Post-release survey to assess impact and potential host range expansion by Amitus hesperidum and Encarsia perplexa, two parasitoids introduced for the biological control of the citrus blackfly, Aleurocanthus woglumi in Dominica

Four years after the release of two exotic parasitoids, Amitus hesperidum Silvestri (Hymenoptera: Platygasteridae) and Encarsia perplexa Huang and Polaszek (Hymenoptera: Aphelinidae) for the classical biological control of the citrus blackfly (CBF), Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae) in Dominica, a survey was conducted to assess establishment as well as potential nontarget effects especially on Aleyrodidae and other related taxa. CBF populations were low to non-existent in 50 of 51 field sites examined. At the site where CBF was encountered, both E. perplexa and A. hesperidum were present and CBF populations were declining. The two parasitoids were not among the several species collected on nontarget Aleryodidae and Hemiptera. It is concluded that E. perplexa and A. hesperidum have kept CBF populations under effective biological control in Dominica and there is no evidence of any nontarget effects on other Aleyrodidae or their natural enemies. Handling Editor: Dirk Babendreier.     

Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.     

Relative Contributions of Cystathionine ��-Synthase and ��-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H₂S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H₂S generation. CBS from both organisms shows a marked preference for H₂S generation by β-replacement of cysteine by homocysteine. The alternative H₂S-generating reactions, i.e. β-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H₂S production by CBS would account for ∼25–70% of the total H₂S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H₂S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H₂S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H₂S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.Hydrogen sulfide (H₂S)² elicits an array of physiological effects, including modulation of blood pressure and reduction of ischemia reperfusion injury (1, 2). Other novel effects of H₂S include induction of a state of suspended animation in mouse by decreasing oxygen consumption and drastically reducing the metabolic rate (3) and synchronizing ultradian metabolic oscillation in yeast (4). Under conditions of metabolic cycling in yeast, H₂S production is catalyzed by sulfite reductase in the sulfur assimilation pathway (4). Inhibition of sulfite reductase reduces H₂S production and in turn perturbs metabolic oscillations. H₂S is a specific and potent inhibitor of cytochrome c oxidase in the electron transport chain (3).Although concentrations of H₂S have been reported to range from 50 to 160 μm in brain (5–7) and 30–50 μm in the peripheral system (8), these appear to be grossly overestimated (9). Significantly lower H₂S concentrations of 17 and 14 nm in liver and brain, respectively, have been reported recently (9). The very significant discrepancy between these and the previous estimates of H₂S levels presumably derives from the earlier use of acidic conditions that led to the release of acid-labile sulfur from iron-sulfur centers.In mammals, the primary catalysts for H₂S generation are reported to be the two pyridoxal phosphate (PLP)-dependent enzymes involved in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (10, 11). The trans-sulfuration pathway operates in the reverse direction in mammals serving to convert homocysteine to cysteine (Fig. 1), although in yeast and bacteria the pathway is involved in sulfur assimilation from sulfate to cysteine. CBS is widely assumed to be the major contributor to H₂S