An efficient and convenient microwave-assisted chemical synthesis of (thio)xanthones with additional in vitro and in silico characterization

Xanthones and their thio-derivatives are a class of pleiotropic compounds with various reported pharmacological and biological activities. Although these activities are mainly determined in laboratory conditions, the class itself has a great potential to be utilized as promising chemical scaffold for the synthesis of new drug candidates. One of the main obstacles in utilization of these compounds was related to the difficulties in their chemical synthesis. Most of the known methods require two steps, and are limited to specific reagents not applicable to a large number of starting materials. In this paper a new and improved method for chemical synthesis of xanthones is presented. By applying a new procedure, we have successfully obtained these compounds with the desired regioselectivity in a shorter reaction time (50s) and with better yield (>80%). Finally, the preliminary in vitro screenings on different bacterial species and cytotoxicity assessment, as well as in silico activity evaluation were performed. The obtained results confirm potential pharmacological use of this class of molecules.     

Defective activation of ERK in macrophages lacking the p50/p105 subunit of NF-kappaB is responsible for elevated expression of IL-12 p40 observed after challenge with Helicobacter hepaticus

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-kappaB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-kappaB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-kappaB with H. hepaticus.     

Oligomeric forms of peptide fragment PrP(214-226) in solution are preferentially recognized by PrP(Sc)-specific antibody

A specific monoclonal antibody (mAb) V5B2 that discriminates between brain tissue of Creutzfeldt-Jakob disease patients and that from normal controls without proteinase K digestion has been prepared using a 13-residue synthetic peptide P1 from the primary structure of human PrP. In the light of the specific interaction between mAb V5B2 and the pathological isoform of PrP (PrP(Sc)), we investigated the solution behavior of antigen P1 and its interactions with mAb V5B2. Our results show that V5B2 recognizes epitope P1 in dimeric/oligomeric forms in solution and in the fibril-like aggregates, as well as in PrP(Sc) aggregates, and demonstrate that the specific epitope is present in all of these forms, but not in PrP(C).     

Myelin biogenesis: sorting out protein trafficking

Myelin biogenesis is a complex process involving coordinated exocytosis, endocytosis, mRNA transport and cytoskeletal dynamics. Recent studies indicate that soluble neuronal signals may control the surface expression of proteolipid protein, a process that involves reduced endocytosis and/or increased transport carrier recruitment from an intracellular pool.     

900 MHz modulated electromagnetic fields accelerate the clathrin‐mediated endocytosis pathway

We report new data regarding the molecular mechanisms of GSM‐induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 µs pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM–EMF/electric pulses in the presence of drugs inhibiting the clathrin‐ or the caveolin‐dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5–29.5 °C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin‐dependent endocytosis is stimulated by the GSM–EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin‐coated vesicles. Bioelectromagnetics 30:222–230, 2009. © 2008 Wiley‐Liss, Inc.     

Measuring Evolutionary Constraints Through the Dimensionality of the Phenotype: Adjusted Bootstrap Method to Estimate Rank of Phenotypic Covariance Matrices

The potential and direction of phenotypic evolution is constrained by the distribution of genetic variation for the traits as described by the phenotypic (P) and genetic covariance matrices (G). The rank of the covariance matrix reflects the number of independent variational dimensions of the phenotype. Covariance matrices with less than full rank indicate lack of variation in some directions of the phenotype space and thus are an indication of absolute evolutionary constraints. Because selection acts upon phenotypic variation, the rank of P represents the upper limit of the dimensionality in G, relevant for selection response. The limitations of current methods to estimate matrix rank motivated us to analyze and adjust a bootstrap method and evaluate its performance by simulation. The results show that the modified bootstrap method (ABRE) gives reliable and rather conservative rank estimates when the sample size is sufficient for the number of variables studied (the sample size is at least five-fold the number of variables). Applying the method to various datasets suggests high phenotypic dimensionality in all cases. The analysis thus provides no evidence for absolute evolutionary constraints. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determining Protein Complex Connectivity Using a Probabilistic Deletion Network Derived from Quantitative Proteomics

Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.     

Analysis of the Association between CIMP and BRAFV600E in Colorectal Cancer by DNA Methylation Profiling

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF^V600E) is tightly associated with CIMP, raising the question of whether BRAF^V600E plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF^V600E. We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF^V600E causes DNA hypermethylation by stably expressing BRAF^V600E in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF^V600E is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF^V600E and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF^V600E-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF^V600E-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF^V600E in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis.     

Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles in Sulfolobus sp. emerge from a specific budding process with similarity to the endosomal sorting pathway.