The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems

Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide–membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide–membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis).     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, β-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPase-activating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), papillary thyroid carcinomas (37, 47, 57), and melanomas (56). Rap1GAP downregulation has been shown to arise as a consequence of proteasomal degradation (46), loss of heterozygosity (37, 53), and promoter methylation (56, 57). Mutations of unknown significance in RAP1GAP have been identified in breast cancer (42). Although downregulation of Rap1GAP is frequent in human tumors, the functional significance of decreased Rap1GAP expression is unknown. Up to now, studies assessing the role of Rap1GAP in tumor cells have relied exclusively on overexpression experiments. Overexpression of Rap1GAP in oropharyngeal squamous cell (54) and pancreatic (53) carcinoma lines impaired tumor formation in mouse xenograft models. In vitro, overexpression of Rap1GAP impaired tumor cell proliferation (34, 47, 53, 54, 56) and enhanced apoptosis (34, 53, 56). In some instances, overexpression of Rap1GAP inhibited tumor cell migration and invasion (3, 47, 53, 56), while in others, it enhanced invasion (34). While these studies provide insight into cellular processes that can be deregulated by overexpression, they do not assess the significance of depletion of endogenous Rap1GAP in human tumors.Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The majority of CRC deaths arise as a consequence of distant metastases, most frequently to the liver. While the genetic basis of CRC is well understood (19, 48), less is known about the events that trigger the transition to metastatic disease. We report that Rap1GAP is highly expressed in normal colonic epithelium and that its expression is profoundly decreased in primary colorectal carcinomas. As one strategy to assess the significance of Rap1GAP depletion, the expression of Rap1GAP was silenced in human colon carcinoma cells. Silencing of Rap1GAP induced marked increases in Rap1 and Rap2 activity, the first evidence that Rap1GAP is an essential negative regulator of Rap GTPases in colon cancer. Rap1 regulates inside-out signaling through integrins (reviewed in references 8, 9, and 11) and is a target of outside-in signaling via cadherins (reviewed in reference 30). Downregulation of Rap1GAP induced profound alterations in cell/matrix and cell/cell adhesion. Suppressing Rap1GAP expression enhanced adhesion and spreading on collagen. Unexpectedly, based on the role of Rap1 in promoting cell/cell adhesion, silencing of Rap1GAP impaired cell/cell adhesion. These findings demonstrate a requirement for regulated Rap activity in the maintenance of epithelial cell structure and demonstrate a heretofore unappreciated role for Rap1GAP in the regulation of cell/cell adhesion. As the dissemination of tumor cells requires the weakening of cell/cell adhesion and an enhanced ability to adhere to collagen-rich interstitial matrices, our studies identify a potential mechanism through which loss of Rap1GAP contributes to tumor progression.     

Toxins that Modulate Ionic Channels as Tools for Exploring Insulin Secretion

Glucose-induced insulin secretion is a cardinal process in glucose homeostasis and metabolic expenditure. Uncoupling of the insulin response to glucose variations may lead to type-2 diabetes mellitus. Thus the identification of more specific drugs to facilitate the study of insulin secretion mechanisms and to develop new pharmacological agents for therapeutics is fundamental. Venomous organisms possess a great diversity of toxic molecules and some of them are neurotoxins that affect membrane excitability. This article reviews properties of those toxins affecting ion channels pivotal for insulin secretion and the usefulness of such compounds in the study of pancreatic beta-cell physiology. Here we examine the major contributions of toxinology to the understanding of the ionic phase of insulin secretion, to the determination of ion channel composition in different insulin secreting cell-line models as well as from primary cultures of different mammal species. Finally, we present a summary of the many diverse toxins affecting insulin release and a brief discussion of the potential of novel toxins in therapeutics.     

Locomotor anatomy and biomechanics of the Dmanisi hominins

The Dmanisi hominins inhabited a northern temperate habitat in the southern Caucasus, approximately 1.8 million years ago. This is the oldest population of hominins known outside of Africa. Understanding the set of anatomical and behavioral traits that equipped this population to exploit their seasonal habitat successfully may shed light on the selection pressures shaping early members of the genus Homo and the ecological strategies that permitted the expansion of their range outside of the African subtropics. The abundant stone tools at the site, as well as taphonomic evidence for butchery, suggest that the Dmanisi hominins were active hunters or scavengers. In this study, we examine the locomotor mechanics of the Dmanisi hind limb to test the hypothesis that the inclusion of meat in the diet is associated with an increase in walking and running economy and endurance. Using comparative data from modern humans, chimpanzees, and gorillas, as well as other fossil hominins, we show that the Dmanisi hind limb was functionally similar to modern humans, with a longitudinal plantar arch, increased limb length, and human-like ankle morphology. Other aspects of the foot, specifically metatarsal morphology and tibial torsion, are less derived and similar to earlier hominins. These results are consistent with hypotheses linking hunting and scavenging to improved walking and running performance in early Homo. Primitive retentions in the Dmanisi foot suggest that locomotor evolution continued through the early Pleistocene.     

Revisiting the Association of Cationic Groove-Binding Drugs to DNA Using a Poisson-Boltzmann Approach

Proper modeling of nonspecific salt-mediated electrostatic interactions is essential to understanding the binding of charged ligands to nucleic acids. Because the linear Poisson-Boltzmann equation (PBE) and the more approximate generalized Born approach are applied routinely to nucleic acids and their interactions with charged ligands, the reliability of these methods is examined vis-à-vis an efficient nonlinear PBE method. For moderate salt concentrations, the negative derivative, SK_pred, of the electrostatic binding free energy, ΔG_el, with respect to the logarithm of the 1:1 salt concentration, [M⁺], for 33 cationic minor groove drugs binding to AT-rich DNA sequences is shown to be consistently negative and virtually constant over the salt range considered (0.1-0.4 M NaCl). The magnitude of SK_pred is approximately equal to the charge on the drug, as predicted by counterion condensation theory (CCT) and observed in thermodynamic binding studies. The linear PBE is shown to overestimate the magnitude of SK_pred, whereas the nonlinear PBE closely matches the experimental results. The PBE predictions of SK_pred were not correlated with ΔG_el in the presence of a dielectric discontinuity, as would be expected from the CCT. Because this correlation does not hold, parameterizing the PBE predictions of ΔG_el against the reported experimental data is not possible. Moreover, the common practice of extracting the electrostatic and nonelectrostatic contributions to the binding of charged ligands to biopolyelectrolytes based on the simple relation between experimental SK values and the electrostatic binding free energy that is based on CCT is called into question by the results presented here. Although the rigid-docking nonlinear PB calculations provide reliable predictions of SK_pred, at least for the charged ligand-nucleic acid complexes studied here, accurate estimates of ΔG_el will require further development in theoretical and experimental approaches.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.