production in the brain because of its relatively high expression in this organ (10). However, a recent study reported that 3-mercaptopyruvate sulfurtransferase together with cysteine aminotransferase might also generate H₂S in brain (12). The relative contributions of these enzymes and of CSE, which is also present in brain (13, 14), to H₂S production remain to be assessed. Genetic disruption of CSE in mouse leads to cardiac deficits, including pronounced hypertension and reduced endothelium-dependent vasorelaxation, consistent with a major role for CSE in the peripheral system (1). However, brain H₂S levels are reportedly unchanged in CSE^−/− mice.Open in a separate windowFIGURE 1.Diversity of reactions catalyzed by the trans-sulfuration pathway. The turnover numbers (v/[E]) estimated at physiological substrate concentrations, i.e. 10 μm homocysteine, 100 μm cysteine, 560 μm serine, and 5 μm cystathionine, are shown in parentheses for each reaction. The thick arrows highlight reactions that are sensitive to elevated levels of homocysteine. The fold change represents the fold increase in the turnover number of a given reaction under conditions of severe hyperhomocysteinemia (200 μm homocysteine).Despite the growing recognition of the varied physiological effects of H₂S, our understanding of its regulation and mechanism of its biosynthesis is poor. We have recently reported on the complex kinetics of H₂S generation by human CSE (15). The profligacy of the human enzyme affords H₂S generation by a multiplicity of routes involving cysteine and/or homocysteine as substrates. Kinetic simulations predict an increasingly important contribution of homocysteine to H₂S generation with increasing grade of hyperhomocysteinemia, a risk factor for cardiovascular and neurodegenerative diseases (16–18). In addition to H₂S, a variety of products is generated in these reactions, including two novel sulfur metabolites, lanthionine and homolanthionine, which represent the condensation products between 2 mol of cysteine and homocysteine, respectively. Although the steady-state kinetic parameters for H₂S generation from cysteine and homocysteine have been reported for human CBS (hCBS) (19), a comparable detailed kinetic analysis of H₂S generation by CBS by multiple pathways and their sensitivity to the grade of hyperhomocysteinemia is not known. Furthermore, the relative contributions of CBS and CSE to H₂S and lanthionine generation at physiologically relevant concentrations of substrate are not known.Human CBS is a unique heme containing PLP-dependent enzyme (20) that catalyzes the β-replacement of serine by homocysteine to produce cystathionine. The latter is further metabolized by CSE in an α,γ-elimination reaction to produce cysteine. Although yeast and human CBS are highly homologous and catalyze the same chemical reaction with similar kinetic parameters, the yeast enzyme lacks heme and is not allosterically regulated by S-adenosylmethionine (AdoMet) (21).In this study, we have elucidated the kinetics of H₂S biogenesis by yeast and human CBS and used simulations to estimate the relative contributions of CBS and CSE to H₂S production at physiologically relevant concentrations of substrate. We find that CBS and CSE share a common feature, i.e. catalytic promiscuity. However, in contrast to CSE, which is proficient at catalyzing reactions at the β- and γ-carbons of substrates (15), CBS activity is confined to chemical transformations at the β-position. Our studies provide new insights into the existence of alternative trans-sulfuration reactions that can be a source of diverse sulfur metabolites, viz. H₂S, lanthionine, and homolanthionine increasing the diversity of the sulfur metabolome.     

