Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.     

Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease

Via cytoplasmic signal transduction pathways, cytokines induce a variety of biological responses and modulate the outcome of inflammatory diseases and malignancies. Crohn's disease is a chronic inflammatory bowel disease of unknown etiology. Perturbation of the intestinal cytokine homeostasis is believed to play a pivotal role, but the pathogenesis of Crohn's disease is not fully understood. Here, we study intestinal T cells from Crohn's disease and healthy volunteers. We show that STAT3 and STAT4 are constitutively activated in Crohn's patients but not in healthy volunteers. The activation is specific, because other STAT proteins are not constitutively activated. Furthermore, the STAT3 regulated protein, SOCS3, is also constitutively expressed in Crohn's patients but not in healthy volunteers. Taken together, these data provide evidence of abnormal STAT/SOCS signaling in Crohn's disease. This aberrant activation, so far noted only in malignant cells, establish a new critical approach for better understanding the immunopathogenesis of Crohn's disease.     

Phenological variation within and among populations of Plathymenia reticulata in Brazilian Cerrado, the Atlantic Forest and transitional sites

BACKGROUND AND AIMS: Plathymenia reticulata (Leguminosae) is a Brazilian tree that occurs in two biomes: Cerrado, a woody savanna vegetation, and the Atlantic Forest, a tropical forest. In this study, phenological patterns and their variability within and among populations located in these biomes and in transitional zones between them were assessed. METHODS: During a 15-month period, individuals from two populations in Cerrado, two in the Atlantic Forest, and six in transitional zones (three in a cerrado-like environment and three in forest fragments) were evaluated in Minas Gerais State, Brazil. The individuals were evaluated monthly according to the proportion of the canopy in each vegetative phenophase (leaf fall, leaf flush and mature leaves) and each reproductive phenophase (floral buds, flowers, immature fruits and mature fruit/seed dispersal). In order to assess the phenological variability within and among populations, habitats and biomes, the Shannon-Wiener diversity index, the Morisita-Horn similarity index and genetic population approach of partitioning diversity were used. KEY RESULTS: Populations of P. reticulata, in general, showed similar phenology; the main differences were related to leaf fall, a process that starts months earlier in the Cerrado than in transitional sites, and even later in forest areas. Considerable synchrony was observed for reproductive phenology among populations and between biomes. Most phenological diversity was due to differences among individuals within populations. CONCLUSION: In spite of environmental differences, P. reticulata from the Atlantic Forest and Cerrado showed similar phenological behavior with only about 10% of the total diversity being attributed to differences between biomes.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

A Taxonomic Search Engine: Federating taxonomic databases using web services

Background  

The taxonomic name of an organism is a key link between different databases that store information on that organism. However, in the absence of a single, comprehensive database of organism names, individual databases lack an easy means of checking the correctness of a name. Furthermore, the same organism may have more than one name, and the same name may apply to more than one organism.     

Wildlife strike risk assessment in several Italian airports: lessons from BRI and a new methodology implementation

The presence of wildlife in airport areas poses substantial hazards to aviation. Wildlife aircraft collisions (hereafter wildlife strikes) cause losses in terms of human lives and direct monetary losses for the aviation industry. In recent years, wildlife strikes have increased in parallel with air traffic increase and species habituation to anthropic areas. In this paper, we used an ecological approach to wildlife strike risk assessment to eight Italian international airports. The main achievement is a site-specific analysis that avoids flattening wildlife strike events on a large scale while maintaining comparable airport risk assessments. This second version of the Birdstrike Risk Index (BRI2) is a sensitive tool that provides different time scale results allowing appropriate management planning. The methodology applied has been developed in accordance with the Italian Civil Aviation Authority, which recognizes it as a national standard implemented in the advisory circular ENAC APT-01B.     

Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture biofilm

doi: 10.1111/j.1741‐2358.2010.00378.x Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture biofilm Objective: To evaluate the antimicrobial action of effervescent tablets and ultrasound on Candida spp. and mutans streptococci from denture biofilm. Background: It is not uncommon for edentulous patients to be elderly and find it difficult to brush their dentures. Hence, auxiliary methods are required for cleansing dentures as well as treating oral infections. Materials and methods: Seventy‐seven complete denture wearers were randomly assigned into four groups: (A) Brushing with water (control); (B) Effervescent tablets; (C) Ultrasonic device (Ultrasonic Cleaner, model 2840 D); (D) Effervescent tablets and ultrasonic device. All groups brushed their dentures with a specific brush and water, three times a day, before applying their treatments. Denture biofilm was collected at baseline and after 21 days. The samples were collected by brushing the dentures with saline and the detached microbial cells were quantified by plating. Counts [log (CFU+1) ml^?1] of total aerobes, Candida spp. and mutans streptococci were compared by one‐way anova or Kruskal–Wallis test (α = 0.05). Results: No significant difference was found among the methods from C. albicans (p = 0.76), C. tropicalis (p = 0.94) and C. glabrata (p = 0.80). Lower counts were found for methods B and D when compared with the other methods against mutans streptococci (p < 0.001). Method B showed lower total aerobic counts than A, whereas C and D showed intermediate results (p = 0.011). Conclusion: The effervescent tablets significantly reduced mutans streptococci and total aerobes from denture biofilm. However, they was not as effective against C. albicans. Ultrasonic cleansing presented a discrete antimicrobial effect and was less effective than the tablets for complete denture disinfection.     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.