In Seok Bang

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Peptide-based polyclonal antibody against mosquito 14-3-3ζ recognizes 14-3-3 homolog from dipteran and lepidopteran insects

The 14‐3‐3 proteins are known to play an important regulatory role in apoptosis, and various cell signaling cascades. However, no investigation on mosquito 14‐3‐3 has been reported. To investigate the role of 14‐3‐3 proteins in mosquito midgut cells undergoing apoptosis, we decided to take advantage of Anopheles gambiae genome data, and were able to find Ag14‐3‐3ζ cDNA and protein sequences from Ensembl (http://www.ensembl.org). Further in silico analysis using BLAST search revealed that Ag14‐3‐3ζ protein is a polypeptide of 248 amino acids, and shares high identity with 14‐3‐3ζ homologues from Aedes aegypti (100%), Drosophila melanogaster (96%) and Bombyx mori (93%). Due to the perfect match and high homology, we hypothesized that Ag14‐3‐3ζ peptide antibody may recognize 14‐3‐3ζ homologs from other anopheline mosquitoes and insects. We thus generated 14‐3‐3ζ polyclonal antibody against a unique region located in the C‐terminal end of Ag14‐3‐3ζ after in silico epitope analysis. As expected, zoo‐western blot analysis of 14‐3‐3 proteins revealed that a polyclonal antibody against Ag14‐3‐3ζ peptide recognizes 14‐3‐3 homologs from dipteran and lepidopteran insects. To our knowledge, this is the first report on polyclonal antibody production against mosquito 14‐3‐3ζ. The mosquito‐based 14‐3‐3ζ antibody will be very useful for studying the functional characterization of 14‐3‐3ζ in the context of host–pathogen interactions in midgut and other immune cells.

Molecular cloning and expression pattern of 14-3-3ζ from the malaria vector, Anopheles sinensis

14‐3‐3 proteins are known to play a pivotal role in cell survival, apoptosis and signal transduction. The 14‐3‐3ζ isoform has been cloned and characterized from many eukaryotic organisms, including the fruit fly and silkworm. However, no study on mosquito 14‐3‐3 has been reported to date. In an attempt to investigate the function of 14‐3‐3 in midgut epithelial cells undergoing apoptosis, a cDNA library was generated from the malaria vector, Anopheles sinensis, which was treated with apoptosis‐inducing Actinomycin‐D. We were able to identify and obtain A. sinensis 14‐3‐3ζ cDNA (Ansi14‐3‐3ζ) from expressed sequence tags (EST) analysis after conducting massive sequencing of the A. sinensis cDNA library. Ansi14‐3‐3ζ has very high homology to 14‐3‐3 homologs of various insects, such as Anopheles gambiae (100%), Aedes aegypti (100%), Drosophila melanogaster (96%), Bombyx mori (93%), Apis mellifera (93%) and Mus musculus (81%), indicating that mosquito 14‐3‐3ζ is a highly conserved gene in diverse organisms. Analysis of temporal expression patterns showed that Ansi14‐3‐3ζ mRNA is highly expressed in egg, early pupae and adult stages and is also expressed, although at low levels, in fourth instar larvae and late pupae. In response to two immune elicitors (lipopolysaccharide and laminarin), no striking induction of 14‐3‐3ζ mRNA was observed in A. sinensis. Further studies of the precise biological function, inducibility and subcellular distribution of 14‐3‐3ζ are required in Plasmodium invasion‐induced apoptotic midgut cells in A. sinensis in the context of the Time Bomb model.

Cytoprotective Effects and Gene Expression Patterns Observed Based on the Antioxidant Activity of Lonicera japonica Extract

In this study, based on the antioxidative effects in organic solvent fractions obtained from the main methanolic extract of L. japonica, the protective cellular effects and gene expression patterns of ethyl acetate fractions on H₂O₂-induced Raw 264.7 cell death (IC 50 ) were analyzed. The antioxidant activity of the fractions measured using DPPH free radical scavenging activity increased in a dose-dependent manner, and the ED 50 exhibited the highest 39.56 μg/ml in the ethyl acetate fraction. In addition, the ethyl acetate fractions’ cell viability on H₂O₂-induced Raw 264.7 cell damage increased in a concentration- dependent manner, showed a visible cell survival rate of 82.49% at a concentration of 100 μg/ml. The gene expression patterns related to the ethyl acetate fractions’ cytoprotective effect in H₂O₂-induced Raw 264.7 cell damage presented similar patterns to those of BHA. In comparative analysis for antioxidant activity-related genes affected by ethyl acetate fractions and BHA in H₂O₂-induced Raw 264.7 cells, both ethyl acetate fractions and BHA showed very similar gene expression patterns, but the gene expression level of the heme oxygenase 1 (Hmox1) gene making antioxidant enzymes in cells was four times higher in ethyl acetate fractions than BHA. In inflammation-related genes in H₂O₂induced Raw 264.7 cells, the T-box transcription factor (Tbx21) gene was expressed about two times more frequently in the ethyl acetate fraction treatment group, while it was expressed half as frequently in the BHA treatment group.Antioxidant Activity of Lonicera japonica Extract Won June Cho, Hee Seung Yoon, Yong Hyun Kim, Jung Min Kim, Il Jae Yoo, Man-Deuk Han and In Seok Bang 1 * Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University, Asan 336-795, Korea Department of Biology, Soonchunhyang University, Asan 350-646, Korea NAR Center, Inc., Daejeon Oriental Hospital of Daejeon University, Daejeon 301-724, Korea Institute of Nanoproduct Safety Research, Hoseo University, Asan 336-795, Korea

