Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.     

The Yersinia pseudotuberculosis Yut protein,a new type of urea transporter homologous to eukaryotic channels and functionally interchangeable in vitro with the Helicobacter pylori UreI protein

Urea uptake in eukaryotes and prokaryotes occurs via diffusion or active transport across the cell membrane. Facilitated diffusion of urea in both types of organisms requires a single-component channel. In bacteria, these transport systems allow rapid access of urease to its substrate, resulting in ammonia production, which is needed either for resistance to acidity or as a nitrogen source. In Yersinia pseudotuberculosis, a ureolytic enteropathogenic bacterium, a gene of unknown function (yut) located near the urease locus was found to encode a putative membrane protein with weak homology to single-component eukaryotic urea transporters. When expressed in Xenopus oocytes, Yut greatly increases cellular permeability to urea. Inactivation of yut in Y. pseudotuberculosis results in diminished apparent urease activity and reduced resistance to acidity in vitro when urea is present in the medium. In the mouse model, bacterial colonization of the intestine mucosa is delayed with the Yut-deficient mutant. Although structurally unrelated, Yut and the Helicobacter pylori UreI urea channel were shown to be functionally interchangeable in vitro and are sufficient to allow urea uptake in both bacteria, thereby confirming their function in the respective parent organisms. Homologues of Yut were found in other yersiniae, Actinobacillus pleuropneumoniae, Brucella melitensis, Pseudomonas aeruginosa and Staphylococcus aureus. The Y. pseudotuberculosis Yut protein is therefore the first member of a novel class of bacterial urea permeases related to eukaryotic transporters.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.Non-commun abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer     

Analysis of a ribosomal RNA operon in the actinomycete Frankia.

The organisation of ribosomal RNA-encoding (rrn) genes has been studied in Frankia sp. strain ORS020606. The two rrn clusters present in Frankia strain ORS020606 were isolated from genomic banks in phage lambda EMBL3 by hybridization with oligodeoxyribonucleotide probes. The 5'-3' gene order is the usual one for bacteria: 16S-23S-5S. The two clusters are not distinguishable by restriction enzyme mapping inside the coding section, but vary considerably outside it. Sequencing showed that the 16S-rRNA-encoding gene of ORS020606 is very closely related to that of another Alnus-infective Frankia strain (Ag45/Mut15) and highly homologous to corresponding genes of Streptomyces spp. Two possible promoter sequences were detected upstream from the 16S gene, while no tRNA-encoding gene was detected in the whole operon. Regions with a high proportion of divergence for the study of phylogenetic relationships within the genus were looked for and found in the first intergenic spacer, in the 23S and in the 16S gene.     

Near normal levels of isoprenoid lipids in severe mevalonic aciduria

The levels of cholesterol and dolichyl phosphate in the liver of an abortus with severe mevalonic aciduria were found to be approximately 60% of the mean of 5 age matched controls, while the level of squalene was within the normal range. Thus, despite a level of mevalonate kinase reported to be less than 1% of normal (Hoffmann, H. et al. (1986) N. Engl. J. Med. 314, 1610-1614), the liver was able to synthesize normal or near normal levels of isoprenoid lipids.     

Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells,tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.     

SIMS REIN: a multi-source information system for end-stage renal disease

In France, the prevalence of End-Stage Renal Disease (ESRD) is not precisely known. The sources of information are scattered and not coordinated. Consequently, care is ill adapted to meet the demand. The Multi-Source Information System is the basis of the Renal Epidemiology and Information Network (REIN). It is dedicated to improve and organise our medical and epidemiological knowledge of ESRD and to aid public health decision-making in this area. The proposed approach is based on the datawarehouses. This model allows a unified vision of scattered data into distinct databases, for a better management, be it particular (patient follow-up) or global (regional follow-up), with a finality of aid in decision-making. Several categories of problems were considered: the global conception of the information system, the organisation of the datawarehouse, which offers different viewpoints of the data, the integration of heterogeneous data coming from different sources, data exchange and definition of a specific ontology.