-wide SNP map for Candida albicans to study mitotic recombination and chromosome loss. C. albicans is a diploid yeast which propagates primarily by clonal mitotic division. It is the leading fungal pathogen that causes infections in humans, ranging from mild superficial lesions in healthy individuals to severe, life-threatening diseases in patients with suppressed immune systems. The SNP map contains 150 marker sequences comprising 561 SNPs and 9 insertions-deletions. Of the 561 SNPs, 437 were transition events while 126 were transversion events, yielding a transition-to-transversion ratio of 3:1, as expected for a neutral accumulation of mutations. The average SNP frequency for our data set was 1 SNP per 83 bp. The map has one marker placed every 111 kb, on average, across the 16-Mb genome. For marker sequences located partially or completely within coding regions, most contained one or more nonsynonymous substitutions. Using the SNP markers, we identified a loss of heterozygosity over large chromosomal fragments in strains of C. albicans that are frequently used for gene manipulation experiments. The SNP map will be useful for understanding the role of heterozygosity and genome rearrangement in the response of C. albicans to host environments.