Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

Analysis of gene expression in operons of Streptomyces coelicolor

Background  

Recent studies have shown that microarray-derived gene-expression data are useful for operon prediction. However, it is apparent that genes within an operon do not conform to the simple notion that they have equal levels of expression.     

The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay

Salmonella species trigger host membrane ruffling to force their internalization into non-phagocytic intestinal epithelial cells. This requires bacterial effector protein delivery into the target cell via a type III secretion system. Six translocated effectors manipulate cellular actin dynamics, but how their direct and indirect activities are spatially and temporally co-ordinated to promote productive cytoskeletal rearrangements remains essentially unexplored. To gain further insight into this process, we applied mechanical cell fractionation and immunofluorescence microscopy to systematically investigate the subcellular localization of epitope-tagged effectors in transiently transfected and Salmonella-infected cultured cells. Although five effectors contain no apparent membrane-targeting domains, all six localized exclusively in the target cell plasma membrane fraction and correspondingly were visualized at the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated effector pool was detectable in the cell cytosol. Using parallel in vitro assays, we demonstrate that the prenylated cellular GTPase Cdc42 is necessary and sufficient for membrane association of the Salmonella GTP exchange factor and GTPase-activating protein mimics SopE and SptP, which have no intrinsic lipid affinity. The data show that the host plasma membrane is a critical interface for effector-target interaction, and establish versatile systems to further dissect effector interplay.     

The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology

An essential early event in Shigella and Salmonella pathogenesis is invasion of non-phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full-length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N-terminal domain predicted to form a trimeric coiled-coil, and is predominantly alpha-helical. Upon lipid interaction, two transmembrane domains (residues 313-333 and 399-419) penetrate the bilayer, allowing the intervening hydrophilic region (334-398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB-derived 162 residue alpha-helical polypeptide (IpaB(418-580)) is a potent inhibitor of IpaB-directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10(-8) M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB(418-580) failed to prevent the contact-dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact-dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.     

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide

Aims: We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. Methods: Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNF, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. Results: Isolated KC produced a basal amount of TNF, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNF (P<0.001), IL-6 (P<0.01), IL-10 (P<0.001), IL-12 (P<0.01), and NO (P<0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P<0.05) and increased IL-13 (P<0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNF production (P<0.05), without modifying TNF mRNA expression and decreased iNOS expression and NO (P≈0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. Conclusions: These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

Functional analysis of an arthritogenic synovial fibroblast

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.     

Genetic and molecular coordinates of neuroendocrine lung tumors,with emphasis on small-cell lung carcinomas

The aim of this review is to present the advances in our understanding of the progression of tumorigenesis in neuroendocrine lung tumors. Current information on established and putative diagnostic and prognostic markers of neuroendocrine tumors are evaluated, with a special reference to small-cell lung carcinoma, due to its higher incidence and aggressive behavior. The genetic and molecular changes that accompany these neoplasms are highlighted, and factors that influence cell-cycle progression, apoptosis, drug resistance, and escape from immune surveillance are critically assessed.