European pome fruit genetic resources evaluated for disease resistance

Pome fruit genetic resources collections constitute a highly valuable resource not only for fruit breeding but also for direct use by nurseries, growers, and home gardeners. In order to use these resources efficiently and sustainably, reliable evaluation data on fruit and tree characteristics must be generated. Here we focus on pome fruit genetic resources evaluated phenotypically and genotypically for susceptibility to apple scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha), fire blight (Erwinia amylovora), pear rust (Gymnosporangium sabinae) and storage diseases (e.g., Penicillium expansum). Examples are presented of several ongoing projects throughout Europe, with the aim to evaluate fruit genetic resources for disease susceptibility and potential use in breeding and for commercial use. The COST action 864 has fostered international cooperation in the evaluation of pome fruit genetic resources, and some of these evaluations therefore involve research groups from several of the participating countries.     

Complete denture wearing and fractures among edentulous patients treated in university clinics

doi: 10.1111/j.1741‐2358.2011.00551.x
Complete denture wearing and fractures among edentulous patients treated in university clinics Objective: The prevalence of wearing and fracture of complete dentures was evaluated among edentulous patients treated in two dental schools in Brazil. Background: Acceptance and wearing of complete dentures are related to adaptive behaviour of edentulous patients. However, one reason that could interfere with the wearing dentures is their potential to fracture, which is still a common complication in denture rehabilitation practice. Material and methods: Two hundred and twenty‐four edentulous patients rehabilitated with complete dentures from 2000 to 2005 in Araçatuba and Araraquara Dental School, University of State of São Paulo, were assessed in 2006 and 2007 to answer a questionnaire about wearing and fracture of their dentures. Statistical analysis were performed using Epi Info software and chi‐squared test to compare maxillary and mandibular data (α = 0.05). Results: Almost 26% of the patients did not wear their dentures, and among the remainder, the majority wore the maxillary denture. About 30% of the dentures were fractured, with higher prevalence in the maxillary arch (p = 0.003). Conclusions: Discontinuation of wearing dentures was quite high, especially considering the treatment which was carried out in university clinics. Prevalence of fractures was also high, greater for the maxillary denture, and was one of the main reasons for non‐wearing of complete dentures.     

Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.     

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.     

Transformations of count data for tests of interaction in factorial and split-plot experiments

In applied entomological experiments, when the response is a count-type variable, certain transformation remedies such as the square root, logarithm (log), or rank transformation are often used to normalize data before analysis of variance. In this study, we examine the usefulness of these transformations by reanalyzing field-collected data from a split-plot experiment and by performing a more comprehensive simulation study of factorial and split-plot experiments. For field-collected data, significant interactions were dependent upon the type of transformation. For the simulation study, Poisson distributed errors were used for a 2 by 2 factorial arrangement, in both randomized complete block and split-plot settings. Various sizes of main effects were induced, and type I error rates and powers of the tests for interaction were examined for the raw response values, log-, square root-, and rank-transformed responses. The aligned rank transformation also was investigated because it has been shown to perform well in testing interactions in factorial arrangements. We found that for testing interactions, the untransformed response and the aligned rank response performed best (preserved nominal type I error rates), whereas the other transformations had inflated error rates when main effects were present. No evaluations of the tests for main effects or simple effects have been conducted. Potentially these transformations will still be necessary when performing these tests.     

Long-term mechanical loading of chondrocyte-chitosan biocomposites in vitro enhanced their proteoglycan and collagen content

One major problem in cartilage tissue engineering is the insufficient biochemical composition of the generated biocomposites. The aim of this study was to improve the collagen and proteoglycan deposition in tissue engineering constructs by application of long-term mechanical loading in culture. Chondrocyte-seeded chitosan biocomposites revealed a homogenous cell distribution, high viability (>95%) and maintenance of a rounded cell shape typical for chondrocytes over 3 weeks of load-free culture. Cyclic compression of chitosan biocomposites (0.1 Hz, amplitude 5-15%, 45 min on and 315 min off) was applied after two different preculture times (3, 21 days) for 3 weeks. At day 42 this resulted in enhanced mRNA levels for aggrecan and a significantly higher specific proteoglycan (5-fold, p<0.0002) and collagen type II (2-fold, p<0.02) deposition compared to unloaded controls. In sum, the chitosan scaffold was highly attractive for cartilage tissue engineering approaches and mechanical loading allowed to further improve the biochemical composition of these biocomposites.     

Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis in mammalian hosts

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.     

Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation

Serotonin (5-hydroxytryptamine, 5-HT) is, without doubt, the neurotransmitter for which the number of receptors is the highest. Fifteen genes encoding functional 5-HT receptors have been cloned in mammalian brain. 5-HT₃ receptors are ionotropic receptors, whereas all the others are metabotropic G-protein-coupled receptors (GPCRs). 5-HT receptor diversity is further increased by post-genomic modifications, such as alternative splicing (up to 10 splice variants for the 5-HT₄ receptor) or by mRNA editing in the case of 5-HT_2C receptors. The cellular and behavioral implications of 5-HT_2C receptor editing are of great physiological importance. Signaling of 5-HT receptors involves a great variety of pathways, but only some of these have been demonstrated in neurons. The classical view of neurotransmitter receptors localized within the synaptic cleft cannot be applied to 5-HT receptors, which are mostly (but not exclusively) localized at extra-synaptic locations either pre- or post-synaptically. 5-HT receptors are engaged in pre- or post-synaptic complexes composed of many GPCR-interacting proteins. The functions of these proteins are starting to be revealed. These proteins have been implicated in targeting, trafficking to or from the membrane, desensitization, and fine-tuning of signaling.