Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in na?ve insects and in insects manipulated with bacterial and hormonal treatments.     

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.     

Chlorothalonil-biotransformation by glutathione S-transferase of Escherichia coli

It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al, 2003). GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification. In an attempt to elucidate the relation between chlorothalonil-detoxification and GST, the GST of Escherichia coli was expressed and purified. The drug-hypersensitive E. coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil. The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione. Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Photoluminescence imaging of europium (III)‐doped γ‐Al2O3 nanofiber structures

Aluminium oxide (Al₂O₃) has widely been used for catalysts, insulators, and composite materials for diverse applications. Herein, we demonstrated if γ‐Al₂O₃ was useful as a luminescence support material for europium (Eu) (III) activator ion. The hydrothermal method and post‐thermal treatment at 800°C were employed to synthesize Eu(III)‐doped γ‐Al₂O₃ nanofibre structures. Luminescence characteristics of Eu(III) ions in Al₂O₃ matrix were fully understood by taking 2D and 3D‐photoluminescence imaging profiles. Various sharp emissions between 580 to 720 nm were assigned to the ⁵D₀→⁷F_J (J = 0, 1, 2, 3, 4) transitions of Eu(III) activators. On the basis of X‐ray diffraction crystallography, Auger elemental mapping and the asymmetry ratio, Eu(III) ions were found to be well doped into the γ‐Al₂O₃ matrix at a low (1 mol%) doping level. A broad emission at 460 nm was substantially increased upon higher (2 mol%) Eu(III) doping due to defect creation. The first 3D photoluminescence imaging profiles highlight detailed understanding of emission characteristics of Eu(III) ions in Al oxide‐based phosphor materials and their potential applications.     

Metagenome microarray for screening of fosmid clones containing specific genes

A critical step in the process of metagenome analysis is to screen for clones that contain specific genes among a large number of clones. To form one of the sequence-based screening tools of a metagenome library, we designed a format of microarray [metagenome microarray (MGA)] that is arrayed with fosmid library clone DNA samples on a glass slide. We evaluated the MGA using random prime labeled fluorescent probes prepared from PCR products of the target gene and found that we could obtain specific hybridization signals only for the fosmid clone that contained the target gene. We found that the detection limit of the MGA was c. 10 ng microL(-1) of fosmid clone DNA, and that the MGA-based hybridization was quantitative within a concentration range of 10-200 ng microL(-1) of fosmid clone DNA. We used the MGA successfully to identify two fosmid clones that contained 16S rRNA genes from a fosmid library from the sediment of the East Sea, Korea. In conclusion, we have demonstrated that the MGA can be used for screening for fosmid clones containing specific genes in a metagenome library, and that this technology has potential application as a high-throughput metagenome screening tool.     

Sunxiuqinia dokdonensis sp. nov., isolated from deep sub-seafloor sediment

A novel facultatively anaerobic strain DH1^T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea. The new strain was characterized using polyphasic approaches. The isolate was Gram-stain-negative, motile by gliding, non-spore-forming rods, oxidase-negative, and catalase-positive; and formed colonies of orange-red color. The NaCl range for growth was 0.5–7.0% (w/v) and no growth was observed in the absence of NaCl. The isolate grew optimally at 30°C, with 2% (w/v) NaCl and at pH 7. The cell-wall hydrolysates contained ribose as a major sugar. The DNA G+C content was 40.8 mol%. The closest related strains are Sunxiuqinia faeciviva JAM-BA0302^T and Sunxiuqinia elliptica DQHS-4^T (97.9 and 96.3% sequence similarity, respectively). The level of DNA-DNA relatedness between strain DH1^T and S. faeciviva JAM-BA0302^T was around 41% (but only 6% between DH1T and S. elliptica DQHS-4^T). The major cellular fatty acids of the isolate were contained iso-C_15:0 (25.9%), anteiso-C_15:0 (16.7%), and summed feature 9 (comprising C_16:0 3-OH and/or unknown fatty acid of dimethylacetal ECL 17.157; 13.2%). The predominant menaquinone was MK-7. On the basis of polyphasic evidence from this study, the isolate was considered to represent a novel species of the genus Sunxiuqinia, for which the name Sunxiuqinia dokdonensis sp. nov. is proposed; the type strain is DH1^T (=KCTC 32503^T =CGMCC 1.12676^T =JCM 19380^T).