The aim of this study is to analyze the functional activity of an endo-β-1, 4-glucanase from the wood dwelling lower termite Coptotermes gestroi. Full length cDNA sequences of the endo-β-1,4-glucanase were obtained by primer walking in conjunction with Rapid Amplification cDNA Ends. With the obtained full length sequences, primers for amplifying open reading frame (ORF) excluding the signal peptide and glycophosphatidylinositol anchor were designed. Amplified endo-β-1,4-glucanase fragment was cloned and expressed using pET30(+) expression vector in BL21 E.coli strain. Expression of endo-β-1,4-glucanase was confirmed by Western blotting and the result revealed that only full ORF was expressed. The cellulase activity of protein preparations from the induced and non-induced cells was analyzed with Congo Red assay with the cellulase from Aspergillus niger (Sigma Aldrich) as a positive control. The activity of C. gestroi endo-β-1,4-glucanase was significantly higher than those observed in the positive control and the enzyme preparation from non-induced cells. Therefore, this study confirmed that C. gestroi endo-β-1,4-glucanase had a function of cellulose hydrolysis.

In this study, we investigated the C-V diagrams and metal surface related to the electrochemistry characterization of metal(nickel, SUS-304). We determined electrochemical measurement by using cyclic voltammetry with a three-electrode system. A measuring range was reduced from initial potential to -1350mV, continuously oxidized to 1650 mV and measured to the initial point. The scan rate were 50, 100, 150, 200 and 250 mV/s. As a result, the C-V characterization of metal using N,N-dimethylacetamide and N,N-dimethylformamide inhibitors appeared irreversible process caused by the oxidation current from the cyclic voltammogram. After adding organic corrosion inhibitors, adsorption film constituted, and the passive phenomena happened. According to the results by cyclic voltammetry method, it was revealed that the addition of inhibitors containing amide functional group enhances the corrosion resistance properties.

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. Except paraoxon, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulfide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagenlike tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

Ultraviolet curable coating solution was prepared with poly(ethylene glycol) acrylate oligomer and various mono and multi-functional acrylate monomers. The optical properties of UV cured coating layer on PET film with acrylate coating solution containing metal oxides, such as fumed silica and alumina, were also investigated to reduce light reflection on films. Poly(ethylene glycol) diacrylate which has 575 of average molecular weight was used as oligomer acrylate, and pentaerythritol triacrylate and dipentaerythritolpenta-/hexa acrylate were used as multi-functional acrylate monomers. Also, butyl acrylate was used to improve the adhesion as well as to reduce glass transition temperature to give a better flexability. 1-hydroxy cyclohexyl phenyl ketone was used as photoinitiator. We found out the metal oxides in acrylate coating solution showed a homogeneous dispersion from energy dispersive spectroscopy data. Transmittance and light reflection of coated PET film was measured with UV/vis spectrometer and gloss meter, respectively. When 1.00 g of both metal oxides was added into coating solution, the transmittance and the glossiness were reduced from 90% to 30% and from 190 GU to 35 GU, respectively. However, adding up to 1.00 g of the metal oxide into coating solution did not affect on the hardness of coating layer and adhesion between coated layer and PET film. Conclusively, we can control transmittance and light reflection of coated film by adjusting the amounts of metal oxide in coating solution.

A thioredoxin peroxidase (TPx) gene was cloned from the bumblebee, Bumbus ignitus. The B. ignitus TPx (BiTPx) contains an open reading frame of 585 bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BiTPx cDNA showed 90% identity to Apis melifera (AmTPx-1), 80% to Aedes aegypti (AaTPx), and 78% - 47% to other insect 2-Cys TPx. Northern blot analysis revealed the presence of BiTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BiTPx in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting the BiTPx is not secretable. The cDNA encoding BiTPx was expressed as a 27-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BiTPx was shown to reduce H2O2 in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the geneitself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface asa GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by α3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing α3β4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

3The goal of this study was to develop high value mushroom foods possessing functionality. The effect of the Pholiota adiposa on the alcohol fermentation and its antihypertensive angiotensin I-converting(ACE) inhibitory activity of Korean traditional rice wine, Yakju was investigated. The ACE inhibitory activity of traditional rice wine was increased about 8% by the addition of 0.1% of the edible mushroom Pholiota adiposa ASI 24012 fruiting bodies into the mash containing 1% Lycii fructus, cooked rice, koji and antihypertensive S. cerevisiae and its Pholiota adiposa PAD-022 which showed anticholesteromia HMGCoA reductase inhibitory activity. Its quality characteristics and stability were investigated during the storage of room temperature and 40℃ for 3 months. It showed very high acceptability and also was very stable at 40℃ for 3 months[This study was supported by a grant from ARPC, 2005-2007].

