Sugars and acid invertase mediate the physiological response of Schenkia spicata root cultures to salt stress

A heterotrophic model system was established in our studies in order to differentiate the effect of high salt concentrations in external medium on growth and sugar metabolism in roots from the effect of reduced sugar availability resulting from decreased photosynthesis under salinity. Soluble sugar content and the activity of acid invertase in root cultures of salt-tolerant (ST) and salt-sensitive (SS) Schenkia spicata (L.) Mansion genotypes were investigated during exposure to different NaCl concentrations (0-200mM). Their response to severe salinity was characterized by a metabolic adjustment that led to the accumulation of sucrose (Suc) in root tissues. There was clear evidence that cell wall invertase (CW-Inv) is the major contributor to the Suc/hexose ratio in roots during exposure to elevated salinity. The results of CW-Inv activity and immunodetection assays in our study suggest that the regulation of CW-Inv expression is most likely achieved in a salt stress dependent manner. Also, NaCl modulated soluble acid invertase (SA-Inv) expression differentially in SS and ST genotypes of S. spicata. Regardless of the salt treatment, genotype, or the amount of enzyme, SA-Inv activity was generally low, indicating regulation at the posttranslational level. The results suggest no direct role of SA-Inv in the regulation of the root tissue carbohydrate pool and therefore in the control of the availability of glucose and fructose for the primary metabolism and/or osmotic adjustment in the present heterotrophic model system.     

The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila

The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF2^2(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it’s functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.     

Etanercept reduces matrix metalloproteinase-9 level in children with polyarticular juvenile idiopathic arthritis and TNF-α-308GG genotype

Genetic contribution of tumor necrosis factor polymorphism (TNF-α-308G/A) in patients with juvenile idiopathic arthritis (JIA) on response to TNF blocking agents, as well as matrix metalloproteinase-9 (MMP-9) production, is not yet well established. We have investigated whether the TNF-α-308G/A polymorphism can influence MMP-9 level and clinical response to etanercept (TNF receptor II-Fc fusion protein) in JIA patients, after 1 year of treatment. A total of 66 patients with polyarticular JIA and 65 healthy children were screened for the polymorphism using the polymerase chain reaction–restriction fragment length polymorphism method. JIA patients donated paired blood samples prior to and 12 months after etanercept therapy. Plasma MMP-9 level was determined using an enzyme-linked immunosorbent assay kit. Clinical assessment was performed according to ACR Pedi 50 improvement criteria. The frequency of the A allele was significantly higher in JIA patients compared to controls (39% vs. 26%, P?=?0.026). Patients with the ?308GG genotype achieved an ACR Pedi 50 response significantly more frequently than those with the ?308AA genotype (P?=?0.035). MMP-9 level in patients with the genotype ?308GG was significantly decreased after 1 year of treatment with etanercept compared to the value from before (P?=?0.036). On the other hand, there was a decrease of MMP-9 levels after treatment, but not statistically significant in patients with the genotypes ?308GA/AA. We conclude that etanercept reduces MMP-9 level in children with polyarticular JIA and TNF-α-308GG genotype. Our results correlate with findings that the ?308A allele is associated with a lower response to etanercept treatment.     

Localization of Rheb to the endomembrane is critical for its signaling function

Rheb, a small GTPase, has emerged as a key molecular switch that directly regulates the activity of the mammalian target of rapamycin (mTOR). Similar to other members of the Ras superfamily, Rheb has a C-terminal CaaX box that is subject to farnesylation. This study reports that farnesylation is a key determinant of Rheb's subcellular localization and directs its association with the endomembrane. Timed imaging of live cells expressing EGFP-Rheb reveals that following brief association with the ER, Rheb localizes to highly ordered, distinct structures within the cytoplasm that display characteristics of Golgi membranes. Failure of Rheb to localize to the endomembrane impairs its ability to interact with mTOR and activate downstream targets. Consistent with the notion that the endomembrane may serve as a platform for the assembly of a functional Rheb/mTOR complex, treatment of cells with brefeldin A interferes with transmission of Rheb signals to p70S6K.     

