Retrospective study on necrotizing enteritis in piglets in Switzerland

The re-emergence of necrotizing enteritis (NE) in Swiss pig breeding farms raised concern that, besides C. perfringens type C strains, additional C. perfringens toxinotypes might cause this disease. Therefore we retrospectively investigated the association of NE with C. perfringens type C or different C. perfringens toxinotypes. We evaluated pathological lesions, routine diagnostic bacteriology results, and multiplex real-time PCR analyses from DNA extracts of archived intestinal samples of 199 piglets from our diagnostic case load. 96.5% of NE cases and 100% of herds affected by NE were positive for C. perfringens type C genotypes. Animals without necrotizing enteritis revealed a significantly lower detection rate of type C genotypes. Non affected piglets showed a high prevalence for beta-2-toxin positive C. perfringens type A strains. Collectively, our data indicate that outbreaks of NE in piglets in Switzerland cannot be attributed to newly emerging pathogenic toxinotypes, but are due to a spread of pathogenic C. perfringens type C strains.

Low resolution brain electromagnetic tomography (LORETA) functional imaging in acute, neuroleptic-naive, first-episode, productive schizophrenia

Functional imaging of brain electrical activity was performed in nine acute, neuroleptic-naive, first-episode, productive patients with schizophrenia and 36 control subjects. Low-resolution electromagnetic tomography (LORETA, three-dimensional images of cortical current density) was computed from 19-channel of electroencephalographic (EEG) activity obtained under resting conditions, separately for the different EEG frequencies. Three patterns of activity were evident in the patients: (1) an anterior, near-bilateral excess of delta frequency activity; (2) an anterior-inferior deficit of theta frequency activity coupled with an anterior-inferior left-sided deficit of alpha-1 and alpha-2 frequency activity; and (3) a posterior-superior right-sided excess of beta-1, beta-2 and beta-3 frequency activity. Patients showed deviations from normal brain activity as evidenced by LORETA along an anterior-left-to-posterior-right spatial axis. The high temporal resolution of EEG makes it possible to specify the deviations not only as excess or deficit, but also as inhibitory, normal and excitatory. The patients showed a dis-coordinated brain functional state consisting of inhibited prefrontal/frontal areas and simultaneously overexcited right parietal areas, while left anterior, left temporal and left central areas lacked normal routine activity. Since all information processing is brain-state dependent, this dis-coordinated state must result in inadequate treatment of (externally or internally generated) information.

Smell and taste of chewing gum affect frequency domain EEG source localizations

We investigated brain electric field signatures of subjective feelings after chewing regular gum or gum base without flavor. 19-channel eyes-closed EEG from 20 healthy males before and after 5 minutes of chewing the two gum types in random sequence was source modeled in the frequency domain using the FFT-Dipole-Approximation. 3-dimensional brain locations and strengths (Global Field Power, GFP) of the equivalent sources of five frequency bands were computed as changes from pre-chewing baseline. Gum types differed (ANOVA) in pre-post changes of source locations for the alpha-2 band (to anterior and right after regular gum, opposite after gum base) and beta-2 band (to anterior and inferior after regular gum, opposite after gum base), and of GFP for delta-theta, alpha-2 and beta-1 (regular gum: increase, gum base: decrease). Subjective feeling changed to more positive values after regular gum than gum base (ANOVA).—Thus, chewing gum with and without taste-smell activates different brain neuronal populations.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

Zazen meditation and no-task resting EEG compared with LORETA intracortical source localization

Meditation is a self-induced and willfully initiated practice that alters the state of consciousness. The meditation practice of Zazen, like many other meditation practices, aims at disregarding intrusive thoughts while controlling body posture. It is an open monitoring meditation characterized by detached moment-to-moment awareness and reduced conceptual thinking and self-reference. Which brain areas differ in electric activity during Zazen compared to task-free resting? Since scalp electroencephalography (EEG) waveforms are reference-dependent, conclusions about the localization of active brain areas are ambiguous. Computing intracerebral source models from the scalp EEG data solves this problem. In the present study, we applied source modeling using low resolution brain electromagnetic tomography (LORETA) to 58-channel scalp EEG data recorded from 15 experienced Zen meditators during Zazen and no-task resting. Zazen compared to no-task resting showed increased alpha-1 and alpha-2 frequency activity in an exclusively right-lateralized cluster extending from prefrontal areas including the insula to parts of the somatosensory and motor cortices and temporal areas. Zazen also showed decreased alpha and beta-2 activity in the left angular gyrus and decreased beta-1 and beta-2 activity in a large bilateral posterior cluster comprising the visual cortex, the posterior cingulate cortex and the parietal cortex. The results include parts of the default mode network and suggest enhanced automatic memory and emotion processing, reduced conceptual thinking and self-reference on a less judgmental, i.e., more detached moment-to-moment basis during Zazen compared to no-task resting.

Accelerated discovery of novel benzodiazepine ligands by experiment-guided virtual screening.

High throughput discovery of ligand scaffolds for target proteins can accelerate development of leads and drug candidates enormously. Here we describe an innovative workflow for the discovery of high affinity ligands for the benzodiazepine-binding site on the so far not crystallized mammalian GABAA receptors. The procedure includes chemical biology techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine-reactive groups, knowledge of amino acid residues contributing to the drug-binding pocket, and crystal structures either of proteins homologous to the target protein or, better, of the target itself. Part of the protocol is virtual screening that without additional rounds of optimization in many cases results only in low affinity ligands, even when a target protein has been crystallized. Here we show how the integration of functional data into structure-based screening dramatically improves the performance of the virtual screening. Thus, lead compounds with 14 different scaffolds were identified on the basis of an updated structural model of the diazepam-bound state of the GABAA receptor. Some of these compounds show considerable preference for the α3β2γ2 GABAA receptor subtype.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction

G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.