In the present study, we investigated the expression patterns of p63, a member of the p53 gene family, in hair follicle cells at different stages of the hair cycle and examined the relation with cell proliferation activity. For this study, immunohistochemistry for p63 and Ki-67, a marker of cell proliferation, was performed in skin obtained from C3H/he mice with depilation. In the anagen stage, p63 was strongly expressed in the cells of bulge areas and epithelial strand, matrix cells of the hair bulbs and outer root sheath cells, but inner root sheath cells and dermal papilla cells were negative for p63. These expression patterns of p63 were similarly noted in hair follicles in the early catagen stage. In the late catagen and telogen stages of hair follicles, outer root sheath cells, seboblasts and duct cells were immunoreactive for p63. On the other hand, Ki-67-positive cells were selectively observed among the p63 positive cell components, although p63 positive cells were not always proliferative. Most of the matrix cells in the hair bulbs were positive for Ki-67. Ki-67-positive cells were also frequently evident in the cells of epithelial strands in the early anagen stage. Outer root sheath cells were often positive for Ki-67 in the anagen and early catagen stages, but very rare in the late catagen and telogen stages. In summary, p63 was expressed in the bulge stem cells, epithelial strand cells, matrix cells and outer root sheath cells of hair follicles at any stage of the cycle, which was associated with the movement of hair progenitor cells for regeneration. Ki-67-positive cells were evident among the p63-expressing cell components. Our results strongly suggest that p63 plays an important role in stem cell regulation, at least associated with cell proliferation, for the regeneration of hair follicles.

We have analyzed the transcriptome of Scolopendra subspinipes mutilans using RNA sequencing and identified severalantimicrobial peptide candidates. Among the peptides, named scolopendrasins, were selected based on the physicochemicalproperties of antimicrobial peptides via an in silico analysis. As a result, we evaluated the antimicrobial activities ofscolopendrasins against Gram positive and negative bacteria including Candida albicans by radial diffusion assay and colonycount assay. We also investigated the cytotoxicity of scolopendrasins through hemolysis assay. We found that the actionof scolopendrasins involved binding to the surface of microorganisms via a specific interaction with lipopolysaccharides,lipoteichoic acid, and peptidoglycans, which are components of the bacterial membrane. These results will provide a basisfor developing therapeutic agents such as peptide antibiotics.

The dynastid beetle Allomyrina dichotoma has been used as a herbal medicine. Recently, we performed de novo RNAsequencing of Allomyrina dichotoma and identified several antimicrobial peptide candidates based on in silico analysis.Among them, cationic antimicrobial peptide, Allomyrinasin, was selected and we assessed the anti-inflammatory activitiesof Allomyrinasin against mouse macrophage Raw264.7 cells. The results showed that Allomyrinasin decreased the nitricoxide production of the lipopolysaccharide-induced Raw264.7 cells. In addition, quantitative RT-PCR, ELISA and Westernblot analysis revealed that Allomyrinasin reduced cytokine expression levels in the Raw264.7 cells. Taken together, thesedata indicated that Allomyrinasin had anti-inflammatory activity in the lipopolysaccharide-induced Raw264.7 cells.

The objective of this study was to evaluate the effect of the Allomyrina dichotoma larva (AD) on allergy and inflammation.We examined inhibitory effect of AD on allergic reactions in mast cells (RBL-2H3) activated by Compound 48 / 80and inflammatory response in macrophages (Raw 264.7) activated by LPS. Anti-allergy and anti-inflammatory actions ofAD water extract had no cytotoxicity. At these concentrations AD inhibited ẞ-hexosaminidase, tumor necrosis factor- α(TNF- α), interleukin-4 (IL-4), interleukin-6(IL-6) and cyclooxygenase-2(COX-2). AD also inhibited the expression of inducibleNO synthase (iNOS). AD reduced the release of inflammatory cytokines including IL-4, IL-6, TNF-α, and ẞ-hexosaminidase.These results suggest that AD may be potential anti-allergy and anti-inflammatory therapeutic agent.

