N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo. phosphatidylinositol 4,5-bisphosphate; cytoskeleton; membrane traffic; Arp2/3; actin comets     

Speciation with gene flow in whiptail lizards from a Neotropical xeric biome

Two main hypotheses have been proposed to explain the diversification of the Caatinga biota. The riverine barrier hypothesis (RBH) claims that the São Francisco River (SFR) is a major biogeographic barrier to gene flow. The Pleistocene climatic fluctuation hypothesis (PCH) states that gene flow, geographic genetic structure and demographic signatures on endemic Caatinga taxa were influenced by Quaternary climate fluctuation cycles. Herein, we analyse genetic diversity and structure, phylogeographic history, and diversification of a widespread Caatinga lizard (Cnemidophorus ocellifer) based on large geographical sampling for multiple loci to test the predictions derived from the RBH and PCH. We inferred two well‐delimited lineages (Northeast and Southwest) that have diverged along the Cerrado–Caatinga border during the Mid‐Late Miocene (6–14 Ma) despite the presence of gene flow. We reject both major hypotheses proposed to explain diversification in the Caatinga. Surprisingly, our results revealed a striking complex diversification pattern where the Northeast lineage originated as a founder effect from a few individuals located along the edge of the Southwest lineage that eventually expanded throughout the Caatinga. The Southwest lineage is more diverse, older and associated with the Cerrado–Caatinga boundaries. Finally, we suggest that C. ocellifer from the Caatinga is composed of two distinct species. Our data support speciation in the presence of gene flow and highlight the role of environmental gradients in the diversification process.     

Chromosomal characterization of two species of genus <Emphasis Type="Italic">Steatogenys</Emphasis> (Gymnotiformes: Rhamphichthyoidea: Steatogenini) from the Amazon basin: sex chromosomes and correlations with Gymnotiformes phylogeny

Gymnotiformes are an important component of the Neotropical ichthyofauna and they are known for their ability to generate and detect electrical discharges. Phylogenetic relationships of Gymnotiformes are still not well understood. However, the monophyly of the superfamily Rhamphichthyoidea is well accepted, despite the position of tribe Steatogenini (Steatogenys, Hypopygus and Stegostenopos) within this superfamily is unclear. The genus Steatogenys includes three species that, together with Hypopygus and Stegostenopos, form tribe Steatogenini. Cytogenetic information is currently only available for Hypopygus lepturus. Here, we describe the karyotypes of Steatogenys elegans from four localities and S. duidae from two localities. S. elegans was found to have 2n = 50, ZZ/ZW (12m-sm/38st-a), while S. duidae had 2n = 50 (50m-sm). In S. elegans, constitutive heterochromatin (CH) was observed in the centromeric regions of all chromosomes, in the interstitial region of 1q, and in two blocks of Wq. In S. duidae, CH was observed in the centromeric and pericentromeric regions of all chromosomes, and in the interstitial regions of 2q, 3q, 5q, and 7q. Nucleolar organizer regions (NORs) were identified in the distal regions of one chromosome pair in each species. The CMA₃ fluorochrome (specific to G-C rich regions) coincided with the NORs in both species, and with the HC of S. elegans except on chromosome pair 5 and the W. The DAPI fluorochrome (specific to A-T rich regions) coincided with the CH of both species, and was very intense for chromosome pair 5 and the W of S. elegans. Our observations suggest that the ZZ/ZW system observed in S. elegans likely evolved through CH addition followed by a paracentric inversion. The chromosomal data described herein are consistent with the phylogenetic hypothesis that tribe Steatogenini should be positioned within family Ramphychthyidae.     

β-Gal gene expression MRI reporter in melanoma tumor cells. Design, synthesis, and in vitro and in vivo testing of a Gd(III) containing probe forming a high relaxivity, melanin-like structure upon β-Gal enzymatic activation

The aim of this work is to design and test an MRI probe (Gd-DOTAtyr-gal) able to report on the gene expression of β-galactosidase (β-Gal) in melanoma cells. The probe consists of a Gd-DOTA reporter bearing on its surface a tyrosine-galactose-pyranose functionality that, upon the release of the sugar moiety, readily transforms, in the presence of tyrosinase, into melanin oligomeric/polymeric mixture. The formation of Gd-DOTA-containing melanin oligomers and polymers is accompanied by a marked increase of the water proton relaxation rate. The steps involving the release of the galactose-pyranose group and the formation of the melanin-like structure have been carefully investigated in vitro by relaxometric and UV-vis measurements. Cellular uptake studies of Gd-DOTAtyr-gal by melanoma cells have shown that the probe enters the cells, and it appears not to be confined in endosomal vesicles. Using B16-F10LacZ transfected cells, the fast formation of paramagnetic melanin-Gd(III)-containing species has been assessed by the measurement of increased longitudinal relaxation rates of the cellular pellets suspensions. The in vitro results have been confirmed in in vivo MRI investigations on murine melanoma tumor bearing mice. Upon direct injection of Gd-DOTAtyr-gal, a good contrast is observed after 5 h post injection in B16-F10LacZ tumors, but not in B16-F10 tumors lacking the β-Gal enzyme. Gd-DOTAtyr-gal in combination with tyrosinase introduces a novel approach for the detection of β-Gal expression by MRI in vivo.     

Review of Florida Red Tide and Human Health Effects

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue-one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.     

Evaluation of the in vitro activity of dermaseptin 01, a cationic antimicrobial peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 μg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 μg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 μg/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.     

Pannexin channels are not gap junction hemichannels

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers, or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term "hemichannels" to describe pannexin oligomers while others use the term "channels" instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus have an assembly and functional state as gap junctional intercellular channels. Here we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channel in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature.     

Tirucallane triterpenes from the leaf extract of Entandrophragma angolense

From the leaves of Entandrophragma angolense, three triterpenoidal compounds were isolated and structurally elucidated by mass and NMR spectroscopy. They belong to the tirucallane group but two of them possess the rare seco-ring-A feature. The phytochemical data are discussed from a chemotaxonomic and biogenetic points of view.     

Proinflammatory factors present in sera from patients with acute dengue infection induce activation and apoptosis of human microvascular endothelial cells: possible role of TNF-alpha in endothelial cell damage in dengue

There is evidence that severe dengue disease is associated with alterations of the microvascular endothelium. We examined the hypothesis that activation and damage of microvascular endothelial cells (EC) could be induced by inflammatory mediators present in dengue patient's sera. We cultured human microvascular EC (HMEC-1) in vitro with sera from patients with acute dengue infection. Sera from patients with acute dengue induced an increase in ICAM-1 expression on HMEC-1. This effect was greater with samples from the acute febrile phase than with samples from the convalescent phase of the disease. Acute dengue sera had elevated levels of TNF-alpha and the endothelial activating effect of acute dengue sera was inhibited up to 80% by pre-treatment with monoclonal antibodies against TNF-alpha. Furthermore, acute dengue sera induced apoptosis in HMEC-1. These findings support the pathophysiologic significance of microvascular EC and serum inflammatory mediators in dengue.