Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins'' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein''s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.During the last few decades, the insecticidal toxins produced by Bacillus thuringiensis have been used increasingly in the forms of formulated sprays and transgenic plants for the highly focused biological control of insect pests (29). At the same time, the mechanism by which these proteins form pores in the apical membrane of midgut epithelial cells of targeted insects has been studied extensively (7, 29). In the case of the three-domain Cry toxins, specificity is mostly attributable to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets (16, 27). On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 (17, 20).Several lines of evidence indicate that helices α4 and α5 play a particularly important role in these processes (3). Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were most efficient when α4 and α5 were connected by a segment corresponding to the α4-α5 loop of the toxin (13, 14). A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized (2, 9, 10, 15, 18, 23, 25, 30, 31, 33). Finally, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin (23).Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 (31) but in most of its hydrophilic residues (15). Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or size of the pores but from a reduced pore-forming capacity of the toxin (15, 31). In order to better understand the role of α4 in the mechanism of pore formation, the present study was carried out with a series of previously characterized Cry1Aa mutants in which most of the residues from this helix were replaced by cysteines (15). By subjecting these mutants to circular dichroism (CD), protease sensitivity, pore formation inhibition, and electrophoretic mobility analyses, our data suggest that the mutations in α4 which alter the pore-forming ability of Cry1Aa do so mainly by preventing the proper oligomerization or membrane insertion of the toxin.     

Neuro-inflammation induced in the hippocampus of 'binge drinking' rats may be mediated by elevated extracellular glutamate content

The neuropathological and immune changes induced in the brain by 'binge drinking' have been investigated in a rat model. Evidence of neuro-inflammation was identified in the 'binge drinking' rat model of alcohol abuse after 3 weeks of administration of 2 or 3 g/kg ethanol (EtOH), three times per day for two consecutive days, followed by 5 days of abstinence: Firstly, alveolar macrophages, isolated from these animals, showed significant increases in inducible nitric oxide synthase, as assayed by nitrite release, both before and after lipopolysaccaharide stimulation. Secondly, significant numbers of activated microglia were present in the dentate gyrus region of the hippocampus of the 'binge drinking' model, after major histocompatibility complex class II staining, by comparison with the control. Microdialysis studies in the ventral hippocampus identified a significant increase in the basal extracellular concentration of glutamate, in both the 2 and 3 g/kg administered 'binge drinking' rats. In contrast, no changes in the hippocampal extracellular concentrations, of GABA and taurine, or the dopamine and serotonin metabolites were observed under basal conditions. A further dose of EtOH induced a significant decrease in the concentrations of both 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid, whereas glutamate, taurine and GABA levels were unaffected. There was no evidence that EtOH preference was initiated by the 'binge drinking' regimen. Our results suggest that the possible toxicity associated with 'binge drinking' maybe directed by the elevated glutamate levels, which in turn, activate phagocytic cells to release their inflammatory cytokines and chemokines, ultimately leading to neuro-inflammation.     

Asymptotic properties of infinite Leslie matrices

The stable population theory is classically applicable to populations in which there is a maximum age after which individuals die. Demetrius [1972. On an infinite population matrix. Math. Biosci. 13, 133-137] extended this theory to infinite Leslie matrices, in which the longevity of individuals is potentially infinite. However, Demetrius had to assume that the survival probability per time step tends to 0 with age. We generalise here the conditions of application of the stable population theory to infinite Leslie matrix models and apply these results to two examples, including or not senescence.     