Changes in antioxidant systems in soybean as affected by Sclerotinia sclerotiorum (Lib.) de Bary

Changes in antioxidant systems in soybean [Glycine max (L.) Merr., Fabaceae] genotypes infected with Sclerotinia sclerotiorum were studied 12, 24, 48 and 72 h after inoculation. Generation of superoxide and hydroxyl radicals was evaluated together with the production of malonyldialdehyde, main end product of lipid peroxidation. Several enzymatic and non-enzymatic parameters were monitored as well, such as the activity of antioxidant enzymes superoxide dismutase and pyrogallol and guaiacol peroxidases, reduced glutathione, soluble proteins and total carotenoids content. Results showed that genotypes expressed oxidative burst as well as different antioxidant systems in response to biotic stress caused by pathogen invasion. It has been confirmed that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect soybean plants against infection by the necrotrophic pathogen such as S. sclerotiorum. Still, some genotypes showed distinctive and combined activity of several biochemical parameters which may point to further directions in exploring host-pathogen relations and lead to selection and production of new genotypes with higher levels of tolerance     

Biological activities of phenolic compounds and ethanolic extract of <Emphasis Type=Italic>Halacsya sendtneri</Emphasis> (Boiss) Dőrfler

The objective of this study was to evaluate the efficacy of the ethanolic extract of endemic plant Halacsya sendtneri in inhibiting the growing of the test fungi and bacteria as well as to determine its genotoxic potential and toxicity using the Allium anaphase-telophase assay. Minimum inhibitory concentrations (MIC) were determined for 15 indicator strains of pathogens, representing both bacteria and fungi. The highest susceptibility to the ethanolic extract of H.sendtneri was exhibited by Pseudomonas glycinea (FSB4), (MIC=0.09 mg/ml) among the bacteria, and by Phialophora fastigiata (FSB81), (MIC=1.95 mg/ml) among the fungi. The composition of H. sendtneri extracts was also determined using HPLC analysis. Rosmarinic acid was found to be the dominant phenolic compound. The Allium anaphase-telophase genotoxicity assay revealed that the ethanolic extract of H. sendtneri at concentrations of 31.5 mg/l and below does not produce toxic or genotoxic effects. This is the first report of chemical constituents, genotoxic and antimicrobial activities of the endemic species, H. sendtneri.     

Skills or strength—how tortoises cope with dense vegetation?

Dense vegetation cover undoubtedly offers certain advantages for small and slow-moving animals, but its disadvantages concerning some aspects of spatial ecology (e.g. movements) were neglected in previous studies. Tortoises could get stuck in vegetation by protuberant part of the shell and thus succumb to overheating, dehydration or predators. To examine how vegetation cover shapes behavioural responses of ‘trapped’ tortoises, we tested adults of six populations from habitats with contrasting vegetation cover. The tortoises were fitted with a non-stretchable rope, representing a piece of vegetation, stuck on the protruding front part of the plastron. Results suggested the existence of two distinct releasing techniques. First, and only successful in this study, is frequent changing of the movement direction, with a minimal pulling force, until the obstacle detached. The other involved the maximal pulling force aimed at ripping out the constraint. Tortoises from shrub habitats had more releasing success, used less pulling force and needed shorter time period to release, contrary to tortoises from herbaceous habitats. Although sexes showed similar releasing success, females obtained lower number of direction changes and higher yanking force compared to males, suggesting slightly different liberating strategies between the sexes. For immobilized tortoises without suitable shelter from overheating and dehydration, appropriate behavioural response could be vital, especially during drought years, due to increased physiological stresses. Variability of behavioural patterns among tortoise populations, described in this study, could have an adaptive significance.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations

The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.     

An Autoinhibited Noncanonical Mechanism of GTP Hydrolysis by Rheb Maintains mTORC1 Homeostasis

Rheb, an activator of mammalian target of rapamycin (mTOR), displays low intrinsic GTPase activity favoring the biologically activated, GTP-bound state. We identified a Rheb mutation (Y35A) that increases its intrinsic nucleotide hydrolysis activity ～10-fold, and solved structures of both its active and inactive forms, revealing an unexpected mechanism of GTP hydrolysis involving Asp65 in switch II and Thr38 in switch I. In the wild-type protein this noncanonical mechanism is markedly inhibited by Tyr35, which constrains the active site conformation, restricting the access of the catalytic Asp65 to the nucleotide-binding pocket. Rheb Y35A mimics the enthalpic and entropic changes associated with GTP hydrolysis elicited by the GTPase-activating protein (GAP) TSC2, and is insensitive to further TSC2 stimulation. Overexpression of Rheb Y35A impaired the regulation of mTORC1 signaling by growth factor availability. We demonstrate that the opposing functions of Tyr35 in the intrinsic and GAP-stimulated GTP catalysis are critical for optimal mTORC1 regulation.