The gut microbiota in the metagenomics era: sometimes a friend, sometimes a foe

The normal intestinal microflora (microbiota) represents a complex, dynamic, and diverse collection of microorganisms, which usually inhabit the gastrointestinal tract. Normally, between this flora and the human host a mutually beneficial long-term symbiotic relationship is established, where the host contributes essential nutrients necessary for the survival of the microbiota and the latter fulfils multiple roles in host nutrition and development. Several achievements have recently converged to renew interest in studying the normal gut microbiota: the development of molecular methods of studying the microbial communities, the improved understanding of host-microbe interactions in health and disease, and the potential for therapeutic manipulation of the microbiota. We present recent data concerning the molecular technologies of studying the microbiota and new findings regarding the composition of the normal flora. We underline the beneficial activities of the gut flora on the human host. We emphasize the recent findings in the alterations of the microbiota in various medical conditions (celiac disease, irritable bowel syndrome, obesity, colorectal cancer, allergic disorders, and especially inflammatory bowel diseases). The results of these new studies suggest that changes of the microbiota could be linked to the etiopathogenesis of these diseases. These outstanding findings could be used for further diagnostic tools and/or therapy.     

Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.     

Phloem transport of sulfur in Ricinus

Mature leaves of Ricinus communis fed with ³⁵SO ₄ ^2- in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of ³⁵S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the ³⁵S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.Abbreviations CySH cysteine - GSH glutathione - GSSG glutathione disulfide - NEM N-ethylmaleimide - CyS-SCy cystine     

Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter , Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae . Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacte r sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs . Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.