The Family X DNA Polymerase from Deinococcus radiodurans Adopts a Non-standard Extended Conformation

Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX_Dr). Deletion of PolX_Dr has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3′→5′ exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX_Dr solved at 2.46-Å resolution reveals that PolX_Dr has a novel extended conformation in stark contrast to the closed “right hand” conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX_Dr the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for the maintenance of cellular life. These functions are carried out by various DNA replicating engines, most of them acting as multiprotein complexes. Deinococcus radiodurans, a Gram-positive bacterium, is characterized by an extraordinary resistance to ionizing radiation and desiccation. After radiation induced cutting of its 3.28-megabase genome into hundreds of small fragments, it is capable of reassembling it completely (1). Different hypotheses have been suggested to explain this radioresistance. A recently proposed mechanism involves the creation of long linear DNA intermediates by an extended synthesis-dependent strand annealing process, where overlapping chromosomal fragments are used both as primers and as templates for synthesis of complementary single strands (2). Recircularization of chromosomes would be assured by homologous recombination. Although DNA polymerase I is one of the main enzymes involved in this process, it was shown that other proteins affect double strand break repair efficiency in D. radiodurans. One of these is an X family DNA polymerase (PolX_Dr)⁵ (3). Cells devoid of PolX_Dr protein show increased sensitivity to γ-irradiation and a longer delay in the restoration of an intact genome after irradiation. It was therefore proposed that PolX_Dr has an important role in double strand break repair in D. radiodurans. The contribution of PolX_Dr may become essential for instance when damage gets too important or, alternatively, it may act in different repair pathways from polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces cerevisiae Pol4 and human polymerase λ (4) have been proposed to play important roles in different DNA repair processes, including non-homologous end-joining (5). It was shown that PolX_Dr also has strong 3′→5′ exonuclease activity that is stimulated by Mn²⁺ (6). This activity is associated with proofreading mechanisms in other polymerase families and encoded by protein domains or subunits distinct from the polymerase catalytic domain (7). Curiously the exonuclease activity of PolX_Dr is modulated upon encounter of a stem-loop structure. The combination of both activities leads to the hypothesis that PolX_Dr might be involved in DNA repair, potentially non-homologous end-joining, by processing damaged DNA or repair intermediates, thus generating substrates for other repair proteins (6). Very recently an orthologue of PolX from Bacillus subtilis was characterized. It was shown that PolX_Bs is a template-directed DNA polymerase acting on DNA gaps with a downstream 5′ phosphate group, suggesting it may play a role in base excision repair (8).DNA polymerases all combine a catalytic palm domain, a thumb domain, binding double-stranded DNA, and a finger domain that fixes the incoming nucleotide. The polymerase domain of the X family belongs to the Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid identity with the DNA polymerase domains of Polλ, Pol4, and Polβ. PolX_Dr has a second domain at the C terminus called PHP, with strong sequence identity with the histidinol phosphatase involved in histidine transport in bacteria. Due to its similarity to histidinol phosphatase and the presence of a trinuclear zinc site, the PolX_Dr PHP domain is thought to function as phosphoesterase (9). In the context of DNA polymerases, this activity might be responsible for the degradation of pyrophosphate, thus driving the polymerization reaction, or contributes to a nuclease reaction that would be involved in proofreading the newly synthesized strand. The deletion of the PHP domain also had a negative effect on survival of γ-irradiated cells suggesting that this domain possesses a function in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure modulated but not the general 3′→5′ exonuclease activity (6). No activity could be demonstrated for the PHP domain alone.In this report we present the crystal structure of PolX_Dr at 2.46-Å resolution. Surprisingly, PolX_Dr adopts a stretched out conformation instead of the commonly observed closed right hand conformation. In the active site of the polymerase catalytic domain, the two universally conserved aspartates are replaced by two glutamates, whereas the active site of the PHP domain is obstructed by its interaction with the polymerase domain.     

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.     

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

Mapping field-scale spatial patterns of size and activity of the denitrifier community

There is ample evidence that microbial processes can exhibit large variations in activity on a field scale. However, very little is known about the spatial distribution of the microbial communities mediating these processes. Here we used geostatistical modelling to explore spatial patterns of size and activity of the denitrifying community, a functional guild involved in N-cycling, in a grassland field subjected to different cattle grazing regimes. We observed a non-random distribution pattern of the size of the denitrifier community estimated by quantification of the denitrification genes copy numbers with a macro-scale spatial dependence (6–16 m) and mapped the distribution of this functional guild in the field. The spatial patterns of soil properties, which were strongly affected by presence of cattle, imposed significant control on potential denitrification activity, potential N₂O production and relative abundance of some denitrification genes but not on the size of the denitrifier community. Absolute abundance of most denitrification genes was not correlated with the distribution patterns of potential denitrification activity or potential N₂O production. However, the relative abundance of bacteria possessing the nosZ gene encoding the N₂O reductase in the total bacterial community was a strong predictor of the N₂O/(N₂ + N₂O) ratio, which provides evidence for a relationship between bacterial community composition based on the relative abundance of denitrifiers in the total bacterial community and ecosystem processes. More generally, the presented geostatistical approach allows integrated mapping of microbial communities, and hence can facilitate our understanding of relationships between the ecology of microbial communities and microbial processes along environmental gradients.     

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.