A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.During the past decade protein degradation has become increasingly recognized as a critical mechanism by which cells regulate a number of fundamental processes (reviewed in references 37, 57, and 59). Degradation frequently involves one of a variety of E3 ubiquitin ligase complexes in which a substrate recognition component introduces the target protein for ubiquitination and subsequent degradation by proteasomes (reviewed in reference 59). Several types of these complexes involve a member of the cullin family (reviewed in reference 59), and a considerable amount of information is known about those containing Cul2 or Cul5. In these cases the substrate recognition module is linked via elongins B and C to a subcomplex containing Cul2 or Cul5 and the RING protein Rbx1 (34, 58). This complex interacts with an E2 conjugating enzyme, often either Cdc34 or Ubc5, to conjugate ubiquitin chains to the substrate (44). With both Cul2- and Cul5-based complexes interaction with elongins B and C occurs via a single BC box sequence (42). The presence of either Cul2 or Cul5 is generally determined through the presence in the substrate recognition protein of specific Cul2- or Cul5-box sequences (35).Many viruses have evolved to encode products that inhibit cellular E3 ligases to protect important viral or cellular species or, in some cases, that highjack these cellular complexes to target key substrates for degradation, including components of cellular host defenses, to facilitate the infectious cycle (reviewed in reference 4). These strategies are quite common among the small DNA tumor viruses (7), and one of the most studied examples is the complex formed by the human adenovirus E4orf6 and E1B55K proteins. These proteins have been known for some time to interact (69) and to reduce the levels of the p53 tumor suppressor in infected cells (14, 47, 48, 62, 72, 73). In addition, they were shown to function in concert to block nuclear export of cellular mRNAs late in infection (2, 6, 29, 60) and to enhance the selective export of late viral mRNAs (2, 26, 29, 60, 78). Our group showed that the human adenovirus serotype 5 (Ad5) E4orf6 product interacts with several proteins (13), including components of what was at the time a unique Cul5-based E3 ubiquitin ligase containing elongins B and C and Rbx1 that degrades p53 (61). Curiously, Ad5 E4orf6 contains three BC boxes that we believe make it highly efficient in highjacking cellular elongin B/C complexes (8, 17, 41). The mechanism of selective recruitment of Cul5 by the Ad5 complex remains unknown as E4orf6 lacks a Cul5-box (17, 41). E1B55K seems to function as the substrate recognition module and, of considerable interest, both its association with E4orf6 and induction of selective late viral mRNA transport was found to depend on formation of the E3 ubiquitin ligase complex, suggesting that additional degradation substrates must exist (8, 9). This idea is not surprising since viruses, especially the small DNA tumor viruses, often evolve gene products that target multiple critical cellular pathways (32). In fact two additional E1B55K-binding substrates have now been identified, Mre11 from the MRN DNA repair complex (8, 75), and DNA ligase IV (3), the degradation of which prevent formation of viral genome concatemers, thus enhancing packaging of progeny DNA. Degradation of p53 has been suggested to promote enhanced progeny virus production by preventing the early apoptotic death of infected cells due to the stabilization of p53 by the viral E1A products (reviewed in reference 66). Nevertheless, degradation of these substrates seems unlikely to explain the observed effects on mRNA transport, suggesting that still more substrates remain to be identified. Although the studies described in the present report were in part launched to identify such substrates, as will become clear below, these targets remain to be identified.In an attempt to identify new substrates of the Ad5 E4orf6/E1B55K E3 ubiquitin ligase complex, a proteomics-based approach was initiated involving two-dimensional difference gel electrophoresis (2D-DIGE) analysis and subsequent mass spectrometry. As is well known, this technique has the advantage of improved sensitivity and accuracy provided by its ability to separate samples under two different conditions on a single gel together with a reference sample, thus reducing significantly the analytical coefficient of variation. It allows the quantification of differentially abundant proteins in complex biological samples, providing a tool to detect decreases in the levels of proteins in the cell due to targeted proteolytic degradation. We report here our attempts to identify substrates of the Ad5 E4orf6/E1B55K complex by comparing the proteomes of human non-small cell lung carcinoma H1299 cells expressing, by means of adenovirus vectors, both E1B55K and E4orf6 proteins or E4orf6 protein alone. Ten candidate proteins were identified, most having functions seemingly unrelated to our current understanding of the roles of the E4orf6/E1B55K complex. At least three showed promising features characteristic of substrates, and one has now been confirmed in a parallel study to be a bone fide E4orf6/E1B55K substrate (20). We suggest that this approach could be utilized to identify candidate substrates, among relatively high abundance proteins, that are degraded by other viral cullin-based E3 ubiquitin ligase complexes.     

Structure and diversity of the Mesozoic wood genus Xenoxylon in Far East Asia: implications for terrestrial palaeoclimates

Although the faunal elements of Far East Asian Mesozoic terrestrial biota have attracted much attention in recent years, their palaeoecology remains poorly known. In particular, features of the palaeoclimate are highly controversial. To address this point we used the Mesozoic fossil wood Xenoxylon , a genus recognized as an indicator of wet temperate biotopes and which is common in the area during the Carnian–Maastrichtian interval. We re-appraised bibliographic data and gathered new data for Xenoxylon in the Mesozoic of Far East Asia. This demonstrated that previous taxonomic approaches to the genus have been so far idiosyncratic. We examined the anatomical diversity of morphogenus Xenoxylon in Far East Asia and compared it to that of samples from Europe. This indicates that in an area centred on north-eastern China, Xenoxylon reached a level of anatomical diversity unmatched elsewhere in the world. We hypothesize that this diversity witnesses the persistence of palaeoecological conditions particularly suitable for Xenoxylon and that a wet temperate climate prevailed over most of the area throughout the Carnian–Maastrichtian interval. It is in this setting that the famous Jehol Biota probably evolved.     

