Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.     

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.     

Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8(+) T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4(+) T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.     

No contribution of GSTM1 and GSTT1 null genotypes to the risk of neutropenia due to benzene exposure in Southeastern Brazil

Exposure to benzene has been associated with haematological diseases such as neutropenia (NEB) and acute myeloid leukaemia (AML). We tested whether the null genotypes of the GSTM1 and GSTT1 genes, involved in benzene inactivation, altered the risk for NEB in southeastern Brazil. Genomic DNA from 55 NEB patients and 330 controls was analysed by multiplex-polymerase chain reaction. The frequency of the GSTM1, GSTT1 and combined null genotypes was similar in patients and controls (GSTM1, 27.3% vs. 38.8%, p = 0.16; GSTT1, 25.5% vs. 19.7%, p = 0.24; GSTM1/GSTT1, 12.7% vs. 6.7%, p = 0.26; respectively). The distribution of genotype classes in NEB patients was similar to normal controls, suggesting that GSTM1 and GSTT1 null genotypes make no specific contribution to the risk of NEB. As the GSTM1 and GSTT1 null genotypes were previously associated with increased risk for AML in Brazil and elsewhere, we hypothesise that different thresholds of chemical exposure relative to distinct GSTM1 and GSTT1 genotypes may determine whether AML or NEB manifests in benzene exposed individuals from southeastern Brazil. Although indicative, our results still require support by prospective and large scale epidemiological studies, with rigorous assessment of daily chemical exposures and control of the possible contribution of other polymorphic genes involved in benzene metabolism.     

Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.     

Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum

The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.     

Gene expression patterns to define stages of post-harvest senescence in Alstroemeria petals

Petal senescence in many species is regulated by ethylene but some flowers, such as those on the monocotyledonous plant Alstroemeria, var. Rebecca are ethylene insensitive. Changes in gene expression during the post-harvest senescence of Alstroemeria flowers were investigated using several different techniques. Suppressive subtractive hybridization (SSH) was used to obtain cDNA libraries enriched for genes expressed at selected stages of petal senescence. Sequencing of the EST clones obtained resulted in over 1000 sequences that represent approximately 500 different genes. Analysis of the potential functions of these genes provides a snapshot of the processes that are taking place during petal development. Both cell wall related genes and genes involved in metabolism were present at a higher proportion in the earlier stages. Genes encoding metal binding proteins (mostly metallothionein-like) were the major component of senescence enhanced libraries. This limited the diversity of genes identified showing differential expression at the later stages. Changes in the expression of all genes were analysed using microarray hybridization, and genes showing either up or down-regulation were identified. The expression pattern of a selection of genes was confirmed using Northern hybridization. Northern hybridization confirmed the up-regulation of metallothioneins after floral opening, however, this was not detected by the microarray analysis, indicating the importance of using a combination of methods to investigate gene expression patterns. Considerably more genes were up-regulated than down-regulated. This may reflect the need during Alstroemeria petal senescence for the expression of a whole new set of genes involved with degradation and mobilization. The potential uses of expression profiling to improve floral quality in breeding programmes or as a diagnostic tool are discussed.