Genomic analysis of Grapevine Retrotransposon 1 (Gret1) in Vitis vinifera

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret1 distribution between cultivars. The combined cytological and molecular results suggest that Gret1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.     

Identification of all FK506-binding proteins from Neurospora crassa

Immunophilins are intracellular receptors of immunosuppressive drugs, carrying peptidyl-prolyl cis-trans isomerase activity, with a general role in protein folding but also involved in specific regulatory mechanisms. Four immunophilins of the FKBP-type (FK506-binding proteins) were identified in the genome of Neurospora crassa. Previously, FKBP22 has been located in the endoplasmic reticulum as part of chaperone/folding complexes and FKBP13 has been found to have a dual location in the cytoplasm and mitochondria. FKBP11 is apparently located exclusively in the cytoplasm. It is not expressed during vegetative development of the fungus although its expression can be induced with calcium and during sexual development. Overexpression of the respective gene appears to confer a growth advantage to the fungus in media containing some divalent ions. FKBP50 is a nuclear protein and its genetic inactivation leads to a temperature-sensitive phenotype. None of these proteins is, alone or in combination, essential for N. crassa, as demonstrated by the isolation of a mutant strain lacking all four FKBPs.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.     

Development of a molecular detection method for naphthalene degrading pseudomonads

Abstract: A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze-thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten-fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 10³, P. putida G7 cfu (g soil)⁻¹ was detected. This method was successfully employed to detect indigenous naphthalene-degrading bacteria from subsurface sediment collected from different locations of a naphthalene-contaminated site. Similar approaches could be developed for other soil-borne genetic markers.     

IL-10 underlies distinct susceptibility of BALB/c and C57BL/6 mice to Mycobacterium avium infection and influences efficacy of antibiotic therapy

Increased production of IL-10 has been frequently associated with augmented susceptibility to infection. However, the correlation between IL-10 activity and susceptibility to mycobacterial infection is still uncertain. Although studies using transgenic mice overexpressing IL-10 consistently showed an increased susceptibility to mycobacterial infection, experimental approaches in which IL-10 activity was reduced or abrogated originated inconclusive data. We show here that this controversy might be due to the mouse strains used in the various experimental procedures. Our results show that BALB/c mice are more susceptible than C57BL/6 to Mycobacterium avium infection. This increased susceptibility of BALB/c mice is, to a great extent, due to distinct activity of IL-10 between the two mouse strains. In accordance, reduction of IL-10 activity through the administration of anti-IL-10R mAb, or the absence of IL-10 as studied in IL-10 knockout mice, clearly decreased the susceptibility of BALB/c mice to M. avium but had a less obvious effect in C57BL/6 mice. Moreover, abrogation of IL-10 activity in infected BALB/c mice increased the efficacy of antimycobacterial therapy, whereas for the C57BL/6 mice it produced no effect. These observations show that the activity of IL-10 in response to the same mycobacterial stimulus influences not only the susceptibility to infection but also the efficacy of antimycobacterial therapy. This should now be considered in the context of human response to mycobacterial infection, particularly as a possible strategy to improve treatment against infections by mycobacteria.     

Range-wide phylogeography and gene zones in Pinus pinaster Ait. revealed by chloroplast microsatellite markers

Some 1339 trees from 48 Pinus pinaster stands were characterized by five chloroplast microsatellites, detecting a total of 103 distinct haplotypes. Frequencies for the 16 most abundant haplotypes (p(k) > 0.01) were spatially interpolated over a lattice made by 430 grid points. Fitting of spatially interpolated values on raw haplotype frequencies at the same geographical location was tested by regression analysis. A range-wide 'diversity map' based on interpolated haplotype frequencies allowed the identification of one 'hotspot' of diversity in central and southeastern Spain, and two areas of low haplotypic diversity located in the western Iberian peninsula and Morocco. Principal component analysis (PCA) carried out on haplotypes frequency surfaces allowed the construction of a colour-based 'synthetic' map of the first three PC components, enabling the detection of the main range-scale genetic trends and the identification of three main 'gene pools' for the species: (i) a 'southeastern' gene pool, including southeastern France, Italy, Corsica, Sardinia, Pantelleria and northern Africa; (ii) an 'Atlantic' gene pool, including all the western areas of the Iberian peninsula; and (iii) a 'central' gene pool, located in southeastern Spain. Multivariate and AMOVA analyses carried out on interpolated grid point frequency values revealed the existence of eight major clusters ('gene zones'), whose genetic relationships were related with the history of the species. In addition, demographic models showed more ancient expansions in the eastern and southern ranges of maritime pine probably associated to early postglacial recolonization. The delineation of the gene zones provides a baseline for designing conservation areas in this key Mediterranean pine.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.