Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

Genetic and molecular analysis in Cristobalina sweet cherry,a spontaneous self-compatible mutant

Self-compatibility in a naturally self-incompatible species like sweet cherry is a highly interesting trait for breeding purposes and a powerful tool with which to investigate the basis of the self-incompatible reaction in gametophytic systems. However, natural self-compatibility in sweet cherry is a very rare phenomenon. Cristobalina is a local Spanish sweet cherry cultivar that has proven to be spontaneously self-compatible. In this work, the nature of the self-compatibility in Cristobalina has been studied using genetic and molecular approaches. Pollination studies and microscopic observations of pollen tube growth were carried out to confirm the self-compatible character and the results obtained indicate that self-compatibility is caused by a failure of the pollen and not the style factor. Polymerase chain reaction (PCR) analysis of progenies derived from Cristobalina revealed that self-compatibility in this genotype is not related uniquely to one of the two pollen S alleles, but that pollen grains carrying either of the two haplotypes can overcome the incompatibility barrier. Moreover, PCR analysis and microscopic observation of pollen tube growth in progeny derived from Cristobalina also confirmed that the self-compatible descendants can carry either of the two S haplotypes of their progenitor. Isolation and sequencing of the style S-RNases and pollen SFBs revealed that the DNA sequences of these factors are the same as those described in other self-incompatible sweet cherry cultivars with the same S alleles. Possible mechanisms to explain self-compatibility in Cristobalina are discussed.     

Biennial acorn maturation and its relationship with flowering phenology in Iberian populations of <Emphasis Type="Italic">Quercus suber</Emphasis>

Since the XIX century, there is a controversy about the existence of biennial maturation of the acorns in Quercus suber L. While some authors recognised biennial cycles as an adaptation to habitats with short vegetative periods, other authors discarded the biennial pattern. Successive flowering events from spring to autumn and annual acorn ripening are proposed as an explanation of the multiple acorn crops typical of Iberian forests. To clarify this discussion, the presence of annual and biennial acorns was assessed in seven cork oak stands, covering a wide range of environmental conditions. In each stand, 100 individuals were sampled once in spring and once in autumn. Biennial acorns were observed with variable frequencies in all populations. There was a significant and positive relationship between latitude and the percentage of trees with biennial acorns within northern and central populations. On the contrary, this trend was not significant among southern populations. The hypothesis that the presence of biennial acorns in Quercus suber is related to individual female flowering phenology was confirmed in four populations located in the southwestern Iberian Peninsula. Unregarding local differences in the distribution of phenological stages anticipated trees bore significantly less biennial acorns than delayed individuals of the same stand. This result is coherent with the idea that the length of the vegetative period plays a crucial role in the frequency of annual and biennial acorn ripening patterns. The relationship between annual and biennial ripening cycles and the multiple acorn crops is discussed.     

Differential responses of three fungal species to environmental factors and their role in the mycorrhization of<Emphasis Type="Italic"> Pinus radiata</Emphasis> D. Don

Three ectomycorrhizal (ECM) isolates of Rhizopogon luteolus, R. roseolus and Scleroderma citrinum were found to differ markedly in their in vitro tolerance to adverse conditions limiting fungal growth, i.e. water availability, pH and heavy metal pollution. S. citrinum was the most sensitive, R. luteolus intermediate and R. roseolus the most tolerant species. Pinus radiata D. Don seedlings were inoculated in the laboratory and in a containerised seedling nursery with spore suspensions of the three ECM species. Colonisation percentage was considerably lower under nursery conditions, probably due to competition by native fungi. The effects of nursery ECM inoculation on seedling growth depended on the fungal species. Only R. roseolus-colonised plants showed a significantly higher shoot growth than non-mycorrhizal plants. All three fungi induced significantly higher root dry weights relative to control plants. Despite the low mycorrhizal colonisation, mycorrhization with all three species improved the physiological status of nursery-grown seedlings, e.g. enhanced root enzyme activity, shoot nutrient and pigment content, net photosynthesis rate and water use efficiency. Of the three fungal species, R. roseolus was the most effective; this species was also the most adaptable and showed the greatest range of tolerance to adverse environmental conditions in pure culture. It is, therefore, proposed as a promising fungal species for ECM inoculation of P. radiata in the nursery.     

