Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class

Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca²⁺‐dependent hydrolysis of phospholipids. Lys49‐PLA₂s are well‐characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49‐PLA₂ (BthTX‐II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49‐PLA₂ conserves all the residues responsible for Ca²⁺ coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX‐II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX‐II is not catalytic based on an in vitro cell viability assay and time‐lapse experiments on C2C12 myotube cell cultures, X‐ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX‐II is devoid of catalytic activity, as already observed for Lys49‐PLA₂s. Crystallographic studies of the complex BthTX‐II/Ca²⁺ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca²⁺ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX‐II is more phylogenetically related to Lys49‐PLA₂s than to other Asp49‐PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway

Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP⁺/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.     

Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.     

The in vitro leishmanicidal activity of hexadecylphosphocholine (miltefosine) against four medically relevant Leishmania species of Brazil

The in vitro leishmanicidal activity of miltefosine? (Zentaris GmbH) was assessed against four medically relevant Leishmania species of Brazil: Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) chagasi. The activity of miltefosine against these New World species was compared to its activity against the Old World strain, Leishmania (Leishmania) donovani, which is known to be sensitive to the effects of miltefosine. The IC50 and IC90 results suggested the New World species harboured similar in vitro susceptibilities to miltefosine; however, miltefosine was approximately 20 times more active against the Old World L. (L.) donovani than against the New World L. (L.) chagasi species. The selectivity index varied from 17.2-28.9 for the New World Leishmania species and up to 420.0 for L. (L.) donovani. The differences in susceptibility to miltefosine suggest that future clinical trials with this drug should include a laboratory pre-evaluation and a dose-defining step.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

Soil chromium bioremediation: Synergic activity of actinobacteria and plants

The aim of this work was to evaluate a strategy to reduce the bioavailable chromium fraction in soil, using a Cr(VI) resistant microorganism, Streptomyces sp. MC1, under non sterile conditions, with maize plants as bioindicator and/or bioremediator.Soil samples were contaminated with 100, 200 and 400 mg kg⁻¹ of Cr(VI) or Cr(III). Bioavailable chromium (35%) was only detected in samples with Cr(VI). Soil samples with Cr(VI) 200 mg kg⁻¹ were inoculated with Streptomyces sp. MC1, and bioavailable chromium decreased up to 73%.Zea mays seedlings were planted in soil samples contaminated with chromium. Plantlets accumulated chromium mainly as Cr(III), and biomass decreased up to 88%. Streptomyces sp. MC1 was inoculated in soil samples contaminated with 200 mg kg⁻¹ of Cr(VI) and Z.mays seedlings were planted.Streptomyces sp. MC1 caused Z.mays biomass increase (57%), chromium accumulation and bioavailable chromium decreased up to 46% and 96%, respectively.This work constitutes the first contribution of cooperative action between actinobacteria and Z.mays in the bioremediation of Cr(VI) contaminated soil. The large removal capacity of bioavailable chromium by Streptomyces sp. MC1 and Z.mays infers that they could be successfully applied together in bioremediation of soils contaminated with Cr(VI).     

Mitochondrial DNA diversity and population structure among southern right whales (Eubalaena australis)

The population structure and mitochondrial (mt) DNA diversity of southern right whales (Eubalaena australis) are described from 146 individuals sampled on 4 winter calving grounds (Argentina, South Africa, Western Australia, and the New Zealand sub-Antarctic) and 2 summer feeding grounds (South Georgia and south of Western Australia). Based on a consensus region of 275 base pairs of the mtDNA control region, 37 variable sites defined 37 unique haplotypes, of which only one was shared between regional samples of the Indo-Pacific and South Atlantic Oceans. Phylogenetic reconstruction of the southern right whale haplotypes revealed 2 distinct clades that differed significantly in frequencies between oceans. An analysis of molecular variance confirmed significant overall differentiation among the 4 calving grounds at both the haplotype and the nucleotype levels (F(ST) = 0.159; Phi(ST) = 0.238; P < 0.001). Haplotype diversity was significantly lower in the Indo-Pacific (h = 0.701 +/- 0.037) compared with the South Atlantic (h = 0.948 +/- 0.013), despite a longer history of exploitation and larger catches in the South Atlantic. In fact, the haplotype diversity in the Indo-Pacific basin was similar to that of the North Atlantic right whale that currently numbers about 300 animals. Multidimensional scaling of genetic differentiation suggests that gene flow occurred primarily between adjacent calving grounds within an ocean basin, with mixing of lineages from different calving grounds occurring on feeding grounds.     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.