Toluidine blue-O staining of prion protein deposits

Prion diseases or transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases caused by an abnormal form of prion protein (PrP(sc)). In this study, we developed a sensitive histochemical detection of PrP(sc) deposits in a Gertsmann-Str?ussler-Scheinker disease (GSS) patient using toluidine blue-O staining, a specific reagent to stain mucins and mucopolysaccharides. Detection of prion deposits correlated with immunohistochemistry using anti-prion antibodies. Control assays were performed using amyloid-beta (Abeta) plaques from Alzheimer's disease (AD) brains. Our results demonstrated that toluidine blue-O staining allowed to recognize 69.1+/-2.6% of the total plaques recognized by the anti-prion antibody. Furthermore, in the 15 studied brain regions from the GSS patient, toluidine blue-O revealed the same recognition pattern as anti-prion labeling. Toluidine blue-O stained specifically the prion deposits but not the Abeta plaques in AD brains. The specificity of the technique was confirmed in a Creutzfeldt-Jakob disease brain. This method opens several possibilities for postmortem diagnoses. Our results also suggest the relevance of specific post-translational modifications of PrP(sc), identified by toluidine blue-O, that might participate in the transformation of PrP(c) to PrP(sc).     

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance,recovery, and death

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.     

MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac

MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.     

Dna topoisomerase activities in plants: Involvement in cell proliferation

Abstract

The role of DNA topoisomerases in cell processes related to DNA metabolism, their involvement in the regulation of cell proliferation and their distribution in plant tissues are discussed.     

Seed reserve translocation and early seedling growth of eight tree species in a tropical deciduous forest in Mexico

Seed reserves play an essential role during germination and seedling establishment and are particularly important for species that grow in seasonal ecosystems with a short growing season. In this study, we examined (a) how and when the seedlings change their dependence from seed resources to external resources, (b) the lipid, nitrogen, and non-structural carbohydrate reserve translocation from seeds to seedlings over time, and (c) whether reserve translocation may be correlated to cotyledon and leaf lifespan of seedlings for eight tree species in a tropical deciduous forest in north-western Mexico. Our results showed that the cotyledon lifespan was not related to the cotyledon type (photosynthetic or reserve) and that the cotyledon biomass did not decrease significantly until germination. In six of the eight studied species, biomass allocation to the leaves was favored; lipids were the first reserve exhausted before the first leaves were totally expanded in seven of the eight study species. Species with the highest N concentration had expanded leaves and lost their cotyledons faster than species with a low N concentration. Our results suggest that tropical deciduous forest species employ different strategies to survive the dry season and re-sprout in the next growing season mediated by seed reserve concentrations, translocation patterns and subsequent biomass allocation.     

Interactive effects of aphid feeding and virus infection on host gene expression and volatile compounds in salt-stressed soybean, <Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.

Saline soils are becoming an important limiting factor in production agriculture. Soybean cultivars [Glycine max (L.) Merr.] differ in their ability to tolerate salt stress with those that cannot limit ion uptake into leaves being salt sensitive. Those that can partially limit ion uptake into leaves are generally more salt tolerant. Soybean mosaic virus (SMV) is an important viral pathogen of soybean worldwide and is commonly transmitted by the soybean aphid, Aphis glycines Matsumura. In this study, we investigate the interaction of salt stress in soybean with SMV infection and infestation by the soybean aphid by measuring aphid populations in a no-choice assay, gene expression levels, and the induction of volatile organic compounds using static headspace GC–MS analysis. Salt stress and SMV infection both reduced total aphid populations, though SMV did not reduce the total number of aphids per gram of fresh weight. Aphid suppression of a calcium EF hand gene and OPR1 was lost when salt-sensitive soybean plants were salt stressed and when salt-tolerant plants were subjected to all three stressors. The relative levels of SMV in aphid-infested soybeans were increased by salt stress in the salt-sensitive cultivar, whereas SMV levels decreased in the salt-tolerant cultivar. Static headspace collection of volatile organic compounds revealed that salt stress and SMV infection had suppressive activities on aphid-induced terpenes. These results suggest that although salt stress has a negative impact on aphid population size, the changes in volatiles and SMV levels could alter the incidence of SMV in salt-stressed fields.     

Biological activity of <Emphasis Type="Italic">Claviceps gigantea</Emphasis> in juvenile New Zealand rabbits

The Ascomycete fungus Claviceps gigantea infects maize kernels and synthetizes several alkaloids, mostly dihydrolysergamides. There is limited information on the damage these toxins cause in mammals, despite reports from infested areas with 90% presence of the fungus sclerotia. With this background, it was decided to determine the biological activity of chemical compounds present in sclerotia of C. gigantea in rabbits 38 days after weaning. Sclerotia of C. gigantea were collected in fields with high incidence of the disease, ground and analysed for nutrients. Experimental diets were prepared with four treatments, where sclerotial powder was added, substituting for alfalfa flour in increasing proportions [C. gigantea/alfalfa flour (0:100, 5:95, 15:85 and 25:75)]. Total ergot alkaloid content was analysed by high-performance liquid chromatography. Male juvenile rabbits were utilised and distributed in completely randomised design with four replications. Initial weight was recorded in each animal, and experimental diet was offered. In this study, weight of animals, feed consumption and feed conversion were evaluated in individual animals. Blood samples were taken for haemograms, and finally euthanasia was practiced. The consumption of C. gigantea had a negative effect on body weight and feed consumption. The necropsies showed anomalies proportional to the consumption of feed contaminated with the fungus.     

Discovery and molecular characterization of a plasmid localized in Buchnera sp. bacterial endosymbiont of the aphid Rhopalosiphum padi

We have identified and completely sequenced a novel plasmid isolated from the aphid Rhopalosiphum padi. Evidence which suggests that the plasmid occurs localized within the bacterial endosymbionts is presented. The plasmid contains the four genes that constitute the entire leucine operon. This fact makes it really unique since most plasmids are dispensable and lack genes that encode essential anabolic functions. Four more phloem-feeding aphid species also seem to contain homologous plasmids.Although further work is necessary, we hypothesize that this plasmid has appeared during the evolution of the symbiotic association between the aphid and the bacterial endosymbiont. The fact that this plasmid contains the entire leucine operon can be related to physiological evidence showing that the aphid host's diet of plant phloem is deficient in essential amino acids.