Novel quinazolinone derivatives as 5-HT7 receptor ligands

5-HT(7) receptor antagonists generated antidepressant-like effects in animal model and the involvement of the 5-HT(7) receptor in other pathophysiological mechanisms such as thermoregulation, learning and memory, and sleep has been highlighted by various studies. As one of our efforts to discover a new type of 5-HT(7) receptor antagonists, we here report on the synthesis and binding affinities to the 5-HT(7) receptor of the quinazolinone library 1, which was designed with various substituents (X, Y, R(1), and R(2)) on the aromatic rings and different carbon chain length. Total 85 compounds of the quinazolinone library 1 were synthesized and the binding affinities of all the synthesized compounds were obtained by radioligand binding assay for the 5-HT(7) receptor. Among the 85 compounds, 24 compounds show very good binding affinities with IC(50) values below 100 nM. Mainly the compounds with IC(50) values below 100 nM have o-OMe or o-OEt as R(2) substituent. The compound with the best binding affinity is 1-68 of which the IC(50) value is 12 nM. In in vivo animal study, some synthesized compounds really have the antidepressant activity in the forced swimming test in mice.     

Bacillus anthracis spores influence ATP synthase activity in murine macrophages

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be downregulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.     

Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus

The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6xHis) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6xHis tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short beta-sheet structure encompassing residues 7-13 and 19-25 that is stabilized by three disulfide bonds.     

Duplication of phospholipase C-delta gene family in fish genomes

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.     

An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography-chemical ionization-mass spectrometry

We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.     

The statistical neuroanatomy of frontal networks in the macaque

We were interested in gaining insight into the functional properties of frontal networks based upon their anatomical inputs. We took a neuroinformatics approach, carrying out maximum likelihood hierarchical cluster analysis on 25 frontal cortical areas based upon their anatomical connections, with 68 input areas representing exterosensory, chemosensory, motor, limbic, and other frontal inputs. The analysis revealed a set of statistically robust clusters. We used these clusters to divide the frontal areas into 5 groups, including ventral-lateral, ventral-medial, dorsal-medial, dorsal-lateral, and caudal-orbital groups. Each of these groups was defined by a unique set of inputs. This organization provides insight into the differential roles of each group of areas and suggests a gradient by which orbital and ventral-medial areas may be responsible for decision-making processes based on emotion and primary reinforcers, and lateral frontal areas are more involved in integrating affective and rational information into a common framework.     

Catalase from the white-spotted flower chafer, Protaetia brevitarsis: cDNA sequence, expression, and functional characterization

Catalase, which is one of the key enzymes of the cellular antioxidant defense system, prevents free hydroxyl radical formation by breaking down hydrogen peroxide into oxygen and water. Here, we show the cloning and characterization of a catalase gene in a coleopteran insect. This gene was isolated by searching the white-spotted flower chafer Protaetia brevitarsis cDNA library, and the gene itself encodes a protein of 505 amino acids in length, named PbCat. PbCat shows high similarities to the insect catalase genes known to date. The recombinant PbCat, which is expressed as a 56-kDa polypeptide in baculovirus-infected insect Sf9 cells, shows the highest activity at 30 degrees C and pH 7.0. Northern and Western blot analyses revealed the presence of PbCat in all tissues examined, showing its ubiquitous expression. P. brevitarsis larvae in which H(2)O(2) was overloaded, showed a marked up-regulation in PbCat expression. Moreover, P. brevitarsis larvae showed an apparent increase in PbCat expression even after a wounding through injection. These results indicate that PbCat is up-regulated after wounding and oxidative pressure induced by H(2)O(2), reflecting an important role of PbCat in H(2)O(2) scavenging.     

Production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. WN9 KCTC 8951P and its usefulness as a cement mortar admixture

The production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. and its suitability as a viscosity modifying admixture for cement mortar mixing were investigated. After 48 h culture in an optimized medium, cell growth and EPS production were 1,2 g/L and 4.0 g/L, respectively. By adding EPS WN9 to mortar, it was possible to prepare a homogeneous mortar without material segregation and excess air entrapment. The optimal amount of EPS addition to mortar was found to be 0.02 to 0.05%(w/w) of the cement used. Increasing the dosage of EPS WN9 from 0 to 0.05%(w/w) resulted in a setting retardation of 0.14 h to 0.8 h and an increase in the compressive strength of mortar of 10 to 20%.     

Effects of surface charge and ligand type of Au nanoparticles on green fluorescent protein-expressing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effects of the surface charge and ligand type of three types of Au nanoparticles (NPs), namely anionic polyethylene glycol (PEG)-Au NPs, anionic citrate (Cit)-Au NPs, and cationic branched polyethylenimine (bPEI)-Au NPs, on green fluorescent protein (GFP)-expressing Escherichia coli were evaluated through the combined analysis of optical density (OD) and fluorescence intensity (FI). OD and FI can provide information about cell growth and metabolism of bacteria, respectively. The results demonstrated that PEG- and Cit-Au NPs had no major effects on the OD and FI of GFP-expressing bacteria. However, it was found that Cit-Au NPs may slightly influence cell metabolism at higher concentrations, although it is necessary to perform further in-depth study to clarify this issue. Cationic bPEI-Au NPs showed significant effects on cell density and metabolism of E. coli, with an especially strong effect on metabolism. The combined analysis of OD and FI may be useful for monitoring the effects of a wide range of nanomaterials on microorganisms.