Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.     

Cytochrome c biogenesis System I: An intricate process catalyzed by a maturase supercomplex?

Cytochromes c are ubiquitous heme proteins that are found in most living organisms and are essential for various energy production pathways as well as other cellular processes. Their biosynthesis relies on a complex post-translational process, called cytochrome c biogenesis, responsible for the formation of stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of apocytochromes c heme-binding site (C₁XXC₂H) cysteine residues. In some organisms this process involves up to nine (CcmABCDEFGHI) membrane proteins working together to achieve heme ligation, designated the Cytochrome c maturation (Ccm)-System I. Here, we review recent findings related to the Ccm-System I found in bacteria, archaea and plant mitochondria, with an emphasis on protein interactions between the Ccm components and their substrates (apocytochrome c and heme). We discuss the possibility that the Ccm proteins may form a multi subunit supercomplex (dubbed “Ccm machine”), and based on the currently available data, we present an updated version of a mechanistic model for Ccm. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c₂ by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c₂ and c₁, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm K_D values). When present, heme induced conformational changes in class I apocytochromes (e.g. c₂) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c₂ and that heme converts apocytochrome c₂ into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c₂) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.     

Micropilot: automation of fluorescence microscopy-based imaging for systems biology

Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.     

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.