Cytotoxicity of Bacterial Metabolic Products, including Listeriolysin O, on Leukocyte Targets

Bacterial toxins can exhibit anticancer activities. Here we investigated the anticancer effects of the listeriolysin O toxin produced by Listeria monocytogenes. We found that supernatants of Listeria monocytogenes strains (wild type, 1189, and 1190) were cytotoxic to the Jurkat cell line and human peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. The supernatant of strain 1044, not producing listeriolysin O, was inactive. The supernatants of Listeria strains were also cytotoxic toward B cells of chronic leukemia patients, with no significant differences in activities between strains. We also tested supernatants of Bacillus subtilis strains BR1-90, BR1-S, and BR1-89 producing listeriolysin O. BR1-S and BR1-89 were cytotoxic to PBMC and to Jurkat cells, the latter being more sensitive to the supernatants. BR1-90 was not hemolytic or cytotoxic to PBMC, but was cytotoxic to Jurkat cells in the concentration range of 10–30%, suggesting that listeriolysin O is selectively effective against T cells. Overall, the response of human peripheral blood mononuclear and human leukemia cell lines to bacteria supernatants containing listeriolysin O depended on the bacteria strain, target cell type, and supernatant concentration

The slow-releasing and mitochondria-targeted hydrogen sulfide (H2s) delivery molecule ap39 induces brain tolerance to ischemia

This is the final version. Available on open access from MDPI via the DOI in this recordIschemic stroke is the third leading cause of death in the world, which accounts for almost 12% of the total deaths worldwide. Despite decades of research, the available and effective pharmacotherapy is limited. Some evidence underlines the beneficial properties of hydrogen sulfide (H2S) donors, such as NaSH, in an animal model of brain ischemia and in in vitro research; however, these data are ambiguous. This study was undertaken to verify the neuroprotective activity of AP39, a slow-releasing mitochondria-targeted H2S delivery molecule. We administered AP39 for 7 days prior to ischemia onset, and the potential to induce brain tolerance to ischemia was verified. To do this, we used the rat model of 90-min middle cerebral artery occlusion (MCAO) and used LC-MS/MS, RT-PCR, Luminex™ assays, Western blot and immunofluorescent double-staining to determine the absolute H2S levels, inflammatory markers, neurotrophic factor signaling pathways and apoptosis marker in the ipsilateral frontal cortex, hippocampus and in the dorsal striatum 24 h after ischemia onset. AP39 (50 nmol/kg) reduced the infarct volume, neurological deficit and reduced the microglia marker (Iba1) expression. AP39 also exerted prominent anti-inflammatory activity in reducing the release of Il-1β, Il-6 and TNFα in brain areas particularly affected by ischemia. Furthermore, AP39 enhanced the pro-survival pathways of neurotrophic factors BDNF-TrkB and NGF-TrkA and reduced the proapoptotic proNGF-p75NTR-sortilin pathway activity. These changes corresponded with reduced levels of cleaved caspase 3. Altogether, AP39 treatment induced adaptative changes within the brain and, by that, developed brain tolerance to ischemia.National Science Center, PolandMedical Research Council (MRC

Effects of Cocaine-Kindling on the Expression of NMDA Receptors and Glutamate Levels in Mouse Brain

In the present study we examined the effects of cocaine seizure kindling on the expression of NMDA receptors and levels of extracellular glutamate in mouse brain. Quantitative autoradiography did not reveal any changes in binding of [3H] MK-801 to NMDA receptors in several brain regions. Likewise, in situ hybridization and Western blotting revealed no alteration in expression of the NMDA receptor subunits, NR1 and NR2B. Basal overflow of glutamate in the ventral hippocampus determined by microdialysis in freely moving animals also did not differ between cocaine-kindled and control groups. Perfusion with the selective excitatory amino acid transporter inhibitor, pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mM), increased glutamate overflow confirming transport inhibition. Importantly, KCl-evoked glutamate overflow under tPDC perfusion was significantly higher in cocaine-kindled mice than in control mice. These data suggest that enhancement of depolarization stimulated glutamate release may be one of the mechanisms underlying the development of increased seizure susceptibility after cocaine kindling

Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level

<p>Abstract</p> <p>Background</p> <p><it>Rhizopus oryzae </it>is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses.</p> <p>Results</p> <p>Carbohydrate Active enzyme (CAZy) annotation of the <it>R. oryzae </it>identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs) and a high number of glycosyl transferases (GTs) and carbohydrate esterases (CEs). A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars), chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of <it>R. oryzae </it>to its environment.</p> <p>Conclusions</p> <p>CAZy analyses of the genome of the zygomycete fungus <it>R. oryzae </it>and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.</p

Notes for genera: basal clades of Fungi (including Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota)

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1--20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced

Analysis of the interaction between tomato torrado virus proteins using the yeast two-hybrid system

Ten years ago for the first time the new picorna-like virus species - Tomato torrado virus (ToTV) - was found and described on tomato plants. The isolates of this pathogen were reported in Europe, America, and Oceania including Australia. Because of its unique biological and molecular features, ToTV was classified to the new genus Torradovirus, in the Secoviridae family. In Poland, three isolates: Wal’03, Kra, and Ros ToTV were identified on greenhouse tomato cultivars. At present, the biology and the genome structure of this virus are characterised. But there is no data extending beyond the bioinformatics analyses about the function of viral proteins, polyproteins, and non-coding sequences, as well as possible interactions between viral, host and vector factors that may be important for the infection process, encapsidation, transport in plants, and transmission. In this study, we have undertaken a search for the possible protein-protein interaction of ToTV encoded proteins using the yeast two-hybrid (Y2H) system. The viral genome fragments covering full sequences for nine known proteins of ToTV were amplified using specific primers with characteristic recombination sites. This process enabled the construction of basic entry clones for each protein that further facilitated manipulations with prepared constructs using Gateway technology. Two-hybrid assays were performed in the yeast strain and tested interactions of ToTV proteins were analysed in several combinations using auxotrophy markers. Our analyses did not reveal the presence of interactions between ToTV domains. Surprisingly, no interactions were found in the case of various CP subunits as well as between CP subunits and 3A protein, that in some virus families are known to play a role in viral life cycle. This role includes virion assembly or cell-to-cell transport. The lack of interactions may be a result of the limitation of this experimental system, or suggest that these proteins may interact indirectly, or require the presence of genomic RNAs or some host factors