Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE,genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.     

A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes

Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone‐replacement system provides a well‐defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism.     

Screening of Biological Activities of a Series of Chalcone Derivatives against Human Pathogenic Microorganisms

In an effort to develop new antimicrobial agents, a series of chalcone derivatives, 3 – 60 , were prepared by Claisen–Schmidt condensation of appropriate acetophenones and 2‐furyl methyl ketones with appropriate aromatic aldehydes, furfural, and thiophene‐2‐carbaldehyde in an aqueous solution of NaOH and EtOH at room temperature. The synthesized compounds were characterized by means of their IR‐ and NMR‐spectral data, and elemental analysis. All compounds were tested for their antibacterial and antifungal activities by the disc diffusion method. For the most active compounds, also minimum inhibitory concentrations (MICs) were determined.     

The mutagenic and antimutagenic effects of Ecballium elaterium fruit juice in human peripheral lymphocytes

The aim of this study was to investigate the mutagenic and antimutagenic effects of Ecballium elaterium (EE) fruit juice, which has an anti-inflammatory effect, using in vitro human peripheral lymphocytes. To investigate the mutagenic effects of the EE fruit juice, human peripheral lymphocytes were treated with three doses (18, 36, and 72 μl/l) of fruit juice alone for 24 and 48 h. For investigating the antimutagenic effects of the EE fruit juice, the human lymphocytes were also treated with the mixture of the fruit juice and 0.25 μg/ml MMC. The EE fruit juice induced the percentage of total CA when used alone (especially the percentage of structural CA than the percentage of the numerical CA) and synergically induced the percentage of total CA when used as a mixture with MMC. The EE fruit juice did not affect the SCE frequency for 24 and 48 h treatment time. In contrast, EE and MMC as a mixture sinergically induced the SCE frequency at the highest concentration for 48 h treatment time only. EE alone did not decrease the RI while it decreased the MI in a dose-dependent manner. EE and MMC as a mixture have a higher cytotoxic effect than the cytotoxic effects of EE alone. As a result, it can be concluded that EE had no antimutagenic effect while EE had a mutagenic and a cytotoxic effect in human peripheral lymphocytes. This article was submitted by the authors in English.     

Cloning,expression and characterization of endo‐β‐1,4‐mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo‐β‐1,4‐mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA ) and the alcohol oxidase (AOX1 ) promoters, respectively. The highest production of mannanase (352 U mL^?1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL^?1). The fold increase in mannanase production was estimated as ～12‐fold and ～2‐fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (～350 U mg^?1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The InterPro Database, 2003 brings increased coverage and new features

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created in 1999 as a means of amalgamating the major protein signature databases into one comprehensive resource. PROSITE, Pfam, PRINTS, ProDom, SMART and TIGRFAMs have been manually integrated and curated and are available in InterPro for text- and sequence-based searching. The results are provided in a single format that rationalises the results that would be obtained by searching the member databases individually. The latest release of InterPro contains 5629 entries describing 4280 families, 1239 domains, 95 repeats and 15 post-translational modifications. Currently, the combined signatures in InterPro cover more than 74% of all proteins in SWISS-PROT and TrEMBL, an increase of nearly 15% since the inception of InterPro. New features of the database include improved searching capabilities and enhanced graphical user interfaces for visualisation of the data. The database is available via a webserver (http://www.ebi.ac.uk/interpro) and anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).     

The effect of various liposome formulations on insulin penetration across Caco-2 cell monolayer