3The goal of this study was to develop high value mushroom foods possessing functionality. The effect of the Pholiota adiposa on the alcohol fermentation and its antihypertensive angiotensin I-converting(ACE) inhibitory activity of Korean traditional rice wine, Yakju was investigated. The ACE inhibitory activity of traditional rice wine was increased about 8% by the addition of 0.1% of the edible mushroom Pholiota adiposa ASI 24012 fruiting bodies into the mash containing 1% Lycii fructus, cooked rice, koji and antihypertensive S. cerevisiae and its Pholiota adiposa PAD-022 which showed anticholesteromia HMG-CoA reductase inhibitory activity. Its quality characteristics and stability were investigated during the storage of room temperature and 40℃ for 3 months. It showed very high acceptability and also was very stable at 40℃ for 3 months[This study was supported by a grant from ARPC, 2005-2007].

Treponema maltophilum, a Group IV oral spirochete, is associated with periodontitis and endodontic infections. In this study we analyzed the functional role of the major surface protein of this organism (MspA) in human gingival fibroblasts (HGFs). The full-length gene encoding MspA was cloned and expressed in Escherichia coli by using the expression vector pQE-30. The recombinant protein (rMspA) was purified by affinity chromatography with nickel-nitrilotriacetic acid agarose and possible contamination of E. coli endotoxin in rMspA was removed by using polymyxin B-agarose. rMspA significantly induced the expression of pro inflammatory cytokines like IL-6 and IL-8 and intercellular adhesion molecule (ICAM)-1 in HGFs, when analyzed by reverse transcription-PCR, flow cytometry, and enzyme-linked immunosorbent assay. Our results indicate that MspA of T. maltophilum may play an important role in amplifying the local immune response by upregulating the expression of proinflammatory cytokines and ICAM-1.

The study was performed to explore the molecular changes in the vegetative stage (3-and 5-leaf) of sorghum under waterlogging stress. A total of 74 differentially expressed protein spots were analyzed using LTQ-FT-ICR MS. Among them, 12 proteins were up-regulated and 3 proteins were down-regulated. Mass spectrometry (MS) results showed that about 50% of the proteins involved in various metabolic processes. The level of protein expression of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase related to carbohydrate metabolic process increased in both 3 and 5-leaf stage under waterlogging stress. These proteins are known to function as antistress agents against waterlogging stress. The expression of oxygen-evolving enhancer protein 1 protein related to photosynthesis was slightly increased in the treated group than in the control group, however the expression level was increased in the 5-leaf stage compared to the 3-leaf stage. Probable phospholipid hydroperoxide glutathione peroxidase protein and superoxide dismutase protein related to response to oxidative stress showed the highest expression level in 5-leaf stage treatment. This suggests that the production of reactive oxygen species by the waterlogging stress was the most abundant in the 5-leaf treatment group, and the expression of the antioxidant defense protein was increased.

Acyl-acyl carrier protein (ACP) thioesterase (TE) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. A full-length cDNA of an acyl-ACP thioesterase, named CvFatB, was isolated from oil plant Cuphea viscosissima accumulating up to 90% caprylate (8:0) and caprate (10:0) in its seed oil. This cDNA contains a 1,245-bp open reading frame that encodes a protein of 415 amino acids. The deduced sequence also contains two essential residues (H317 and C352) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Overexpression of the CvFatB cDNA in Arabidopsis resulted in increased levels of saturated fatty acid, especially palmitate, and reduced levels of unsaturated fatty acids. The findings suggest that CvFatB from oil plant C. viscosissima can function as a saturated acyl-ACP TE and can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

