Identification of a cross-reactive HLA-DRB1*0301-restricted CD4 T cell response directed against cholesterol-binding cytolysins from two different pathogens

Cholesterol-binding cytolysins constitute an evolutionarily conserved family of pore-forming proteins expressed by different gram-positive pathogens. Listeriolysin O, one well-characterized member of the cytolysin family, is also known to induce specific CD4 and CD8 T cell responses upon infection of mice with Listeria monocytogenes. Here we describe an HLA-DRB1*0301-restricted listeriolysin O-derived T cell epitope that is conserved among several members of the cytolysin family. An HLA-DRB1*0301-restricted CD4+ T cell line, established from spleen lymphocytes of L. monocytogenes-infected HLA-DRB1*0301-transgenic mice, cross-reacted with a homologous peptide from perfringolysin O, a cytolysin expressed by Clostridium perfringens. Ex vivo analysis of infected mice revealed an even broader cross-reaction of T cells with homologous peptides derived from perfringolysin O, streptolysin O, and cereolysin O. Interestingly, a cross-reactive memory CD4+ T cell response against the homologous peptides derived from listeriolysin O and perfringolysin O could also be detected in the blood from healthy HLA-DRB1*0301+ human donors. Remarkably, this response was even present in donors who did not exhibit a memory T cell reactivity against a second, non-conserved HLA-DRB1*0301-restricted LLO-derived CD4 T cell epitope, suggesting that cytolysin-producing bacteria other than L. monocytogenes can stimulate a cross-reactive cytolysin-specific immunity.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Taxonomy and distribution of Poa carpatica in the Western Carpathians

We examined the taxonomic status and distribution of Poa populations from supramontane and subalpine belt of the central Western Carpathians, so far classified as P. nemoralis subsp. carpatica and P. nemoralis subsp. montana. Significant morphological differences from P. nemoralis s. str. as well as combination of shared vs distinct characters allow us to attribute the populations under study to the species P. carpatica (V. Jirásek) Chopyk with two subspecies: P. carpatica subsp. carpatica a P. carpatica subsp. supramontana subsp. nova.     

Observation of single-stranded DNA on mica and highly oriented pyrolytic graphite by atomic force microscopy

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg(2+), by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.     

Crystal structure of the PP2A phosphatase activator: implications for its PP2A-specific PPIase activity

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all alpha-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity.     

Rate constants of oxidation of unsaturated fatty esters studied by chemiluminescence

This study deals with oxidation kinetics of three unsaturated fatty esters: methyl oleate, methyl linoleate and methyl linolenate at temperatures ranging from 90 to 150 °C. The reaction was monitored by chemiluminescence. A kinetic model was derived from a simple mechanistic scheme, in which initiation is due to hydroperoxides decomposition, whereas propagation results from the abstraction of the most labile hydrogen and termination results only from the bimolecular combination of peroxyl radicals. Analysis of induction period duration indicated that hydroperoxides mainly decompose by a bimolecular process. The model well predicts the main features of the experimental chemiluminescence curves. Kinetic parameters of the three unsaturated fatty esters were assessed from inverse method and discussed.     

Draft genome sequence of four coccolithoviruses: Emiliania huxleyi virus EhV-88, EhV-201, EhV-207, and EhV-208

The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome.     

Growth-inhibition effects of pacifastin-like peptides on a pest insect: the desert locust, Schistocerca gregaria

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI. Nevertheless, the growth inhibition effect of this PI mixture attenuated after a few days, Moreover, a comprehensive study of the observed responses after oral administration of PI revealed that S. gregaria larvae can adjust their secreted digestive enzyme activities in two distinct ways depending on the composition/concentration of the PI-mixture.     

Prey-induced changes in the accumulation of amino acids and phenolic metabolites in the leaves of <Emphasis Type="Italic">Drosera capensis</Emphasis> L

Effect of prey feeding (ants Formica fusca) on the quantitative changes in the accumulation of free amino acids, soluble proteins, phenolic metabolites and mineral nutrients in the leaves of carnivorous plant Drosera capensis was studied. Arginine was the most abundant compound in Drosera leaves, while proline was abundant in ants. The amount of the majority of amino acids and their sum were elevated in the fed leaves after 3 and 21 days, and the same, but with further enhancement after 21 days, was observed in ants. Accumulation of amino acids also increased in young non-fed leaves of fed plants. Soluble proteins decreased in ants, but were not enhanced in fed leaves. This confirms the effectiveness of sundew’s enzymatic machinery in digestion of prey and suggests that amino acids are not in situ deposited, but rather are allocated within the plant. The content of total soluble phenols, flavonoids and two selected flavonols (quercetin and kaempferol) was not affected by feeding in Drosera leaves, indicating that their high basal level was sufficient for the plant’s metabolism and prey-induced changes were mainly N based. The prey also showed to be an important source of other nutrients besides N, and a stimulation of root uptake of some mineral nutrients is assumed (Mg, Cu, Zn). Accumulation of Ca and Na was not affected by feeding.