The insects have been investigated as novel sources for foods and biomaterials in several recent studies. However,its effects on hair growth have not been sufficiently researched. To develop novel and natural materials for preventingalopecia and promoting hair growth, we investigated the antioxidant activity and hair growth promoting effect of Tenebriomolitor larvae extract (TME). As a result, DPPH radical scavenging activity was 81.17%, and nitrite scavenging activitywas 43.69%, which were similar to blueberry extract. And TME promoted the proliferation of human DPCs and NIH3T3cells, concentration dependently. In addition, the TME prevented not only DHT-induced DPCs cytotoxicity but also actionof TBM as a potassium channel blocker in NIH3T3 cells. These results suggest that TME can be used as a functionalmaterial for alopecia therapeutic reagent by preventing hair loss and promoting hair growth.

We recently reported the in vitro and in vivo antiobesity effects of Tenebrio molitor larvae, a traditional food in manycountries, but it remains unknown how the larvae affect appetite regulation in mice with diet-induced obesity. We hypothesizedthat the extract of T. molitor larvae mediates appetite by regulating neuropeptide expression. We investigated T. molitorlarvae extract's (TME's) effects on anorexigenesis and endoplasmic reticulum (ER) stress–induced orexigenic neuropeptideexpression in the hypothalami of obese mice. Central administration of TME suppressed feeding by down-regulating theexpression of the orexigenic neuropeptides neuropeptide Y and agouti-related protein. T. molitor larvae extract significantlyreduced the expression of ER stress response genes. These results suggest that TME and its bioactive components arepotential therapeutics for obesity and ER stress–driven disease states.

This study was performed to develop patties with quality characteristics using mealworm powder, followed by assessments of general compositions and the branched-chain amino acid (BCAA) levels of the patties. An analysis of the chromaticity of the patties shows that the lightness and yellowness were decreased, whereas the redness was increased, as the amount of mealworm powder was increased. According to the sensory evaluation, the mealworm patty that contain 20% of mealworm powder (M20) showed an overall high preference level. In terms of the mechanical properties, the hardness, gumminess, and chewiness were significantly increased, whereas the springiness and cohesivensee were decreased, as the amount of mealworm powder was increased. The general composition of the M20 consists of 41.84% (moisture), 8.78% (carbohydrates), 34.42% (crude protein), 13.15% (crude fats), and 1.81% (crude ash). The BCAA contents (leucine, isoleucine, and valine) significantly increased in correspondence with the increases of the as mealworm-powder amount regarding the M20, the BCAA composition consists of the following: leucine (2,906.25 mg/100 g), isoleucine (1,459.09 mg/100 g), and valine (1,813.18 mg/100g). The conclusion of this study suggests that mealworm is a potential food material that could possibly replace meat.

As a part of a study on insect food, the nutritional and harmful components in the mealworm (Tenebrio molitor) were analyzed. In addition, due to a recent introduction of live Chinese mealworm in the Korea market, those components between the Korean and Chinese mealworms were compared. Analysis of the composition of the general components (moisture, crude protein, crude fat, crude ash, crude fiber, and carbohydrates) showed that abundant crude protein (50.32– 52.79%) was present in both Korean and Chinese mealworm powders, and the protein content in the Chinese mealworm powder was higher than that in the Korean mealworm powder by 2.67%. The amino acid compositions were similar, but the fatty acid composition differed with respect to each component in the Korean and Chinese mealworm powders. The unsaturated fatty acid contents were 76.80-80.55% of the total fatty acid content in the mealworms. The linoleic acid contents in the Korean and Chinese mealworms were 20.8±1.1% and 34.69±1.9%; the linolenic acid contents, 0.47% and 1.31%; and the oleic acid contents, 51.40±0.9% and 40.20±1.5%, respectively. With respect to harmful components such as heavy metals and bacteria that cause food poisoning, bacteria such as Escherichia coli O157:H7 and Salmonella spp. were not detected in both Korean and Chinese mealworms, and the mercury content was below the standard value for common food (Korea, 0.03 mg/kg; China, 0.08 mg/kg).