Expression of recombinant hybrid peptide hinnavin II/α-melanocyte-stimulating hormone in Escherichia coli : Purification and characterization

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/α-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni2+-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

Transcriptome Analysis of Human HaCaT Keratinicytes by Ginsenosides Rb1 and Rg1

Abstract This study examined the efficacy and the mechanism of action of biological response modifiers, ginsenosides Rb1 and Rg1 isolated from Panax ginseng C.A. Meyer on human keratinocytes HaCaT cell lines. A non-significant cytotoxic response was obtained in the HaCaT cell lines on treatment with various concentrations ofginsenosides Rb1 and Rg1 for different time durations. Furthermore, the global changes in the mRNA profile of HaCaT cells were investigated using DNA microarrays after stimulation with the ginsenosides Rb1 and Rg1. Ginsenosides Rb1 and Rg1 strongly increased FGF2 in HaCaT cells, and were found to be a candidate gene for antioxidant activity and elasticity. Other key candidate genes for antioxidant activity, such as FANCD2, LEPR, and FAS, also show enhanced regulation in HaCaT cells treated with ginsenoside Rb1. This study will be useful for understanding the regulatory genes involved in skin elasticity and signal transduction pathway stimulated by the ginsenoside Rb1. This paper currently focuses on the key factors regulating the interaction of anti-aging principlesand skin elasticity.

Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N2+-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth, Plutella xylostella

Calreticulin (CRT) plays pivotal roles in Ca2+ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full‐length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.

Molecular Cloning and Effects of Tm14-3-3ζ-Silencing on Larval Survivability Against E. coli and C. albicans in Tenebrio molitor

The 14-3-3 family of proteins performs key regulatory functions in phosphorylation-dependent signaling pathways including cell survival and proliferation, apoptosis, regulation of chromatin structure and autophagy. In this study, the zeta isoform of 14-3-3 proteins (designated as Tm14-3-3ζ) was identified from the expressed sequence tags (ESTs) and RNA sequencing (RNA-Seq) database of the coleopteran pest, Tenebrio molitor. Tm14-3-3ζ messenger RNA (mRNA) is expressed at higher levels in the immune organs of the larval and adult stages of the insect and exhibit almost five-fold induction within 3 h post-infection of the larvae with Escherichia coli and Candida albicans. To investigate the biological function of Tm14-3-3ζ, a peptide-based Tm14-3-3ζ polyclonal antibody was generated in rabbit and the specificity was confirmed using Western blot analysis. Immunostaining and confocal microscopic analyses indicate that Tm14-3-3ζ is mainly expressed in the membranes of midgut epithelial cells, the nuclei of fat body and the cytosol of hemocytes. Gene silencing of Tm14-3-3ζ increases mortality of the larvae at 7 days post-infection with E. coli and C. albicans. Our findings demonstrate that 14-3-3ζ in T. molitor is essential in the host defense mechanisms against bacteria and fungi.

In silico identification, characterization and expression analysis of attacin gene family in response to bacterial and fungal pathogens in Tenebrio molitor : Expression analysis of TmAttacin gene family

Antimicrobial peptides are effector molecules induced after microbial challenges. These form important components of innate host defense against the pathogens by exhibiting wide‐spectrum antimicrobial activities. In this study, we identified three attacin‐like genes from Tenebrio molitor RNASeq database using Tribolium castaneum attacin gene family as query. The T. molitor attacin gene family was annotated as TmAttacin‐1a [comprising of 154 amino acids (aa)], TmAttacin‐1b (150 aa) and TmAttacin‐2 (164 aa), respectively. Temporal expression analysis shows that the TmAttacin‐1a and ‐1b mRNAs are highly expressed in late larval stages, followed by a general decline in the pre‐pupal stages. The mRNA level shows a decline during metamorphosis, and gets slightly overexpressed in pupal‐adult transition stages. On the other hand, TmAttacin‐2 is mainly overexpressed at 1‐day old pupal stage. Spatial expression analysis indicates that TmAttacin‐1a, −1b, and −2 mRNAs are primarily expressed in gut and fat body, but not in hemocytes and Malpighian tubules in T. molitor larvae. Interestingly, TmAttacin‐1b shows more than 20‐fold expression in the ovary, whereas TmAttacin‐1a and −2 show similar expression patterns in gut, fat body, hemocyte, ovary, and testis in T. molitor adults. Induction pattern analysis demonstrates that the intracellular Gram‐positive bacteria, L. monocytogenes elicited the strongest response by inducing ~1,000‐fold expression of TmAttacin‐1a mRNA. The highest level of TmAttacin‐1b mRNA (~350‐fold) was induced by Gram‐negative bacteria, E. coli. However, the TmAttacin‐2 transcripts were not induced by microbial challenges. These results indicate that TmAttacin‐1a and ‐1b may be required for antimicrobial defenses in T. molitor.