A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative

(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent.     

Effects of single-tryptophan mutations on R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The active form of the protein is a homotetramer possessing D(2) symmetry and a single active-site pore. Two tryptophans occur per monomer: W38 and its symmetry-related residues (W138, W238, and W338) occur at the dimer-dimer interfaces, while W45 and its symmetry-related partners (W145, W245, and W345) occur at the monomer-monomer interfaces. Two single-tryptophan mutant genes were constructed to determine the structural and functional consequences of four mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitors ligand binding and a pH dependent tetramer right harpoon over left harpoon 2 dimers equilibrium. In contrast, four simultaneous W38F mutations at the dimer-dimer interfaces result in tetramer destabilization. The ensuing dimer is relatively inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spectra indicates the fluorescent signal of wild-type R67 DHFR is dominated by the contribution from W38. Equilibrium unfolding/folding curves at pH 5.0, where all protein variants are dimeric, indicate the environment monitored by the W38 residue is slightly less stable than the environment monitored by the W45 residue.     

Intracellular changes of trehalose content in toluene tolerant Pseudomonas sp. BCNU 171 after exposure to toluene

Pseudomonas sp. BCNU 171, when grown with 10% (v/v) toluene for 2 h, accumulated approximately 7.7 mM trehalose probably arising by the action of trehalose-6-phosphate synthase (E.C. 2.4.1.15). Trehalose may thus play a significant role in the tolerance of the Pseudomonas sp. to toluene.     

Karyotypes of three somaclonal variants and wild plants ofAllium tuberosum by bicolor FISH

Using the fluorescence in situ hybridization (FISH) technique, we conducted karyotype analyses to identify the lost chromosomes in three somaclonal variants obtained from tissue culture of wildAllium tuberosum (2n = 4X = 32). The three lost chromosomes of the At29 variant (2n = 29) were all chromosome 2, the two for At30 (2n = 30) were chromosomes 7 and 8, and At31 was missing chromosome 2. Chromosome compositions of these variants were confirmed as being fixed lines during two years of greenhouse cultivation. The bicolor FISH technique, involving both 5S and 18S–5.8S–26S ribosomal RNA genes as probes, was used to assign chromosomal locations and to confirm whether the lost chromosomes contained any rRNA markers. The 5S rRNA gene signals in all variants as well as the wild type were detected as two sets, one on the intercalary region of the short arm of chromosome 3, the other on the intercalary region of the long arm of chromosome 6. One 18S–5.8S–26S rRNA gene site on the secondary constriction included a flanking satellite and terminal region on the short arm of chromosome 8. Signals of the 18S–5.8S–26S rRNA gene in At30 showpd in only three chromosomes, indicating that one of the lost chromosomes was chromosome 8. Overall, three marker chromosomes were established by FISH, using rRNA multigene families.     

Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Engineering antibody fragments to fold in the absence of disulfide bonds

Disulfide bonds play a critical role in the stabilization of the immunoglobulin β-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.     

Butein,a tetrahydroxychalcone,suppresses pro-inflammatory responses in HaCaT keratinocytes

Up-regulation of cell adhesion molecules and proinflammatory cytokines contributes to enhanced monocyte adhesiveness and infiltration into the skin, during the pathogenesis of various inflammatory skin diseases, including atopic dermatitis. In this study, we examined the anti-inflammatory effects of butein, a tetrahydroxychalcone, and its action mechanisms using TNF-α-stimulated keratinocytes. Butein significantly inhibited TNF-α-induced ICAM-I expression and monocyte adhesion in human keratinocyte cell line HaCaT. Butein also decreased TNF-α-induced pro-inflammatory mediators, such as IL-6, IP-10 and MCP-1, in HaCaT cells. Butein decreased TNF-α-induced ROS generation in a dose-dependent manner in HaCaT cells. In addition, treatment of HaCaT cells with butein suppressed TNF-α-induced MAPK activation. Furthermore, butein suppressed TNF-α-induced NF-kappaB activation. Overall, our results indicate that butein has immunomodulatory activities by inhibiting expression of proinflammatory mediators in keratinocytes. Therefore, butein may be used as a therapeutic agent for the treatment of inflammatory skin diseases. [BMB Reports 2015; 48(9): 495-500]     

Active Immunization with Extracellular Vesicles Derived from Staphylococcus aureus Effectively Protects against Staphylococcal Lung Infections,Mainly via Th1 Cell-Mediated Immunity

Staphylococcus aureus is an important pathogenic bacterium that causes various infectious diseases. Extracellular vesicles (EVs) released from S. aureus contain bacterial proteins, nucleic acids, and lipids. These EVs can induce immune responses leading to similar symptoms as during staphylococcal infection condition and have the potential as vaccination agent. Here, we show that active immunization (vaccination) with S. aureus-derived EVs induce adaptive immunity of antibody and T cell responses. In addition, these EVs have the vaccine adjuvant ability to induce protective immunity such as the up-regulation of co-stimulatory molecules and the expression of T cell polarizing cytokines in antigen-presenting cells. Moreover, vaccination with S. aureus EVs conferred protection against lethality induced by airway challenge with lethal dose of S. aureus and also pneumonia induced by the administration of sub-lethal dose of S. aureus. These protective effects were also found in mice that were adoptively transferred with splenic T cells isolated from S. aureus EV-immunized mice, but not in serum transferred mice. Furthermore, this protective effect of S. aureus EVs was significantly reduced by the absence of interferon-gamma, but not by the absence of interleukin-17. Together, the study herein suggests that S. aureus EVs are a novel vaccine candidate against S. aureus infections, mainly via Th1 cellular response.