Specific host genes required for the killing of Klebsiella bacteria by phagocytes

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.     

Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC₅₀ of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.     

Participation of the mitochondrial permeability transition pore in nitric oxide-induced plant cell death

In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)-induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine inhibited growth and caused death in suspension-cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells.     

Antiviral activity of lactoferrin towards naked viruses

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.     

Skin colonization byMalassezia furfur in healthy children up to 15 years old

The prevalence ofM. furfur, a lipodependent fungus, in the skin of 0 to 15-year-old healthy children was studied. Sterilized carpet was used to collect skin samples which were cultured in Oxgall medium (Difco) with 1% olive oil and incubated for 10 days at 37 °C.M. furfur was recovered in 17.8% of infants, with similar findings in both sexes. The highest prevalence ofM. furfur colonization was found among children of 0 to 18 months and 11 to 15-year-olds, with 23.3% and 26.7% respectively.Part of the Ms. thesis of Victor Silva Associação deMalassezia furfur com patologias com componente seborréico.     

Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-d-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the nucleus basalis has not yet been investigated. Here, by means of choline acetyl transferase and NR2B or NR2C double staining, we demonstrate that mice express both the NR2C and NR2B subunits in nucleus basalis cholinergic cells. We generated NR2C-2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C-2B mutant was used to examine whether a subunit exchange in cholinergic neurons would affect acetylcholine (ACh) content in several brain structures. We found increased ACh levels in the frontal cortex and amygdala in the brains of NR2C-2B mutant mice. Brain ACh has been implicated in neuroplasticity, novelty-induced arousal and encoding of novel stimuli. We therefore assessed behavioral habituation to novel environments and objects as well as object recognition in NR2C-2B subunit exchange mice. The behavioral analysis did not indicate any gross behavioral alteration in the mutant mice compared with the wildtype mice. Our results show that the NR2C by NR2B subunit exchange in mice affects ACh content in two target areas of the nucleus basalis.     

A carboxysomal carbon-concentrating mechanism in the cyanelles of the 'coelacanth' of the algal world, Cyanophora paradoxa?

Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.     

Simultaneous study of particle reworking, irrigation transport and reaction rates in sediment bioturbated by the polychaetes Heteromastus and Marenzelleria

The present study employed simultaneously methods to investigate particle and solute transport and reaction rates in sandy sediments inhabited by two worms (2500 m^− 2) with different feeding modes. Heteromastus filiformis is a head-down deposit-feeder and the main activities exerted by this worm are transport of particles as faecal pellets from subsurface to surface sediments and burrow ventilation. Marenzelleria viridis is a surface deposit-feeder that actively searches for food by burrowing near the sediment surface, producing a network of ventilated galleries in this zone. M. viridis exhibited 1.5 to 2.2 times higher particle mixing rates (D_b = 3.3 to 4 × 10^− 3 cm^− 2 d^− 1) compared to H. filiformis. In M. viridis treatments, continuous advection (eddy diffusion) was the major factor influencing solute transport resulting in apparent diffusion rates (D_a = 2.2 cm^− 2 d^− 1), which were 3 times higher than molecular diffusion within the sediment. In H. filiformis inhabited sediments, the transport of solutes was discontinuous and driven by a surprisingly high nonlocal exchange (α = 1.1-1.3 d^− 1), emphasizing its strong irrigation effects. Accordingly, the enhancement of solute fluxes was more pronounced for H. filiformis compared to M. viridis. Depth integrated TCO₂ production derived from diagenetic modelling, which takes into account three reaction zones, is in good agreement with rates obtained from measured fluxes, indicating the applicability of both approaches to get reliable rates. However, the reaction rates showed that the presence of animals had a modest effect on microbial carbon oxidation. The results proved that transport conditions are deeply related to feeding modes. Exchange of solutes was the most important transport process by H. filiformis, while M. viridis affected both mixing and solute transport.