The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.     

Electrochemical Polarization-Induced Changes in the Growth of Individual Cells and Biofilms of Pseudomonas fluorescens (ATCC 17552)

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At −0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 μm min⁻¹, rendering daughter cells that reached up to 3.8 μm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 ± 7 min. Eight-hour-old biofilms at −0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 ± 8 min, cells elongated at a lower rate (up to 0.08 μm min⁻¹), rendering shorter daughters (2.5 ± 0.5 μm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at −0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

EARLY TOXIC EFFECTS OF ZINC ON PSII OF SYNECHOCYSTIS AQUATILIS F. AQUATILIS (CYANOPHYCEAE)1

Zinc toxicity on photosynthetic activity in cells of Synechocystis aquatilis f. aquatilis Sauvageau was investigated by monitoring Hill activity and fluorescence. The oxygen‐evolving activity decreased to about 80% of the initial value after exposure to 0.1 mM ZnSO₄ for 1 h. The PSII activity was inhibited by 40% in the presence of zinc concentrations ranging from 0.5 to 5.0 mM, suggesting that the metal effect is limited by zinc uptake. The fluorescence capacity (F_max–F/F_max) decreased from 0.57 to 0.35 and 0.20 in Zn‐treated cells for 15 and 60 min, respectively, thus providing evidence for rapid inactivation of electron transport at PSII. Zinc treatment promoted a rapid increase in PSII fluorescence that was counteracted by addition of 1,4‐benzoquinone, indicating that electron transfer at the reducing side of the PSII reaction center is arrested by zinc. Furthermore, a decline in the fluorescence yield could be observed after 1 h of zinc treatment as well as when Zn‐treated cells were excited in presence of 3‐(3′,4′‐dichlorophenyl)‐1,1‐dimethylurea. Under these conditions, zinc did not affect energy transfer from phycobilisomes to PSII, and the gradual quenching of PSII fluorescence may be due to a decrease in electron flow on the donor side of PSII. However, the 20% increase in the minimal fluorescence intensity (F_o) in parallel to the absence of changes in the maximal fluorescence intensity (F_max), observed in the first hour of zinc treatment, could also suggest a metal‐induced decline in the energy transfer from PSII‐chl a antenna to the PSII reaction center.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

Resource efficiency and economic implications of alternatives to surgical castration without anaesthesia

This paper presents an analysis of the economic implications of alternative methods to surgical castration without anaesthesia. Detailed research results on the economic implications of four different alternatives are reported: castration with local anaesthesia, castration with general anaesthesia, immunocastration and raising entire males. The first three alternatives have been assessed for their impact on pig production costs in the most important pig-producing Member States of the EU. The findings on castration with anaesthesia show that cost differences among farms increase if the anaesthesia cannot be administered by farmers and when the veterinarian has to be called to perform it. The cost of veterinarian service largely affects the total average costs, making this solution economically less feasible in small-scale pig farms. In all other farms, the impact on production costs of local anaesthesia is however limited and does not exceed 1 €ct per kg. General anaesthesia administered by inhalation or injection of Ketamin in combination with a sedative (Azaperone, Midazolan) is more expensive. These costs depend heavily on farm size, as the inhalation equipment has to be depreciated on the largest number of pigs possible. The overall costs of immunocastration - including the cost of the work load for the farmer - has to be evaluated against the potential benefits derived from higher daily weight gain and feed efficiency in comparison with surgical castrates. The economic feasibility of this practice will finally depend on the price of the vaccine and on consumer acceptance of immunocastration. The improvement in feed efficiency may compensate almost entirely for the cost of vaccination. The main advantages linked to raising entire males are due to the higher efficiency of feed conversion, to the better growth rate and to the higher leanness of carcass. A higher risk of boar taint on the slaughter line has to be accounted for. Raising entire males should not generate more than 2.5% of boar taint among slaughter pigs, in order to maintain the considerable economic benefits of better feed efficiency of entire males with respect to castrates.     

Deletion of sigB in Bacillus cereus affects spore properties

In Bacillus cereus and other gram-positive bacteria the alternative sigma factor sigma(B) is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.