Epigallocatechin gallate inhibits endothelial exocytosis

Consumption of green tea is associated with a decrease in cardiovascular mortality. The beneficial health effects of green tea are attributed in part to polyphenols, organic compounds found in tea that lower blood pressure, reduce body fat, decrease LDL cholesterol, and inhibit inflammation. We hypothesized that epigallocatechin gallate (EGCG), the most abundant polyphenol in tea, inhibits endothelial exocytosis, the initial step in leukocyte trafficking and vascular inflammation. To test this hypothesis, we treated human umbilical-vein endothelial cells with EGCG and other polyphenols, and then measured endothelial exocytosis. We found that EGCG decreases endothelial exocytosis in a concentration-dependent manner, with the effects most prominent after 4 h of treatment. Other catechin polyphenols had no effect on endothelial cells. By inhibiting endothelial exocytosis, EGCG decreases leukocyte adherence to endothelial cells. In searching for the mechanism by which EGCG affects endothelial cells, we found that EGCG increases Akt phosphorylation, eNOS phosphorylation, and nitric oxide (NO) production. NOS inhibition revealed that NO mediates the anti-inflammatory effects of EGCG. Our data suggest that polyphenols can decrease vascular inflammation by increasing the synthesis of NO, which blocks endothelial exocytosis.     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

The most widespread desmosomal cadherin, desmoglein 2, is a novel target of caspase 3-mediated apoptotic machinery

Apoptotic cells are known to regulate the ordered dismantling of intercellular contacts through caspase activity. Despite the important role of desmoglein (Dsg) 2 in epithelial cell-cell adhesion, the fate of this widespread desmosomal cadherin during apoptosis is yet poorly understood. Here, by means of pharmacological approaches, we investigated whether Dsg2 was targeted by caspases in HaCaT and HT-29 cell lines undergoing staurosporine (STS)-induced apoptosis. Results showed that STS induced a caspase-dependent form of cell-death in both keratinocytes (HaCaT) and enterocytes (HT-29), that associated with progressive depletion of Dsg2 from cell lysates. The proteolytic processing of full-length Dsg2 resulted in the appearance of a 70-kDa fragment which was released into the cytosol. Consistently, immunofluorescence studies revealed that Dsg2 staining was abolished from cell surface whereas the cytoplasmic region of Dsg2 did localize intracellularly. Plakoglobin (Pg) also underwent cleavage and detached from Dsg2. Apoptotic changes paralleled with progressive loss of intercellular adhesion strength. All these biochemical, morphological, and functional changes were regulated by caspase 3. Indeed, in the presence of the caspase 3-inhibitor z-DEVD-fmk, full-length Dsg2 protein levels were preserved, whereas the amount of the 70-kDa fragment was maintained on control levels. Furthermore, cells pretreated with z-DEVD-fmk retained the membrane labeling of Dsg2. Taken together, our data demonstrate that the apoptotic processing of Dsg2 is mediated by caspase 3 in epithelial cells.     

Mechanisms of β-Adrenergic Modulation of IKs in the Guinea-Pig Ventricle: Insights from Experimental and Model-Based Analysis

Detailed understanding of I_Ks gating complexity may provide clues regarding the mechanisms of repolarization instability and the resulting arrhythmias. We developed and tested a kinetic model to interpret physiologically relevant I_Ks properties, including pause-dependence and modulation by β-adrenergic receptors (β-AR). I_Ks gating was evaluated in guinea-pig ventricular myocytes at 36°C in control and during β-AR stimulation (0.1 μmol/L isoprenaline (ISO)). We tested voltage dependence of steady-state conductance (Gss), voltage dependence of activation and deactivation time constants (τ_act, τ_deact), and pause-dependence of τ_act during repetitive activations (τ_react). The I_Ks model was developed from the Silva and Rudy formulation. Parameters were optimized on control and ISO experimental data, respectively. ISO strongly increased Gss and its voltage dependence, changed the voltage dependence of τ_act and τ_deact, and modified the pause-dependence of τ_react. A single set of model parameters reproduced all experimental data in control. Modification of only three transition rates led to a second set of parameters suitable to fit all ISO data. Channel unitary conductance and density were unchanged in the model, thus implying increased open probability as the mechanism of ISO-induced Gss enhancement. The new I_Ks model was applied to analyze ISO effect on repolarization rate-dependence. I_Ks kinetics and its β-AR modulation were entirely reproduced by a single Markov chain of transitions (for each channel monomer). Model-based analysis suggests that complete opening of I_Ks channels within a physiological range of potentials requires concomitant β-AR stimulation. Transient redistribution of state occupancy, in addition to direct modulation of transition rates, may underlie β-AR modulation of I_Ks time dependence.     

