Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.     

Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow

Background and Aims Hypericum perforatum

(St. John''s wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixis. Here, hypotheses on the evolutionary origin of St. John''s wort are tested and contrasted with the subsequent history of interspecific gene flow.

Methods

Extensive field collections were analysed for quantitative morphological variation, ploidy, chromosome numbers and genetic diversity using nuclear (amplified fragment length polymorphism) and plastid (trnL-trnF) markers. The mode of reproduction was analysed by FCSS (flow cytometric seed screen).

Key Results

It is demonstrated that H. perforatum is not of hybrid origin, and for the first time wild diploid populations are documented. Pseudogamous facultative apomictic reproduction is prevalent in the polyploids, whereas diploids are predominantly sexual, a phenomenon which also characterizes its sister species H. maculatum. Both molecular markers characterize identical major gene pools, distinguishing H. perforatum from H. maculatum and two genetic groups in H. perforatum. All three gene pools are in close geographical contact. Extensive gene flow and hybridization throughout Europe within and between gene pools and species is exemplified by the molecular data and confirmed by morphometric analyses.

Conclusions Hypericum perforatum

is of a single evolutionary origin and later split into two major gene pools. Subsequently, independent and recurrent polyploidization occurred in all lineages and was accompanied by substantial gene flow within and between H. perforatum and H. maculatum. These processes are highly influenced by the reproductive system in both species, with a switch to predominantly apomictic reproduction in polyploids, irrespective of their origin.     

Biogeographic variation in genetic variability,apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range

Background and Aims

St. John''s wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges.

Methods

Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93 %), while lower frequencies of hexaploid (6 %), diploid (<1 %) and triploid (<1 %) individuals were also identified.

Key Results

A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73 %) than sexual (24 %) seed, while hexaploids produced more sexual (73 %) than apomictic (23 %) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe.

Conclusions

The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherd's purse)

Background and Aims

The duration of the plant life cycle is an important attribute that determines fitness and coexistence of weeds in arable fields. It depends on the timing of two key life-history traits: time from seed dispersal to germination and time from germination to flowering. These traits are components of the time to reproduction. Dormancy results in reduced and delayed germination, thus increasing time to reproduction. Genotypes in the arable seedbank predominantly have short time to flowering. Synergy between reduced seed dormancy and reduced flowering time would create stronger contrasts between genotypes, offering greater adaptation in-field. Therefore, we studied differences in seed dormancy between in-field flowering time genotypes of shepherd''s purse.

Methods

Genotypes with early, intermediate or late flowering time were grown in a glasshouse to provide seed stock for germination tests. Secondary dormancy was assessed by comparing germination before and after dark-incubation. Dormancy was characterized separately for seed myxospermy heteromorphs, observed in each genotype. Seed carbon and nitrogen content and seed mass were determined as indicators of seed filling and resource partitioning associated with dormancy.

Key Results

Although no differences were observed in primary dormancy, secondary dormancy was weaker among the seeds of early-flowering genotypes. On average, myxospermous seeds showed stronger secondary dormancy than non-myxospermous seeds in all genotypes. Seed filling was similar between the genotypes, but nitrogen partitioning was higher in early-flowering genotypes and in non-myxospermous seeds.

Conclusions

In shepherd''s purse, early flowering and reduced seed dormancy coincide and appear to be linked. The seed heteromorphism contributes to variation in dormancy. Three functional groups of seed dormancy were identified, varying in dormancy depth and nitrate response. One of these groups (FG-III) was distinct for early-flowering genotypes. The weaker secondary dormancy of early-flowering genotypes confers a selective advantage in arable fields.     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

Stem extension and mechanical stability of Xanthium canadense grown in an open or in a dense stand

Background and Aims

Plants in open, uncrowded habitats typically have relatively short stems with many branches, whereas plants in crowded habitats grow taller and more slender at the expense of mechanical stability. There seems to be a trade-off between height growth and mechanical stability, and this study addresses how stand density influences stem extension and consequently plant safety margins against mechanical failure.

Methods

Xanthium canadense plants were grown either solitarily (S-plants) or in a dense stand (D-plants) until flowering. Internode dimensions and mechanical properties were measured at the metamer level, and the critical buckling height beyond which the plant elastically buckles under its own weight and the maximum lateral wind force the plant can withstand were calculated.

Key Results

Internodes were longer in D- than S-plants, but basal diameter did not differ significantly. Relative growth rates of internode length and diameter were negatively correlated to the volumetric solid fraction of the internode. Internode dry mass density was higher in S- than D-plants. Young''s modulus of elasticity and the breaking stress were higher in lower metamers, and in D- than in S-plants. Within a stand, however, both moduli were positively related to dry mass density. The buckling safety factor, a ratio of critical buckling height to actual height, was higher in S- than in D-plants. D-plants were found to be approaching the limiting value 1. Lateral wind force resistance was higher in S- than in D-plants, and increased with growth in S-plants.

Conclusions

Critical buckling height increased with height growth due mainly to an increase in stem stiffness and diameter and a reduction in crown/stem mass ratio. Lateral wind force resistance was enhanced due to increased tissue strength and diameter. The increase in tissue stiffness and strength with height growth plays a crucial role in maintaining a safety margin against mechanical failure in herbaceous species that lack the capacity for secondary growth.     

Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli

Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam⁺) and GM33 (?dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA⁺ strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ?gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation caused by constitutive versus overproduction of Dam, as well as possible conformation of DNA influence the expression of act and gidA genes in A. hydrophila SSU. Our results indicate that the act gene is under the control of both Dam and GidA modification methylases, and Dam regulates Act production via GidA.     

Deferiprone and idebenone rescue frataxin depletion phenotypes in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA), the most common inherited ataxia, is a neurodegenerative disease caused by a reduction in the levels of the mitochondrial protein frataxin, the function of which remains a controversial matter. Several therapeutic approaches are being developed to increase frataxin expression and reduce the intramitochondrial iron aggregates and oxidative damage found in this disease. In this study, we tested separately the response of a Drosophila RNAi model of FRDA ( Llorens et al., 2007) to treatment with the iron chelator deferiprone (DFP) and the antioxidant idebenone (IDE), which are both in clinical trials. The FRDA flies have a shortened life span and impaired motor coordination, and these phenotypes are more pronounced in oxidative stress conditions. In addition, under hyperoxia, the activity of the mitochondrial enzyme aconitase is strongly reduced in the FRDA flies. This study reports that DFP and IDE improve the life span and motor ability of frataxin-depleted flies. We show that DFP eliminates the excess of labile iron in the mitochondria and thus prevents the toxicity induced by iron accumulation. IDE treatment rescues aconitase activity in hyperoxic conditions. These results validate the use of our Drosophila model of FRDA to screen for therapeutic molecules to treat this disease.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Identifying and characterizing a member of the RhopH1/Clag family in Plasmodium vivax

Plasmodium vivax malaria caused is a public health problem that produces very high morbidity worldwide. During invasion of red blood cells the parasite requires the intervention of high molecular weight complex rhoptry proteins that are also essential for cytoadherence. PfClag9, a member of the RhopH multigene family, has been identified as being critical during Plasmodium falciparum infection. This study describes identifying and characterizing the pfclag9 ortholog in P. vivax (hereinafter named pvclag7). The pvclag7 gene is transcribed at the end of the intraerythrocytic cycle and is recognized by sera from humans who have been infected by P. vivax. PvClag7 subcellular localization has been also determined and, similar to what occurs with PfClag9, it co-localize with other proteins from the Rhoptry high molecular weight complex.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.     

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3)

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.