Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.     

Evolutionary transformations of myoseptal tendons in gnathostomes

Axial undulations in fishes are powered by a series of three-dimensionally folded myomeres separated by sheets of connective tissue, the myosepta. Myosepta have been hypothesized to function as transmitters of muscular forces to axial structures during swimming, but the difficulty of studying these delicate complex structures has precluded a more complete understanding of myoseptal mechanics. We have developed a new combination of techniques for visualizing the three-dimensional morphology of myosepta, and here we present their collagen-fibre architecture based on examination of 62 species representing all of the major clades of notochordates. In all gnathostome fishes, each myoseptum bears a set of six specifically arranged tendons. Because these tendons are not present outside the gnathostomes (i.e. they are absent from lampreys, hagfishes and lancelets), they represent evolutionary novelties of the gnathostome ancestor. This arrangement has remained unchanged throughout 400 Myr of gnathostome evolution, changing only on the transition to land. The high uniformity of myoseptal architecture in gnathostome fishes indicates functional significance and may be a key to understanding general principles of fish swimming mechanics. In the design of future experiments or biomechanical models, myosepta have to be regarded as tendons that can distribute forces in specific directions.     

Perspectives in anti-infective drug design. The late steps in the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate

A mevalonate-independent pathway for the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that has been elucidated during the last decade is essential in plants, many eubacteria and apicomplexan parasites, but is absent in Archaea and animals. The enzymes of the pathway are potential targets for the development of novel antibiotic, antimalarial and herbicidal agents. This review is focused on the late steps of this pathway. The intermediate 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted into IPP and DMAPP via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of the iron-sulfur proteins IspG and IspH. IPP and DMAPP can be interconverted by IPP isomerase which is essential in microorganisms using the mevalonate pathway, whereas its presence is optional in microorganisms using the non-mevalonate pathway. A hitherto unknown family of IPP isomerases using FMN as coenzyme has been discovered recently in Archaea and certain eubacteria.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.     

Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile

ShK, a peptide isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a selective K(v)1.3 inhibitor and to block this channel with equivalent potency as ShK [Kalman et al. (1998) J. Biol. Chem. 273, 32697-32707]. In this study, a large body of evidence is presented which indicates that the potencies of wild-type ShK peptide for both K(v)1.3 and K(v)1.1 channels have been previously underestimated. Therefore, the affinity of ShK-Dap(22) for both channels appears to be ca. 10(2)-10(4)-fold weaker than ShK. ShK-Dap(22) does display ca. 20-fold selectivity for human K(v)1.3 vs K(v)1.1 when measured by the whole-cell voltage clamp method but not in equilibrium binding assays. ShK-Dap(22) has low affinity for K(v)1.2 channels, but heteromultimeric K(v)1.1-K(v)1.2 channels form a receptor with ca. 200-fold higher affinity for ShK-Dap(22) than K(v)1.1 homomultimers. In fact, K(v)1.1-K(v)1.2 channels bind ShK-Dap(22) with only ca. 10-fold less potency than ShK and reveal a novel pharmacology not predicted from the homomultimers of K(v)1.1 or K(v)1.2. The concentrations of ShK-Dap(22) needed to inhibit human T cell activation were ca. 10(3)-fold higher than those of ShK, in good correlation with the relative affinities of these peptides for inhibiting K(v)1.3 channels. All of these data, taken together, suggest that ShK-Dap(22) will not have the same in vivo immunosuppressant efficacy of other K(v)1.3 blockers, such as margatoxin or ShK. Moreover, ShK-Dap(22) may have undesired side effects due to its interaction with heteromultimeric K(v)1.1-K(v)1.2 channels, such as those present in brain and/or peripheral tissues.     

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.     

The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.