Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.     

Localization of genes for bacterial canker resistance in Lycopersicon peruvianum using RFLPs

A backcross population of the L. peruvianum accession LA 2157, which is resistant to bacterial canker caused by Clavibacter michiganensis ssp. michiganensis, with the susceptible L. peruvianum accession LA 2172 was evaluated for the segregation of C. michiganenis resistance and of RFLP markers in order to map the loci involved in this resistance. The development of symptoms of the disease was scored using an ordinal scale. The mapping of the disease resistance was hampered by distorted segregation ratios of a large number of markers and unexpected quantitative inheritance of the resistance. By means of the Kruskal-Wallis rank-sum test, five regions on chromosomes 1, 6, 7, 8 and 10 were identified that may be involved in C. michiganensis resistance.     

The origin of the pelagobenthic metazoan life cycle: what's sex got to do with it?

The biphasic (pelagobenthic) life cycle is found throughoutthe animal kingdom, and includes gametogenesis, embryogenesis,and metamorphosis. From a tangled web of hypotheses on the originand evolution of the metazoan pelagobenthic life cycle, currentopinion appears to favor a simple, larval-like holopelagic ancestorthat independently settled multiple times to incorporate a benthicphase into the life cycle. This hypothesis derives originallyfrom Haeckel's (1874) Gastraea theory of ontogeny recapitulatingphylogeny, in which the gastrula is viewed as the recapitulationof a gastraean ancestor that evolved via selection on a simple,planktonic hollow ball of cells to develop the capacity to feed.Here, we propose an equally plausible hypothesis that the originof the metazoan pelagobenthic life cycle was a direct consequenceof sexual reproduction in a likely holobenthic ancestor. Indoing so, we take into account new insights from poriferan developmentand from molecular phylogenies. In this scenario, the gastruladoes not represent a recapitulation, but simply an embryologicalstage that is an outcome of sexual reproduction. The embryocan itself be considered as the precursor to a biphasic lifestyle,with the embryo representing one phase and the adult anotherphase. This hypothesis is more parsimonious because it precludesthe need for multiple, independent origins of the benthic form.It is then reasonable to consider that multilayered, ciliatedembryos ultimately released into the water column are subjectto natural selection for dispersal/longevity/feeding that setsthem on the evolutionary trajectory towards the crown metazoanplanktonic larvae. These new insights from poriferan developmentthus clearly support the intercalation hypothesis of bilaterianlarval evolution, which we now believe should be extended todiscussions of the origin of biphasy in the metazoan last commonancestor.     

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.     

Understanding photosynthetic biofilm productivity and structure through 2D simulation

We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 μm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass.     

ITS1 region of the rDNA of Pythium proliferatum, a new species: its taxonomy and its comparison with related species

A new species, Pythium proliferatum (F-382), was isolated from soil samples taken in Genlis in the burgundian region of France. The fungus is unique because of the character combinations of its large, spherical to elongated, proliferating sporangia, and its smooth walled oogonia supplied with different types of antheridia like hypogynous, monoclinous sessile, monoclinous stalked, diclinous and wrapping around the oogonia. Almost all types of antheridia that are found in the genus are present in this new species. Morphological features of this new species, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are discussed in this article.     

Diabetic β-Cells Can Achieve Self-Protection against Oxidative Stress through an Adaptive Up-Regulation of Their Antioxidant Defenses

Background

Oxidative stress (OS), through excessive and/or chronic reactive oxygen species (ROS), is a mediator of diabetes-related damages in various tissues including pancreatic β-cells. Here, we have evaluated islet OS status and β-cell response to ROS using the GK/Par rat as a model of type 2 diabetes.

Methodology/Principal Findings

Localization of OS markers was performed on whole pancreases. Using islets isolated from 7-day-old or 2.5-month-old male GK/Par and Wistar control rats, 1) gene expression was analyzed by qRT-PCR; 2) insulin secretion rate was measured; 3) ROS accumulation and mitochondrial polarization were assessed by fluorescence methods; 4) antioxidant contents were quantified by HPLC. After diabetes onset, OS markers targeted mostly peri-islet vascular and inflammatory areas, and not islet cells. GK/Par islets revealed in fact protected against OS, because they maintained basal ROS accumulation similar or even lower than Wistar islets. Remarkably, GK/Par insulin secretion also exhibited strong resistance to the toxic effect of exogenous H₂O₂ or endogenous ROS exposure. Such adaptation was associated to both high glutathione content and overexpression (mRNA and/or protein levels) of a large set of genes encoding antioxidant proteins as well as UCP2. Finally, we showed that such a phenotype was not innate but spontaneously acquired after diabetes onset, as the result of an adaptive response to the diabetic environment.

Conclusions

The GK/Par model illustrates the effectiveness of adaptive response to OS by β-cells to achieve self-tolerance. It remains to be determined to what extend such islet antioxidant defenses upregulation might contribute to GK/Par β-cell secretory dysfunction.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.