METANNOGEN: compiling features of biochemical reactions needed for the reconstruction of metabolic networks

Background  

One central goal of computational systems biology is the mathematical modelling of complex metabolic reaction networks. The first and most time-consuming step in the development of such models consists in the stoichiometric reconstruction of the network, i. e. compilation of all metabolites, reactions and transport processes relevant to the considered network and their assignment to the various cellular compartments. Therefore an information system is required to collect and manage data from different databases and scientific literature in order to generate a metabolic network of biochemical reactions that can be subjected to further computational analyses.     

Basal body positioning is controlled by flagellum formation in Trypanosoma brucei

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.     

Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.     

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.     

Renal Response to L-Arginine in Diabetic Rats. A Possible Link between Nitric Oxide System and Aquaporin-2

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [¹⁴C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.     

A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.