In<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>, the effect of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on ATP,but not on glyceraldehyde-3-phosphate dehydrogenase,depends on the glucose concentration

As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively. Depending on the glucose concentration in the medium, the effect of the addition of H₂O₂ on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed. In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged. The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H₂O₂. Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose.     

Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer--dimer equilibrium

In mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is implicated in the regulation of the cellular levels of asymmetric methylarginines, small molecule metabolites that themselves represent a family of endogenous inhibitors of nitric oxide synthase (NOS). The involvement of DDAH function in the regulation of NOS makes this enzyme a potentially attractive therapeutic target. DDAH from the bacterium Pseudomonas aeruginosa (PaDDAH) is so far the only structurally tractable homologue of mammalian DDAH isoforms. To complement the recent crystal structure of this protein, we show by hydrodynamic measurements that PaDDAH exists in dynamic equilibrium between monomer (ca 29 kDa) and symmetric homodimer (ca 58 kDa) states with a dimer dissociation constant, K(d) approximately 500nM. For the purposes of NMR-based approaches to the study of this enzyme's interactions with substrate and inhibitor ligands, it would be useful to obtain the protein in monomeric form. Through detailed analysis of the homodimer PaDDAH crystal structure we identified key residues involved in the protomer-protomer interface and targeted these for mutation. The hydrodynamic and self-associative properties of a series of PaDDAH interface mutants were analyzed by concentration-dependent analytical size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation. The individual substitution of several of the interface residues shifts the equilibrium position towards the monomer, which allowed the design of a double mutant variant (Arg40-->Glu, Arg98-->His) that behaves exclusively as a stable monomer, yet retains greater than 95% catalytic activity compared to wild-type. Comparative two-dimensional (1)H, (15)N heteronuclear NMR spectra indicate that the double mutant remains a monomer even at approximately 1 mM concentration. Accordingly, the double mutant PaDDAH is an attractive template for further NMR-based investigations of the enzyme mechanism and characterization of ligand-binding and inhibitor-binding profiles. These results indicate that dimerization of PaDDAH is not critical for the maintenance of the biological function of the protein. These results are discussed in the context of known modes of self-association between structurally related, but functionally distinct, members of the beta/alpha-propeller fold class.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Habitat-dependent Ecotype Micro-distribution at the Mid-shore in Natural Populations of <Emphasis Type="Italic">Littorina saxatilis</Emphasis>

On the NW coast of Spain, there is a hybrid zone in natural populations of L. saxatilis, where the largest ecotype is adapted to the desiccation and heat stress of the upper shore, whereas the smaller one is adapted to the wave impacts of the lower shore. The two ecotypes meet and hybridise in a mid-shore area producing intermediate individuals or hybrids. It has been postulated that this hybrid zone is maintained by a selection-gradient model that assumes habitat-dependent selection for the different environments of upper and lower shore areas. In this study, we focus on the hybrid (mid-shore) area, where a transition occurs between upper-shore and lower-shore environments, in order to ascertain whether the habitat-dependent selection is maintained at the micro-habitat scale. We present data on snail density at several levels of the mid-shore (varying accordingly in the relative frequency of mussels and barnacles) obtained for three seasons in three consecutive years and three localities. In the mid-shore, the abundance of the RB ecotype increases with increasing tidal height, whereas the abundance of the SU ecotype increases with decreasing tidal height, suggesting that habitat-dependent selection is maintaining the ecotype micro-distribution.     

CFTR haplotype distribution in the Brazilian Western Amazonian region

Two hundred twenty-one individuals from four groups located around the Brazilian town of Porto Velho, Rond?nia, were studied in relation to four sites located within or near the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The allele frequencies, when considered individually, do not depart markedly from frequencies obtained from other populations of mainly European descent. However, when haplotypes were estimated, two of the groups departed markedly from other Brazilian and non-Brazilian samples. This finding is probably related to the complex multiethnic origin of these groups.     

Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (H_e) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (H_o) and expected heterozygosities (H_e) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( _IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (_IT ) was 0.98 and the genetic diversity indexes among populations, _ST and _ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.