Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

Background  

The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification.     

Formation of transmembrane ionic channels of primary amphipathic cell-penetrating peptides. Consequences on the mechanism of cell penetration

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs) CH3-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH, CH3-CO-GALFLAFLAAALS LMGLWSQPKKKRKV-NH-CH2-CH2-SH, and CH3-CO-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH called Pbeta, Palpha and Pep-1, respectively, to promote pore formation is examined both in Xenopus oocytes and artificial planar lipid bilayers. A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. This work shows that the cell-penetrating peptides Pbeta and Pep-1 are able to induce formation of transmembrane pores in artificial bilayers and that these pores are most likely at the basis of their ability to facilitate intracellular delivery of therapeutics. In addition, their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process.     

NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD⁺ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H₂O₂ produced during the oxidative process or added exogenously.

Structured summary

MINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystallography (MI:0114)     

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

Minimal cut sets in biochemical reaction networks

MOTIVATION: Structural studies of metabolic networks yield deeper insight into topology, functionality and capabilities of the metabolisms of different organisms. Here, we address the analysis of potential failure modes in metabolic networks whose occurrence will render the network structurally incapable of performing certain functions. Such studies will help to identify crucial parts in the network structure and to find suitable targets for repressing undesired metabolic functions. RESULTS: We introduce the concept of minimal cut sets for biochemical networks. A minimal cut set (MCS) is a minimal (irreducible) set of reactions in the network whose inactivation will definitely lead to a failure in certain network functions. We present an algorithm which enables the computation of the MCSs in a given network related to user-defined objective reactions. This algorithm operates on elementary modes. A number of potential applications are outlined, including network verifications, phenotype predictions, assessing structural robustness and fragility, metabolic flux analysis and target identification in drug discovery. Applications are illustrated by the MCSs in the central metabolism of Escherichia coli for growth on different substrates. AVAILABILITY: Computation and analysis of MCSs is an additional feature of the FluxAnalyzer (freely available for academic users upon request, special contracts for industrial companies; see web page below). Supplementary information: http://www.mpi-magdeburg.mpg.de/projects/fluxanalyzer     

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.     

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression,antibiotic biosynthesis,and morphological development in Streptomyces avermitilis

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5′-TTKAMKHSTTSAV-3′, was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency