Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.     

Conservation status and ex situ cultivation efforts of endemic flora of the Juan Fernández Archipelago

Was realized field studies and ex situ propagation on the vascular flora of Juan Fernández Archipelago during 15 year period. To evaluate the conservation status of a total of 133 species and subspecies of vascular endemic plants I used a IUCN classification founding: 2 species extinct, 1 extinct in it natural habitat, 52 critically endangered, 37 endangered and 9 vulnerable. Thus, 73.8% are contained in a threat category; only 24 taxon can be considered to be of a lesser conservation concern. The largest threat of extinction is a reduction in individuals in local populations resulting in small, isolated populations. This habitat fragmentation and a reduction in endemic flora has also impacted endemic fauna. Besides, during this period was propagated in nurseries a total of 80 of these species and subspecies (60%). It seem clear the necessity to continue to actions conserve this particular ecosystem.     

Nuclear shield: a multi-enzyme task-force for nucleus protection

Background

In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions.

Methodology/Principal Findings

Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization.

Conclusions/Significance

The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes.     

The extended catalysis of glutathione transferase

Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ? [E]. As glutathione transferase lowers the pK_a of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed.     

Developmental stages in diapausing eggs: an investigation across monogonont rotifer species

Monogononts reproduce by parthenogenesis punctuated by events of mixis that generate ‘resting eggs’, which actually are embryos whose development is arrested. We document the nuclei number and position of the resting eggs of nine rotifer species (Brachionus plicatilis, B. calyciflorus, B. manjavacas, Plationus patulus, Epiphanes senta, E. chihuahuaensis, Rhinoglena frontalis, Lecane bulla and Sinantherina socialis) in the attempt to assess the stage at which dormant embryos are arrested. The morphology of the entire embryos was reconstructed visualising nuclear DNA using confocal microscopy, and their developmental stage identified. Two groups of species were identified: dormant embryos of one group possessed on average less than 30 nuclei, with low variation within and between species, and the stage may correspond to early gastrula; embryos in the second group contain relatively more nuclei, averaging around 40–60, with higher variation within species. The two groups of species seem not to reflect any phylogenetic relationship. Consequences of the dormant embryo stages are discussed.     

The role of serotonin in a bdelloid life cycle

Serotonin is a neurotransmitter present throughout animal kingdom that controls a number of crucial functions in different taxa. In this study we document the role of serotonin in a bdelloid rotifer, Macrotrachela quadricornifera. Following life table experimental protocol, we recorded age-specific fecundity and survival rates of cohorts that received WAY 100135 maleate, a selective antagonist of the serotonin receptor 1A. The antagonist was provided from the age of 4 days (age at first reproduction) until death to one cohort group, and during 15 days to another group. Both groups were cultivated regularly, and their life history traits were recorded. Treated rotifers continued to produce eggs, but most eggs were never oviposited and eventually ruptured the mother’s body wall. The retained eggs, being parthenogenetic began embryogenesis and some could hatch inside the mother’s body cavity (pseudocoel). When WAY was suspended, most treated rotifers oviposited all their eggs. About 75% of these eggs developed, but development was 1–2 days longer than in controls. Most newborns reached sexual maturity, although their maturity was delayed as well. Eggs produced after WAY suspension hatched as much as the controls (~100%). WAY possibly did not affect egg production, but prevented regular oviposition. Nevertheless, viability was reduced and development took longer during WAY treatment. Results suggest that in bdelloid rotifers serotonin is involved in the control of egg laying and development, among other features, as it does in most animals, from nematodes to chordates.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Degradation of aromatic hydrocarbons by white-rot fungi in a historically contaminated soil

Phanerochaete chrysosporium NRRL 6361 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in an aged contaminated soil that also contained high concentrations of heavy metals. After 24 days fungal incubation, carbon-CO2 liberated, an indicator of microbial activity, reached a plateau. At the end of the incubation time (30 days), fungal colonization was clearly visible and was confirmed by ergosterol and cell organic carbon determinations. In spite of unfavorable pH (around 7.4) and the presence of heavy metals, both fungi produced Mn-peroxidase activity. In contrast, laccase and aryl-alcohol oxidase were detected only in the soil treated with P. pulmonarius CBS 664.97 and lignin-peroxidase in that with P. chrysosporium NRRL 6361. No lignin-modifying enzyme activities were present in non-inoculated soil incubated for 30 days (control microcosm). Regardless of the fungus employed, a total removal of naphtalene, tetrachlorobenzene, and dichloroaniline isomers, diphenylether and N-phenyl-1-naphtalenamine, was observed. Significant release of chloride ions was also observed in fungal-treated soil, in comparison with that recorded in the control microcosm. Both fungi led to a significant decrease in soil toxicity, as assessed using two different soil contact assays, including the Lepidium sativum L. germination test and the Collembola mortality test.     

A 10-miRNA risk score-based prediction model for pathological complete response to neoadjuvant chemotherapy in hormone receptor-positive breast cancer

Patients with hormone receptor(HR)-positive tumors breast cancer usually experience a relatively low pathological complete response(p CR) to neoadjuvant chemotherapy(NAC). Here, we derived a 10-micro RNA risk score(10-mi RNA RS)-based model with better performance in the prediction of p CR and validated its relation with the disease-free survival(DFS) in 755 HRpositive breast cancer patients(273, 265, and 217 in the training, internal, and external validation sets, respectively). This model,pres...