The plant-specific transcription factor, LEAFY (LFY) is considered to be a master regulator of flower development in the model plant, Arabidopsis. This protein plays a dual role in plant growth, integrating signals from the floral inductive pathways and acting as a floral meristem identity gene by activating genes for floral organ development. Although LFY occupies an important position in flower development, the functional divergence of LFY homologues has been demonstrated in several plants including monocots and gymnosperms. In particular, the functional roles of LFY genes from orchid species such as Phalaenopsis that contain unique floral morphologies with distinct expression patterns of floral organ identity genes remain elusive. Here, PaLFY, a orthologue of Arabidopsis LFY from Phalaenopsis aphrodite subsp. formosana, a Taiwanese native monopodial orchid was isolated and characterized through analyses of expression and protein activity. PaLFY transcripts accumulated in the floral primordia of developing inflorescences and the PaLFY protein had transcriptional autoactivation activity forming as a homodimer. Furthermore, PaLFY rescues the aberrant floral phenotypes of Arabidopsis lfy mutants. Over-expression of PaLFY alone or together with PaFT1, a P. aphrodite subsp. formosana homologue of Arabidopsis FLOWERING LOCUS T (FT) in rice caused precocious heading. Consistently, higher chlorophyll content in the sepals and morphological changes in epidermal cells were observed in the floral organs of PaLFY knock-down orchids generated by virus-induced gene silencing. Taken together, these results suggest that PaLFY is functionally distinct from RICE FLORICAULA/LEAFY (RFL) but similar to Arabidopsis LFY based on phenotypes of our transgenic Arabidopsis and rice plants.

FT is one of the major floral activator in photoperiod-dependent flowering pathway. To understand the role of FT homologs in flowering time control of short-day plant soybean, we identified ten soybean FT genes and named GmFTs. Phylogenetic analysis revealed that ten GmFT genes were further categorized into three subclades. Gene expression analysis showed that the most GmFT genes are mainly expressed in leaves. The expression of GmFT2a, GmFT2b, GmFT5a, and GmFT6 was strongly induced under the floral inductive short-day condition, but GmFT4 exhibited opposite expression pattern compared to those of GmFT2a, GmFT2b, GmFT5a, and GmFT6. To understand roles of GmFT genes in flowering, we generated Arabidopsis transgenic plant overexpressing GmFT genes. Both 35S:GmFT2a and 35S:GmFT5a transgenic plants showed extremely early flowering. In contrast, overexpression of GmFT4 delayed flowering of transgenic plants compared to wild type Arabidopsis. The results indicated that GmFT2a and GmFT5a might function as floral activators, while GmFT4 has an opposite function in soybean flowering. Moreover, domain swapping approaches between GmFT2a and GmFT4 revealed that the substitution of the segment B region alone, which is located in 4th exon, was sufficient to change the function of GmFT2a to floral repressor and GmFT4 to floral activator. The results suggested that soybean FT homologs have been functionally diversified during evolution and might play different roles in photoperiod-dependent flowering of soybean.

The roots of Platycodon grandiflorum are massively used in traditional herbal medicine as a remedy for pulmonary disease and respiratory disorders. However, in spite of its potential medicinal significance, the molecular mechanism of its roots is still unknown. In the present study, high throughput proteome approach was conducted to profile proteins from 3, 4 and 5 months aged diploid and tetraploid roots of Platycodon grandiflorum. Two dimensional gels stained with CBB, a total of 68 differential expressed proteins were identified from diploid root out of 767 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 29 differential expressed protein spots (≥ 2-fold) were analyzed using LTQ-FTICR MS whereas a total of 24 protein spots were up regulated and 5 protein spots were down-regulated. On the contrary, in the case of tetraploid root, a total of 86 differential expressed proteins were identified from tetraploid root out of 1033 protein spots of which a total of 39 differential expressed protein spots (≥ 2-fold) were analyzed using LTQ-FTICR MS whereas a total of 21 protein spots were up regulated and a total of 18 protein spots were down-regulated. It was revealed that the identified proteins from the explants were mainly associated with the nucleotide binding, oxidoreductase activity, transferase activity. In that way, the exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.