A Novel Pzg-NURF Complex Regulates Notch Target Gene Activity

Drosophila putzig was identified as a member of the TRF2–DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. Here, we show that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig–NURF chromatin complex required for epigenetic activation of Notch targets.     

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren’s syndrome salivary glands

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren’s syndrome (SS) remain to be elucidated. It is known that the TACE–AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins’ level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren’s syndrome.     

Preliminary characterization of some Streptomyces species isolated from a composting process and their antimicrobial potential

The aim of this study was to screen Streptomycetes isolates with antimicrobial and antiviral activity, in a search for new metabolites. The isolates were obtained from a composting process, and identified based on morphological characteristics and molecular biological methods. The antimicrobial activity was determined using the double-layer agar method against 53 test organisms (bacteria, yeasts, and filamentous fungi). All isolates were grown in submerged culture, in mineral salts-starch-casein (SC) broth and ISP2 media, and the filtrate cultures were used in the assays for antibacterial and antiviral activity. Bovine Herpes virus (BoHV-I) was used for the antiviral activity. The morphological and molecular characteristics confirmed that all 25 isolates belonged to the genus Streptomyces. In the assay for antimicrobial activity, 80% of the Streptomyces isolates were able to inhibit at least one of the test organisms. Of these, 80% were active against bacteria and 45% against fungi. Eight of the isolates showed a broad spectrum of inhibitory activity; of these, the isolate Streptomyces spp. 1S was able to inhibit 46 of the test organisms, and, most importantly, the 16 Gram-negative strains were inhibited. Of the 25 isolates, 44.4% of the isolates were able to grow and produce bioactive metabolites when grown in submerged culture. Four extracts showed a cytopathic effect in 10 CCID₅₀ MDBK cell, even though no viricidal effect was observed. The results obtained with these isolates indicated good biotechnological potential of these Streptomyces strains.     

Novel purine and pyrazolo[3,4-d]pyrimidine inhibitors of PI3 kinase-α: Hit to lead studies

Series of purine and pyrazolo[3,4-d]pyrimidine inhibitors of phosphatidylinositol-3-kinases (PI3K) have been prepared. The optimized purine inhibitors show good potency in a PI3K p110α (PI3K-α) fluorescence polarization assay with good selectivity versus PI3K p110γ (PI3K-γ) and the mammalian target of rapamycin (mTOR). The related pyrazolo[3,4-d]pyrimidines show potent PI3K-α and mTOR inhibition with good selectivity versus PI3K-γ. Representative compounds showed activity in a cellular proliferation assay against Caco-2 colorectal, LoVo colorectal and PC3MM2 prostate adenocarcinoma cancer cells. Signaling through the PI3K pathway was confirmed via inhibition of phospho-AKT in MDA-361 cells.     

Novel benzofuran-3-one indole inhibitors of PI3 kinase-α and the mammalian target of rapamycin: Hit to lead studies

A series of benzofuran-3-one indole phosphatidylinositol-3-kinases (PI3K) inhibitors identified via HTS has been prepared. The optimized inhibitors possess single digit nanomolar activity against p110α (PI3K-α), good pharmaceutical properties, selectivity versus p110γ (PI3K-γ), and tunable selectivity versus the mammalian target of rapamycin (mTOR). Modeling of compounds 9 and 32 in homology models of PI3K-α and mTOR supports the proposed rationale for selectivity. Compounds show activity in multiple cellular proliferation assays with signaling through the PI3K pathway confirmed via phospho-Akt inhibition in PC-3 cells.     

Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate‐active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin‐containing proteins bear catalytic modules from numerous carbohydrate‐active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip‐cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32‐kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate‐binding module 6 or carbohydrate‐binding module 22 xylan‐binding modules along dockerins. This newly identified xyl‐doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip‐cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Phylogeny of magpie‐robins and shamas (Aves: Turdidae: Copsychus and Trichixos): implications for island biogeography in Southeast Asia

Aim Magpie‐robins and shamas are forest and woodland birds of south Asia. There are two genera: Trichixos for the monotypic T. pyrrhopygus, and Copsychus for other species. Two species are widespread, whereas the others are restricted to specific islands. Endemicity is highest in the Philippines. Using phylogenetic methods, we examined how this group came to its unusual distribution. Location Mainland Asia from India to southern China, and islands from Madagascar to the Philippines. Particular emphasis is placed on the Greater Sundas and Philippines. Methods The phylogeny was estimated from DNA sequences of 14 ingroup taxa representing all nine currently recognized Copsychus and Trichixos species. The entire mitochondrial ND2 gene and portions of nuclear myoglobin intron 2 (Myo2) and transforming growth factor beta 2 intron 5 (TGFβ2‐5) were sequenced for all but two species. The phylogeny was reconstructed using maximum likelihood and Bayesian methods. The timing of divergence events was estimated using a relaxed molecular clock approach, and ancestral areas were examined using stochastic modelling. Results The group comprises three main clades corresponding to ecological types: Trichixos, a primary‐forest specialist; Copsychus magpie‐robins, open‐woodland and coastal species; and Copsychus shamas, thick‐forest species. Trichixos appears to be sister to the magpie‐robins, rendering Copsychus polyphyletic. The dating of phylogenetic nodes was too ambiguous to provide substantial insight into specific geographical events responsible for divergence within the group. Some patterns are nevertheless clear. Copsychus shamas reached the Philippines, probably in two separate invasions, and split into endemic species. Copsychus malabaricus and C. saularis expanded widely in the Greater Sundas and mainland Southeast Asia without species‐level diversification. Main conclusions Magpie‐robins are excellent dispersers and have diversified into distinct species only on isolated oceanic islands. Trichixos, a poor disperser, is restricted to mature forests of the Malay Peninsula, Sumatra and Borneo. Copsychus shamas are intermediate in habitat preference and dispersal capabilities. Their endemism in the Philippines may be attributed to early colonization and specialization to interior forests. In the Greater Sundas, C. malabaricus and C. saularis populations split and came together on Borneo to form two separate subspecies (of each species), which now hybridize.     

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 °C. Structural models of the complex with d(CGTACG)₂ were developed on the basis of DOSY, 2D NOESY and ³¹P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.     

Phosphorylation of the β1 Integrin Cytoplasmic Domain: Toward an Understanding of Function and Mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the β1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of β1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in β1 phosphorylation and selective loss of β1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with β1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with β1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with β1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with β1. A possible role for regulated β1 phosphorylation in cell migration is discussed.