Molecular

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients' wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hy...

Mutations in the beta-amyloid precursor protein gene (APP) have been found in familial early-onset Alzheímer’s disease (AD). DNA variants at several genes have been linked to the risk of developing the most common late-onset form of AD (LOAD). A few studies analyzed the contribution of APP variants to LOAD, with negative or conflicting results. We determined the variation in the 18 APP exons and flanking intronic sequences in a total of 350 LOAD patients from Spain. A total of 13 nucleotide changes were found and 6 were new and not found among 340 healthy controls, including the only missense change (D243N). The in silico analysis suggested that none of them would have an effect on pre-mRNA splicing or protein folding (D243N). Patients and controls were also genotyped for three APP promoter polymorphisms, and none of them was significantly associated with LOAD. We concluded that APP variants would not contribute to the risk of developing LOAD in our population.

An efficient and direct procedure has been developed for the preparation of amidoalkyl naphthols by a one-pot condensation of aryl aldehydes, β-naphthol and urea or amides, in the presence of potassium dodecatungstocobaltate trihydrate [K5CoW12O40 · 3H2O, (1 mol%)] as a heterogeneous catalyst. The reactions were carried out in 1,2-dichloroethane at room temperature or under solvent-free media at elevated temperature. The present methodology offers several advantages such as excellent yields, simple procedure and the catalyst exhibited remarkable reusability.

Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC, pagD, ugtL, mig-14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co-regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed ‘SlyA-box’. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP activation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

Mahogany (Swietenia macrophylla, Meliaceae) is the most valuable and intensively exploited Neotropical tree. No information is available regarding the genetic structure of mahogany in South America, yet the region harbours most of the unlogged populations of this prized hardwood. Here we report on the genetic diversity within and the differentiation among seven natural populations separated by up to 2100 km along the southern arc of the Brazilian Amazon basin. We analysed the variation at eight microsatellite loci for 194 adult individuals. All loci were highly variable, with the number of alleles per locus ranging from 13 to 27 (mean = 18.4). High levels of genetic diversity were found for all populations at the eight loci (mean HE = 0.781, range 0.754–0.812). We found moderate but statistically significant genetic differentiation among populations considering both estimators of FST and RST, θ = 0.097 and ρ = 0.147, respectively. Estimates of θ and ρ were significantly greater than zero for all pairwise population comparisons. Pairwise ρ-values were positively and significantly correlated with geographical distance under the isolation-by-distance model. Furthermore, four of the populations exhibited a significant inbreeding coefficient. The finding of local differentiation among Amazonian mahogany populations underscores the need for in situ conservation of multiple populations of S. macrophylla across its distribution in the Brazilian Amazon. In addition, the occurrence of microgeographical genetic differentiation at a local scale indicates the importance of maintaining populations in their diverse habitats, especially in areas with mosaics of topography and soil.

Lipase inhibitors have generated a great interest because they could help in the prevention or the therapy of lipase-related diseases. Therefore, the aim of the work was to evaluate by HPLC, and using Candida rugosa lipase as model, the inhibitory effect of several saponins: β-aescin, digitonin, glycyrrhizic acid (GA) and Quillaja saponin (QS); flavonoids: 3-hydroxyflavone, 5-hydroxyflavone, (±)-catechin and kaempferol; and alkaloids: aspidospermine, papaverine, physostigmine, pilocarpine, raubasine, rescinnamine, reserpine and trigonelline.The inhibition produced by most of these compounds is described here for the first time. Saponins appeared very active, being β-aescin and digitonin the most active compounds (IC50 = 0.8–2.4 × 10−5 M). The inhibitory activity of flavonoids was lower than that of saponins (except GA), and (±)-catechin and kaempferol were the most active. Alkaloids was the most heterogeneous group assayed, varying from rescinnamine, with an IC16 similar to that of digitonin, to papaverine and others which showed almost no inhibition.In conclusion, β-aescin, digitonin, kaempferol or (±)-catechin, strong lipase inhibitors with a low toxicity and present herbal drugs used for lipase-related diseases such as acne or ulcer, are promising candidates for the prevention or the treatment of these diseases.

PurposeMelanolipofuscin (MLF) is a complex granule, exhibiting properties of both melanosomes and lipofuscin (LF) granules, which accumulates in retinal pigment epithelial (RPE) cells and may contribute to the etiology of age-related macular degeneration (AMD). MLF accumulation has been reported by Feeney-Burns to more closely reflect the onset of AMD than the accumulation of lipofuscin. In an effort to assess the possible contribution MLF may have to the onset of AMD, we analyzed the phototoxicity and protein composition of MLF and compared those results to that of LF.MethodsSpecifically, we observed the accumulation of MLF in human RPE from different decades of life, and assessed the phototoxicity of these granules. We also employed fluorescence spectroscopy, atomic force microscopy, transmission and scanning electron microscopy and proteomic analysis to examine the composition of MLF granules in an effort to ascertain their origin.ResultsOur results show that MLF granules are phototoxic and their accumulation more closely reflects the onset of AMD than does LF accumulation. Our compositional analysis of MLF has shown that while these granules contain some similarities to LF granules, MLF is substantially different. Of significant interest is the finding that MLF, in contrast to LF, does not contain photoreceptor-specific proteins, suggesting that MLF may not originate from the phagocytosis of photoreceptor outer segments. Instead the presence of RPE- and melanosome-specific proteins would suggest that MLF accumulates as a result of the melanosomal autophagocytosis of RPE cells.ConclusionsOur results provide significant insight into understanding the formation and toxicity of MLF and suggest a possible contribution to the etiology of retinal diseases.

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding