Assessment of Botulinum a Toxin High Affinity SV2 Receptors on Normal Human Urothelial Cells.

The clinical use of Botulinum A toxin (BoNT/A) is based on its ability to block the vesicular release of Acetilcholine (Ach) and other neurotransmitters at the level of the neuronal plasmatic membrane. This process requires the internalization of the neurotoxin within the target cell, what it happens by means of the binding with high affinity receptors. These receptors are the synaptic vesicle proteins type 2 (SV2). To date three different types of SV2 receptors have been identified in synaptic structures and endocrine tissues

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line.

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells

Reducing the Risk of Intraoperative Neurological Complications during Carotid Endarterectomy with Early Distal Control of the Internal Carotid Artery

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Neurophysiological and neuroradiological test for early poor outcome (Cerebral Performance Categories 3\u20135) prediction after cardiac arrest: Prospective multicentre prognostication data

The data presented here are related to our research article entitled \u201cNeurophysiology and neuroimaging accurately predict poor neurological outcome within 24 hours after cardiac arrest: a prospective multicentre prognostication study (ProNeCA)\u201d [1]. We report a secondary analysis on the ability of somatosensory evoked potentials (SEPs), brain computed tomography (CT) and electroencephalography (EEG) to predict poor neurological outcome at 6 months in 346 patients who were comatose after cardiac arrest. Differently from the related research article, here we included cerebral performance category (CPC) 3 among poor outcomes, so that the outcomes are dichotomised as CPC 1\u20132 (absent to mild neurological disability: good outcome) vs. CPC 3\u20135 (severe neurological disability, persistent vegetative state, or death: poor outcome). The accuracy of the index tests was recalculated accordingly. A bilaterally absent/absent-pathological amplitude (AA/AP) N20 SEPs wave, a Grey Matter/White Matter (GM/WM) ratio <1.21 on brain CT and an isoelectric or burst suppression EEG predicted poor outcome with 49.6%, 42.2% and 29.8% sensitivity, respectively, and 100% specificity. The distribution of positive results of the three predictors did not overlap completely in the population of patients with poor outcome, so that when combining them the overall sensitivity raised to 61.2%

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

<p>Abstract</p> <p>Background</p> <p>Conflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.</p> <p>Methods</p> <p>The rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.</p> <p>Results</p> <p>Initial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.</p> <p>Conclusion</p> <p>CAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.</p

Highly Regio- and Stereoselective Diels-Alder Cycloadditions via Two-Step and Multicomponent Reactions Promoted by Infrared Irradiation under Solvent-Free Conditions

Infrared irradiation promoted the Diels-Alder cycloadditions of exo-2-oxazolidinone dienes 1–3 with the Knoevenagel adducts 4–6, as dienophiles, leading to the synthesis of new 3,5-diphenyltetrahydrobenzo[d]oxazol-2-one derivatives (7, 9, 11 and 13–17), under solvent-free conditions. These cycloadditions were performed with good regio- and stereoselectivity, favoring the para-endo cycloadducts. We also evaluated the one-pot three-component reaction of active methylene compounds 20, benzaldehydes 21 and exo-2-oxazolidinone diene 2 under the same reaction conditions. A cascade Knoevenagel condensation/Diels-Alder cycloaddition reaction was observed, resulting in the final adducts 13–16 in similar yields. These procedures are environmentally benign, because no solvent and no catalyst were employed in these processes. The regioselectivity of these reactions was rationalized by Frontier Molecular Orbital (FMO) calculations