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.     

Single site pacing through the anterior interventricular vein in a patient with a mechanic tricuspid valve

Transvenous endocardial pacing through classical implantation of a pace/sensing lead in the right ventricle is strictly contraindicated in patients with a mechanical tricuspid valve. Usually permanent pacing is achieved by an epimyocardial surgical approach. We hereby describe the implantation of a single site left ventricle pacing lead in the anterior interventricular vein in a 60 year-old woman with symptomatic bradycardia, permanent atrial fibrillation, and mechanical tricuspid valve. The described use of left ventricle pacing through a coronary vein lead, in a patient with favorable venous anatomy, provided (through a minimal invasive approach) effective with a low and stable threshold.     

Chromosomal analysis in Cathartidae: distribution of heterochromatic blocks and rDNA, and phylogenetic considerations

Three species of Cathartidae (Sarcoramphus papa, Cathartes aura and Cathartes burrovianus) were cytogenetically characterized by G- and C-banding. 18S–28S rDNA was used as a probe to map major ribosomal clusters. These species showed very similar karyotypes, with 2n = 80, 10 pairs of macrochromosomes, a submetacentric Z and a metacentric W chromosome. However, differences were found in the amount and distribution of heterochromatic blocks: S. papa showed heterochromatin only in the pericentromeric region and in chromosome W, while both species of Cathartes had heterochromatic blocks also in the long arm of two acrocentric pairs. Ribosomal clusters were found in a small pair in all three species. Karyotype analysis in Cathartidae revealed that this family has retained similarities to the putative avian ancestral karyotype, and placed Cathartidae in a more basal position in relation to Accipitridae and Falconidae. However, the cytogenetic data still cannot clarify the phylogenetic relationship between this family and other groups, such as Ciconiidae, considered its sister-group according to nucleic acid hybridization studies. J. C. Pieczarka and C. Y. Nagamachi—Researcher from CNPq, Brazil.     

Targeting integrins: insights into structure and activity of cyclic RGD pentapeptide mimics containing azabicycloalkane amino acids

A small library of cyclic RGD pentapeptide mimics incorporating stereoisomeric 5,6- and 5,7-fused bicyclic lactams was synthesized. This library was found to contain high-affinity ligands for the alpha(v)beta3 integrin. The aim of this study was to investigate activity, selectivity, and structure of these ligands in order to identify new specific alpha(v)-integrin antagonists that could be evaluated as tumor angiogenesis inhibitors. In vitro screening, including receptor-binding assays to purified alpha(v)beta3, alpha(v)beta5, and alpha5beta1 integrins, and platelet aggregation assay, revealed ST1646 as a potent, highly selective alpha(v)beta3/alpha(v)beta5 integrin antagonist. Structure determination of the cyclic RGD pentapeptide mimics performed by a combination of NMR spectroscopy, and molecular mechanics and dynamics calculations showed a strong dependence of the RGD cyclopeptide conformation on lactam ring size and stereochemistry. ST1646 revealed the highest ability within the library to adopt the proper RGD orientation required for binding to the alpha(v)beta3 integrin, as deduced from the recently solved crystal structure of the extracellular segment of integrin alpha(v)beta3 in complex with a cyclic pentapeptide ligand.     

Expression of COX-2 and hsp72 in peritoneal macrophages after an acute ochratoxin A treatment in mice

Ochratoxin A (OTA) is a secondary fungal metabolite produced by Aspergillus and Penicillium strains that elicits a broad spectrum of toxicological effects in animals and man. A single oral OTA administration (10 mg/kg) in mice induced after 24 h oxidative damage and polymorphonuclear leukocyte (PMN) infiltration in parenchymal organs. In fact, OTA treatment increased lipid peroxidation (via malondialdehyde formation) in kidney and liver and PMN accumulation in duodenum, as shown by myeloperoxidase activity. Following in vivo OTA treatment an increase of cyclooxygenase-2 and of heat shock protein 72 expression was evidenced in peritoneal macrophage lysates by Western blot. That OTA modulates these proteins involved in the inflammatory process indicates that the mycotoxin is able to activate immune cells. This study suggests that the oxidative stress, the neutrophil accumulation in parenchymal tissues and the modulation of inflammatory parameters in peritoneal macrophages induced by OTA are involved in its toxicity, and represent early events related to several aspects of OTA mycotoxicosis.