The aim of the study was to determine the penetration properties of various insulin containing liposome formulations through Caco-2 cell monolayer and to compare the in vitro test results with in vivo tests. The effect of sodium taurocholate as a penetration enhancer when it was added to the liposome formulation was also investigated. In vitro permeation experiments were performed in diffusion cells with the Caco-2 cell monolayer used as the membrane. Permeability values of various insulin containing liposome formulations through Caco-2 cells were determined (log k(insulin-solution) = -2.217 +/- 0.0723 cm.h(-1), log k(insulin-liposome) = -2.141 +/- 0.0625 cm.h(-1), log k(insulin-sodium tauroholate liposome)= -1.952 +/- 0.0623 cm.h(-1)). In vivo tests were performed in mice. Formulations were administered orally and blood glucose levels were determined and penetrations were compared with the Caco-2 cell experiment results. In conclusion, the permeability of insulin was increased across Caco-2 cell monolayer when the liposome sodium taurocholate (NaTC) formulation was used. The oral administration of insulin and NaTC incorporated liposomes significantly decreased blood glucose levels. Furthermore, it was shown that a high in vitro/in vivo correlation was observed using the Caco-2 cell monolayer model.     

Analyzing the Impact of Ramalina digitellata,R. fastigiata,R. fraxinea,and R. polymorpha's Usnic Acid Concentration on Antioxidant,DNA-Protective,Antimicrobial, and Cytotoxic Properties

The present study is focused on the antimicrobial, antioxidant, cytotoxic, and DNA protective effects of methanol extract obtained from R. digitellata, R. fastigiata, R. fraxinea, and R. polymorpha species that are distributed in Turkey. The highest total phenol content was determined in R. digitellata (144.6 mgGAE/g_extract), and the highest total amount of flavonoids was found in R. fastigiata (20.40 mgGAE/g_extract). The content of usnic acid was determined by High-Performance Liquid Chromatography (HPLC) and the highest amount was found in R. digitellata. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2’-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] radical scavenging methods were used for antioxidant activity. R. fraxinea showed the highest DPPH⋅ and ABTS⁺⋅ scavenging activity. In addition, the DNA protective effect was investigated using pBR322 plasmid DNA, and; all studied species were found to have DNA protective effects. The antibacterial activity was investigated using the disc diffusion method, and the R. digitellata methanol extract showed the best results with a 12.35 mm zone on Proteus mirabilis. On the human lung cancer (A549) and breast cancer (MDA-MB-231) cell lines, cytotoxic activity was assessed using an MTT assay. All lichen extracts were found to have a significant cytotoxic effect on both cancer cell lines at 1000 μg/mL concentration. These results suggest that Ramalina species may be potential candidates for developing new phytopharmaceuticals and functional components.     

Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values of methyl benzoates were determined. These compounds inhibited LPO with K_i values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (K_i=0.033±0.004 μM). The most potent inhibitor ( 1 a ) showed with a docking score of −3.36 kcal/mol and an MM-GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives ( 1 a – 16 a ) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).     

Novel Quinazolinone Derivatives: Potential Synthetic Analogs for the Treatment of Glaucoma,Alzheimer's Disease and Diabetes Mellitus

Quinazolinones, which represent an important part of nitrogen-containing six-membered heterocyclic compounds, are frequently used in drug design due to their wide biological activity properties. Therefore, the novel quinazolinones were synthesized from the reaction of acylated derivatives of 4-hydroxy benzaldehyde with 3-amino-2-alkylquinazolin-4(3H)-ones with good yields (85–94 %) and their structures were characterized using Fourier-transform Infrared (FT-IR), Nuclear Magnetic Resonance (¹H-NMR, ¹³C-NMR), and High-Resolution Mass Spectroscopy (HR-MS). As the application of the synthesized compounds, their inhibition properties of the synthesized compounds on α-Glucosidase (α-Glu), Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE), and Carbonic anhydrase I–II (hCA I–II) metabolic enzymes were investigated. All compounds showed inhibition at nanomolar level with the K_i values in the range of 12.73±1.26–93.42±9.44 nM for AChE, 8.48±0.92–25.84±2.59 nM for BChE, 66.17±5.16–818.06±44.41 for α-Glu, 2.56±0.26–88.23±9.72 nM for hCA I, and 1.68±0.14–85.43±7.41 nM for hCA II. Molecular docking study was performed to understand the interactions of the most potent compounds with corresponding enzymes. Also, absorption, distribution, metabolism, excretion, and toxicity (ADME/T) properties of the compounds were investigated.