Advantageous attributes of larval whitefringed weevil, Naupactus leucoloma (Coleoptera: Curculionidae) for bioassaying soil fumigants, and responses to pure and plant-derived isothiocyanates

First instars of the soil-inhabiting whitefringed weevil, Naupactus leucoloma (Boheman), are a particularly good bioassay model for assessing volatile soil fumigants and biofumigants. Eggs are readily obtained and can be stored for long periods with larvae hatched on demand and the first instar is non-feeding, surviving without food or shelter. Longevity varies with temperature, but readily accommodates the period required to conduct bioassays without appreciable mortality of untreated controls. In vitro bioassays of pure methyl isothiocyanate, the active ingredient from metham sodium soil fumigant, and the less volatile 2-phenylethyl isothiocyanate, sensitively detected differences in toxicity and effects of temperature. Bioassay of volatiles emitted from hydrolysed tissue of various isothiocyanate-producing Brassica plants revealed widely varying toxicity effects, indicating that bioassays with N. leucoloma are a sensitive and relevant indicator of the potential of different plants for biofumigation of soil-borne pest organisms.     

Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.     

β1A integrin is a master regulator of invadosome organization and function

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization–depolymerization associated with β1 and β3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using β1^−/− or β3^−/− cells, it seemed that β1A but not β3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the β1A tail. Moreover, our study clearly showed that β1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the β1A cytoplasmic domain allowed specific and immediate loss of function of β1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.     

Herbivore-induced plant volatiles as cues for habitat assessment by a foraging parasitoid

1. Animals usually require information about the current state of their habitat to optimize their behaviour. For this, they can use a learning process through which their estimate is continually updated according to the cues they perceive. Identifying these cues is a long-standing but still inveterate challenge for ecologists. 2. The use of plant cues by aphid parasitoids for the assessment of habitat profitability and the adaptation of patch exploitation was studied. Grounding on predictions from optimal foraging theory, we tested whether parasitoids exploited host patches less intensively after visiting heavily infested plants than after visiting plants bearing fewer aphids. 3. As predicted, after visiting heavily infested plants parasitoids reduced their residence time and attacked fewer hosts in the next patch. This was the case regardless of whether the aphids were actually present on the first plant, indicating that the cue came from the plant. Moreover, the level of infestation of a plant at some distance from the first plant visited affected parasitoid patch exploitation on the second plant in a similar manner, indicating that the cue was volatile. 4. These results highlight a novel role of herbivore-induced volatiles in parasitoid foraging behaviour, different from the widely studied attraction at a distance.     

11beta-Hydroxysteroid Dehydrogenase Type 1 Regulation by Intracellular Glucose 6-Phosphate Provides Evidence for a Novel Link between Glucose Metabolism and Hypothalamo-Pituitary-Adrenal Axis Function

Microsomal glucose-6-phosphatase-alpha (G6Pase-alpha) and glucose 6-phosphate transporter (G6PT) work together to increase blood glucose concentrations by performing the terminal step in both glycogenolysis and gluconeogenesis. Deficiency of the G6PT in liver gives rise to glycogen storage disease type 1b (GSD1b), whereas deficiency of G6Pase-alpha leads to GSD1a. G6Pase-alpha shares its substrate (glucose 6-phosphate; G6P) with hexose-6-phosphate-dehydrogenase (H6PDH), a microsomal enzyme that regenerates NADPH within the endoplasmic reticulum lumen, thereby conferring reductase activity upon 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 11beta-HSD1 interconverts hormonally active C11beta-hydroxy steroids (cortisol in humans and corticosterone in rodents) to inactive C11-oxo steroids (cortisone and 11-dehydrocorticosterone, respectively). In vivo reductase activity predominates, generating active glucocorticoid. We hypothesized that substrate (G6P) availability to H6PDH in patients with GSD1b and GSD1a will decrease or increase 11beta-HSD1 reductase activity, respectively. We investigated 11beta-HSD1 activity in GSD1b and GSD1a mice and in two patients with GSD1b and five patients diagnosed with GSD1a. We confirmed our hypothesis by assessing 11beta-HSD1 in vivo and in vitro, revealing a significant decrease in reductase activity in GSD1b animals and patients, whereas GSD1a patients showed a marked increase in activity. The cellular trafficking of G6P therefore directly regulates 11beta-HSD1 reductase activity and provides a novel link between glucose metabolism and function of the hypothalamo-pituitary-adrenal axis.     

Isolation and crystallization of a unique size category of recombinant Rabies virus Nucleoprotein-RNA rings

In order to study the packaging of rabies virus RNA inside the viral nucleocapsid, rabies nucleoprotein was expressed in insect cells. In the cells, it binds to cellular RNA to form long, helical or short circular complexes, depending on the length of the bound RNA. The circular complexes contained from 9 up to 13 N-protomers per ring. Separation of the rings into defined size classes was impossible through regular column chromatographies or gradient centrifugation. The size classes could be separated by native polyacrylamide gel electrophoresis. A large-scale separation was achieved with a 4% native gel using a preparative electrophoresis apparatus. Crystallization trials were set up with N-RNA rings from three size classes and crystals were obtained in all cases. The best diffracting crystals, diffracting up to 6A, contained rings with 11 N-protomers plus an RNA molecule of 99 nucleotides. The diffraction limit was improved to 3.5A by air dehydration prior to flash freezing.