Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.     

Ab initio molecular dynamics of the reactivity of vitamin C toward hydroxyl and $$ {HO}_2/{O}_2^{-} $$ radicals

Vitamin C is one of the most abundant exogenous antioxidants in the cell, and it is of the utmost importance to elucidate its mechanism of action against radicals. In this study, the reactivity of vitamin C toward OH and \( {HO}_2/{O}_2^{-} \) radicals in aqueous medium was analyzed by ab initio molecular dynamics using CPMD code. The simulations led to results similar to those of static studies or experiments for the pair of \( {HO}_2/{O}_2^{-} \) radicals but bring new insights for the reactivity with hydroxyl radical: the reaction takes place before the formation of an adduct and consists of two steps: first an electron is transferred to hydroxyl radical and then the ascorbyl radical loses a proton.

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Graphical Abstract Reactivity of vitamin C toward hydroxyl and \( {HO}_2/{O}_2^{-} \) radicals

     

Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection

Helicobacter pylori, a microaerophilic gram-negative bacterium, colonizes the human stomach. About 50% of the world's population is infected, and this infection is considered as the major risk factor for the development of gastric adenocarcinomas in 1% of infected subjects. Carcinogenesis is characterized by the process of epithelial-to-mesenchymal transition (EMT), in the course of which fully differentiated epithelial cells turn into depolarized and migratory cells. Concomitant disruption of adherence junctions (AJ) is facilitated by growth factors like hepatocyte growth factor 1 (HGF-1), but has been also shown to depend on ectodomain shedding of E-cadherin. The aim of this study was to investigate the impact of infection with H. pylori of NCI-N87 gastric epithelial cells on the shedding of E-cadherin and HGF-receptor c-Met. Our results show that infection with H. pylori provokes shedding of the surface proteins c-Met and E-cadherin. Evidence is provided that ADAM10 contributes to the shedding of c-Met and E-cadherin.     

Identification of Bacillus anthracis by Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Artificial Neural Networks

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.Members of the genus Bacillus are rod-shaped bacteria that exhibit catalase activity and can be characterized as endospore-forming obligate or facultative aerobes. The genus Bacillus contains two important groups of bacteria named after B. subtilis and B. cereus. The best-characterized member of the former group is B. subtilis, a renowned model organism for genetic research. Other group members, like B. pumilis, B. licheniformis, B. atrophaeus, and B. amyloliquefaciens, exhibit a high degree of phenotypic similarity and are thus not easily distinguishable (15).The B. cereus group comprises a number of closely related bacteria, some of which interfere with human health. Bacteria classified as B. cereus are occasionally associated with food poisoning (16, 28), while B. thuringiensis is primarily an insect pathogen because of its ability to produce toxins that have been widely used for the biocontrol of insect pests (28, 30). A third member of the B. cereus group, B. anthracis, is the causative agent of anthrax and is highly relevant to human and animal health. Other members of the B. cereus group are B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 15).B. anthracis is a possible agent in biological warfare and bioterrorism. Its applicability as a biological warfare agent was made apparent by an accidental release from a Soviet military facility in Sverdlovsk (1, 10). Also, the well-publicized mailing of B. anthracis spores in the United States, which caused 18 confirmed cases of cutaneous and inhalational anthrax and an additional 4 suspected cases of cutaneous anthrax (3, 22), demonstrated that B. anthracis may become a threat from terrorist groups (10).Rapid detection of B. anthracis may be challenging because of its great genetic similarity to other species of the B. cereus group (10) and the difficulties of phenotypic differentiation of B. cereus group members (15). There is some controversy in the literature regarding the taxonomy of the B. cereus group. Indeed, some authors state that B. anthracis, B. cereus, and B. thuringiensis are one species with various virulence plasmids for the toxin pXO1 and the capsule pXO2 of B. anthracis and the insecticidal toxin of B. thuringiensis (10, 19). Other authors do not support this opinion and suggest the presence of even more species within the group (21).Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) intact-cell mass spectrometry (ICMS) has been suggested as a rapid, objective, and reliable technique for bacterial identification (8, 13, 23, 25, 38). As a proteomic technique, ICMS of whole bacterial cells, or cell lysates, relies on the reproducible detection of microbial protein patterns and thus delivers information complementary to genotypic or phenotypic test methods. With the pattern-matching approach, microbial identification is achieved by comparing experimental mass spectra with a collection of mass spectra of known organisms. This requires the compilation of large databases of bacterial reference spectra but has the advantage that an extensive knowledge of biomarker identities is not required. Another advantage of the pattern-matching approach is that genus- and species-specific procedures or consumables are not required, i.e., the same methodology can in principle be applied to all kinds of microorganisms (multiplex advantage).It is thus believed that ICMS offers the possibility to systematically investigate the diversity of bacterial subproteomes, complementing existing methodologies of bacterial characterization. This potential and the need for a rapid, objective, and reliable microbial identification technique that does not rely on nucleic acid detection and the availability of an MS-compatible inactivation protocol for highly pathogenic biosafety level 3 microorganisms and bacterial endospores (26) prompted us to systematically study the MALDI-TOF MS profiles of Bacillus strains and to establish a database of bacterial mass spectra. In the present work, we describe strategies of spectral analysis that allow the identification and validation of group- and species-specific sets of biomarkers. Using unsupervised hierarchical cluster analysis (UHCA) and supervised artificial neural network (ANN) analysis, we also demonstrate how microbial spectra can be employed to establish an MS-based methodology for rapid, objective, and reliable identification of the target species, B. anthracis.     

A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.     

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Exogenous cortisol facilitates responses to social threat under high provocation

Stress is one of the most important promoters of aggression. Human and animal studies have found associations between basal and acute levels of the stress hormone cortisol and (abnormal) aggression. Irrespective of the direction of these changes - i.e., increased or decreased aggressive behavior - the results of these studies suggest dramatic alterations in the processing of threat-related social information. Therefore, the effects of cortisol and provocation on social information processing were addressed by the present study. After a placebo-controlled pharmacological manipulation of acute cortisol levels, we exposed healthy individuals to high or low levels of provocation in a competitive aggression paradigm. Influences of cortisol and provocation on emotional face processing were then investigated with reaction times and event-related potentials (ERPs) in an emotional Stroop task. In line with previous results, enhanced early and later positive, posterior ERP components indicated a provocation-induced enhanced relevance for all kinds of social information. Cortisol, however, reduced an early frontocentral bias for angry faces and - despite the provocation-enhancing relevance - led to faster reactions for all facial expressions in highly provoked participants. The results thus support the moderating role of social information processing in the ‘vicious circle of stress and aggression’.