Essential role of Duox in stabilization of Drosophila wing

NADPH oxidase produces reactive oxygen species (ROS). Drosophila melanogaster has two homologs of NADPH oxidase, dNox and dDuox, with functions that remain unclear in vivo. To clarify these functions, two independent transgenic fly lines expressing dsRNA targeted for different portions of dDuox mRNA were used. In both flies, en-GAL4> UAS-dDuoxIR(976-1145) and en-GAL4> UAS-dDuoxIR(370-518), in which dDuox was knocked down selectively in the posterior area of the wing disc, the posterior compartment of the adult wings became paler and more fragile with wing veins that were indistinct by comparison with the anterior one. Fluorescence staining of the en-GAL4> UAS-dDuoxIR(976-1145) adult wings revealed that the ROS concentration in the posterior compartment was significantly lower than that in the anterior compartment. Moreover, in these flies, the posterior compartment of the wing imaginal disc showed a greater number of apoptotic cells detected by immunostaining with anti-cleaved caspase-3 antibody than those in the anterior compartment. Respective knockdown of tyrosine hydroxylase or dopa-decarboxylase showed paler wing blades in the posterior compartment similar to the phenotype of dDuox-knockdown files. Along with this observation, analysis of the catecholic and dityrosine components in the wings of adult flies proved that dDuox plays important roles in the stabilization of the cuticle structure of the wings via tyrosine cross-linking, the sclerotization and melanization processes possibly through ROS production. These dDuox-knockdown fly lines would be useful tools for further studying dDuox functions during the development of Drosophila.     

Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum

A filamentous bacteriophage (?), ?RS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ?RS603 was found to have a circular single‐stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ?RS603 genome showed strong similarity with those of Ralstonia phages ?RSM1 and ?RSM3, as reported by Askora et al. The ?RS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ?RSM3. ?RS603 had an ORF that was homologous to other Ralstonia phages ?RSS0 and ?RSS1; however, ?RSM1 and ?RSM3 did not.

     

Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (approximately 10 microg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.     

Anti-viral effects of interferon administration on sevenband grouper, Epinephelus septemfasciatus

Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the present study, we report cloning and characterization of the IFN gene from the sevenband grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family. The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic Poly(I:C), which is known as an inducer of IFN. It has also been confirmed that injection of recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to sevenband grouper is effective in preventing severe symptom development after NNV infection.     

Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori

Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility, although this inference remains to be demonstrated. Here, we revised the experimental procedures including that for purification and definitely identified the purified initiatorin protein as an activation factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme. Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate, BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.     

Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism

Quercetin is a typical flavonoid ubiquitously present in fruits and vegetables, and its antioxidant effect is implied to be helpful for human health. The bioavailability of quercetin glycosides should be clarified, because dietary quercetin is mostly present as its glycoside form. Although quercetin glycosides are subject to deglycosidation by enterobacteria for the absorption at large intestine, small intestine acts as an effective absorption site for glucose-bound glycosides (quercertin glucosides). This is because small intestinal cells possess a glucoside-hydrolyzing activity and their glucose transport system is capable of participating in the glucoside absorption. A study using a cultured cell model for intestinal absorption explains that the hydrolysis of the glucosides accelerates their absorption in the small intestine. Small intestine is also recognized as the site for metabolic conversion of quercetin and other flavonoids as it possesses enzymatic activity of glucuronidation and sulfation. Modulation of the intestinal absorption and metabolism may be beneficial for regulating the biological effects of dietary quercetin.     

Comparison of the effects of pioglitazone and rosiglitazone on macrophage foam cell formation

In order to elucidate the antiatherogenic effects of pioglitazone (a peroxisome proliferator-activated receptor [PPAR]gamma agonist with PPARalpha agonistic activity) and rosiglitazone (a more selective PPARgamma agonist), we examined gene expression and cholesteryl ester accumulation in THP-1-derived macrophages. Pioglitazone enhanced the mRNA expression of the proatherogenic factors CD36 and adipophilin, but was approximately 10 times less potent than rosiglitazone. The potencies of the two agents appeared to correspond to their PPARgamma agonistic activities in this respect. However, both agents were similarly potent in enhancing the mRNA expression of the antiatherogenic factors liver X receptor alpha and ATP-binding cassette-transporter A1. Furthermore, both agents enhanced cholesteryl ester hydrolase mRNA expression and inhibited acyl-CoA cholesterol acyltransferase-1 mRNA expression and cholesteryl ester accumulation in macrophages. In this respect, their potencies appeared to correspond to their PPARalpha agonistic activities. These results suggest that pioglitazone has an equally beneficial effect on antiatherogenic events to rosiglitazone, despite being almost 10 times less potent than a PPARgamma agonist.     

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.