Sticky bivalves from the Mesozoic: clues to the origin of the anomalodesmatan arenophilic system

Arenophilic glands represent the only molluscan example of multicellular organs solely concerned with adhesion of foreign particles to the external surfaces of an organism. The glands are exclusive to the bivalve clade Anomalodesmata, but do not occur in all component taxa, having been declared absent in a number of families, including Pholadomyidae. This paper records and describes for the first time the arenophilic apparatus of Pholadomya candida G. B. Sowerby I, 1823, and demonstrates that secretion from these glands is at times preserved in the fossil record. In P. candida , arenophilic glands in the middle mantle folds discharge their products on top of the shell as the animal grows, forming radial lines of secretion that comprise a meandering main strand and numerous thin threads projecting in tufts. The arrangement is similar to that of other families, corroborating the hypothesis that the glands are a synapomorphy of crown-group anomalodesmatans. Instances of preserved secretion in fossil Pholadomya , ranging in age almost to the initial appearance of the genus in the Late Triassic, suggest that fossilized arenophilic secretion may aid systematic studies of problematic fossil groups traditionally included in Anomalodesmata.     

The new chronostratigraphic classification of the Ordovician System and its relations to major regional series and stages and to δ13C chemostratigraphy

The extensive work carried out during more than a decade by the International Subcommission on Ordovician Stratigraphy has resulted in a new global classification of the Ordovician System into three series and seven stages. Formal Global Boundary Stratotype Section and Points (GSSPs) for all stages have been selected and these and the new stage names have been ratified by the International Commission on Stratigraphy. Based on a variety of biostratigraphic data, these new units are correlated with chronostratigraphic series and stages in the standard regional classifications used in the UK, North America, Baltoscandia, Australia, China, Siberia and the Mediterranean‐North Gondwana region. Furthermore, based mainly on graptolite and conodont zones, the Ordovician is subdivided into 20 stage slices (SS) that have potential for precise correlations in both carbonate and shale facies. The new chronostratigraphic scheme is also tied to a new composite δ¹³C curve through the entire Ordovician.     

Redundant Hydrogen Peroxide Scavengers Contribute to Salmonella Virulence and Oxidative Stress Resistance

Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H₂O₂) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H₂O₂ degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF⁻ mutant, which exhibited a high sensitivity to exogenous H₂O₂ and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF⁻ background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H₂O₂ in rich medium. The HpxF⁻ mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H₂O₂ and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.Salmonella is a facultative intracellular pathogen that is associated with gastroenteritis, septicemia, and typhoid fever. This gram-negative bacterium survives and replicates in macrophages during the course of infection and can be exposed to a number of stressful environments during its life cycle (16). One of the host defense mechanisms that Salmonella encounters upon infection is the production of superoxide anion O₂⁻ by the phagocyte NADPH oxidase (1, 25). This radical can pass the outer membrane of the bacteria and represents one of the major weapons used by the macrophage to kill engulfed pathogens (18). Evidence that phagocyte-produced superoxide is a key mechanism for avoiding Salmonella infection is clear: mice and humans who are genetically defective in superoxide production are significantly more susceptible to infection (36, 38). Superoxide dismutases, located in the bacterial periplasm and in the cytoplasm, dismutate superoxide O₂⁻ to hydrogen peroxide H₂O₂ and molecular oxygen. Unlike superoxide, hydrogen peroxide can diffuse readily across bacterial membranes and form HO hydroxyl radicals in the presence of Fe(II) (18). These reactive oxygen species (ROS) can oxidize and damage proteins, nucleic acids, and cell membranes.To scavenge and degrade H₂O₂ molecules generated either as a by-product of aerobic metabolism or by the phagocyte NADPH oxidase, Salmonella has evolved numerous defense mechanisms. The KatE and KatG catalases are involved in H₂O₂ degradation, with katE being described as a member of the RpoS regulon (17, 22) and katG being OxyR dependent (26, 39). Both enzymes share the ability to reduce hydrogen peroxide to water and molecular oxygen, and their role was shown to be predominant at millimolar concentrations of H₂O₂ since they do not require any reductant (32). This observation is of particular importance, since these enzymes are not limited by the availability of a reductant, such as NADH, which cannot be generated fast enough to face a burst of H₂O₂. However, the katG and katE simple mutants, as well as the katE katG double mutant, did not show any increased susceptibility in macrophage or virulence attenuation in mice (5, 27). A possible reason could be the presence of a third nonheme and manganese-dependent catalase called KatN (30). This enzyme may contribute to hydrogen peroxide resistance under certain environmental conditions, but its involvement in virulence remains unknown. Moreover, katE, katG, and katN single mutants did not show any susceptibility to exogenous millimolar H₂O₂, essentially due to the compensatory function of the remaining catalases (5, 30).Another family of enzymes was shown to play an alternative role in H₂O₂ scavenging: the alkyl hydroperoxide reductases. These proteins directly convert organic hydroperoxides to alcohols, e.g., hydrogen peroxide to water. The alkyl hydroperoxide reductase AhpC belongs to the two-cysteine peroxiredoxin family, and the gene encoding this enzyme was identified as a member of the OxyR regulon (26, 39). The redox system consists of two proteins, AhpC and AhpF, with the latter being a thioredoxin reductase-like protein that contains two disulfide centers and transfers electrons from NADH to AhpC (13). AhpC was shown to be a predominant scavenger at low concentrations of H₂O₂, mainly because its catalytic efficiency was better than those of catalases (32). Recently the alkyl hydroperoxide reductase from Helicobacter hepaticus, TsaA (Thiol-Specific Antioxidant), was characterized (24). The tsaA mutant was found to be more sensitive to oxidizing agents like superoxide anion or t-butyl hydroperoxide. Surprisingly, this mutant was more resistant than the wild-type to H₂O₂, essentially because the level of catalase was increased in this background (24). In gastric pathogens, TsaA plays a critical role in the defense against oxygen toxicity that is essential for survival and growth (2). Interestingly, Salmonella contains two genes encoding alkyl hydroperoxide reductases, ahpC and tsaA, whereas a single copy was found in Escherichia coli (ahpC) or in Helicobacter pylori (tsaA).The redundancy of these antioxidant proteins could explain the extremely high resistance of Salmonella to hydrogen peroxide. It has been shown by Imlay and coworkers that in E. coli, three genes were involved in H₂O₂ scavenging: two catalase genes (katE and katG) and an alkyl hydroperoxide reductase gene (ahpC) (32). Simultaneous inactivation of the katE, katG, and ahpCF genes negated H₂O₂ degradation. As a consequence, this triple mutant, called the Hpx⁻ mutant, accumulates intracellular H₂O₂ (32). Moreover, H₂O₂ generated by aerobic metabolism was found to be sufficient to create toxic levels of DNA damage in such a background (28). In the present study, we deleted the Salmonella katE, katG, and ahpCF genes and two more genes absent in E. coli, katN and tsaA, to obtain the HpxF⁻ mutant, which lacks three catalases and two alkyl hydroperoxide reductases. HpxF⁻ cells exhibited the incapacity to degrade micromolar concentrations of H₂O₂, whereas this phenotype was not observed for the Kat⁻ (katE katG katN) and Ahp⁻ (ahpCF tsaA) mutants. Therefore, the HpxF⁻ mutant exhibited a high sensitivity to this compound. Moreover, this mutant did not show any proliferation within macrophages and presented reduced virulence in mice, suggesting that Salmonella catalases and alkyl hydroperoxide reductases form a redundant antioxidant arsenal essential for survival and replication within host cells.     

Effect of stinging nettle habitats on aphidophagous predators and parasitoids in wheat and green pea fields with special attention to the invader Harmonia axyridis Pallas (Coleoptera: Coccinellidae)

The relative occurrence and seasonal abundance of aphids and their natural enemies were visually assessed between May and July 2005–2006 in four types of habitats located in Gembloux (Namur province, Belgium): green pea, wheat and stinging nettle either planted in or naturally growing in woodland adjacent to these crops. Results showed that: (i) Acyrthosiphon pisum Harris, Sitobion avenae F. and Microlophium carnosum Buckton were the most common aphid species, respectively, on green pea, wheat and stinging nettle either in or near field crops; (ii) stinging nettle and field crops shared several important aphidophagous insect species such as the ladybird Coccinella septempunctata L., hoverfly Episyrphus balteatus De Geer and braconid wasp Aphidius ervi Haliday; (iii) the shared beneficial species were typically recorded earlier on stinging nettles than on crops; and (iv) the spatial occurrence of the invasive ladybird Harmonia axyridis Pallas was distinctly associated with stinging nettles, particularly in 2005. Stinging nettles and field crops partially coincide in time, enabling the movement of natural enemies among them. These findings suggest that the presence of stinging nettles in landscapes seems to enhance the local density of aphidophagous insect communities necessary for aphid biocontrol in field crops.     

Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser³⁵-Asp³⁰⁷-His³¹⁰) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.