The antimicrobial peptide cecropin was isolated from the larval hemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. The full-length cDNA of A. yamamai cecropin (Ay-cecA) was cloned by a combination of RT-PCR and 3' RACE based on N-terminal sequence obtained by Edman degradation. The cloned cDNA consists of 419 nucleotides encoding a 64 amino acid precursor containing a 37-residue mature peptide. Like many insect cecropins, Ay-cecA also harbored a glycine residue for C-terminal amidation at the C-end. To understand this peptide better, we successfully expressed bioactive recombinant Ay-cecA in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The fusion CecA-KSI protein was expressed as inclusion body at high level. Recombinant Ay-cecA was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr), and purified by FPLC chromatography. The purified recombinant Ay-cecA showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. The time-kill assay showed that Ay-CecA displayed a time-dependent bactericidal activity, as was also seen after treatment with melittin. our results proved that Ay-cecA can be developed into novel antibacterial agent.

To identify genes that are differentially expressed, we compared the mRNA expression profile of Harmonia axyridis larvae untreated and treated with LPS. We extracted mRNAs from the larvae with or without LPS treatment, and subjected them to ACP RT-PCR analysis using a combination of 120 arbitrary primers (ACP1-ACP120)and oligo (dT) primer (dT-ACP2). After synthesized cloning DNA from 37 DEGs, it practiced the sequencing homology analysis using BLAST search. Among the 37 DEGs differentially expressed, we identified a cDNA showing homology with previously reported antimicrobial peptide. A cDNA encoding a 82-mer propeptide was identified and its predicted molecular mass and pI was 9.25 kDa and 7.54, respectively. A 35-mer mature peptide was also selected and named herein as Hamoniasin. The antimicrobial activity of chemically synthesized peptide (Mou def 1~8) against human bacterial pathogens was investigate. the result showed all bacteria strains were susceptible to Mou def 2,8 with MIC values in the 32 uM range. And biological changes of the respective cells according to peptide (Mou def 8) treatment were compared. MTT assay was tested that treatment of Mou def 8 decreased cell viability in AML-2, Jurkat, U937 (maximum 200ug/ml, 24hours). That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to Mou def 8 treatment.

To examine the expression profile of oxidative stress responsive genes in Spodoptera litura, we constructed a cDNA library from S. litura injected with hydrogen peroxide (H2O2). Using a microarray chip composed of 2,964 cDNAs, we screened gene expression at 1, 3, 5, 7, and 9 h post H2O2 injection. Data were clustered into 15 groups of genes that behave similarly across each time course. Seventy-three genes were identified as being at least 2-fold up- or downregulated after treatment with H2O2 in S. litura. We constructed expressed sequence tags (ESTs) for genes that changed at least 2-fold after treatment with H2O2. The functional classification of these ESTs based on Gene Ontology showed that the ESTs are rich in genes involved in oxidoreductase activity (5.7%), defense (14.3%), cellular process (22.9%), and development (17.1%).

COPRISIN is an antibiotic substance extracted from Copris tripartitus. This study is intended to identify various cell biological stimuli that COPRISIN, widely known as an antibacterial substance, has on human cells and to identify its molecule mechanism. A variety of human cell lines were divided into epithelial cells including kidney cells or womb cells, and immunocyte including T cells or macrophages and, after their being cultivated and maintained, cell biological changes of the respective cells according to COPRISIN treatment were compared. As a result, it was confirmed that, different from other experiment cells, COPRISIN specifically caused cell kill in T cells and macrophages. That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to COPRISIN treatment. An Apoptosis process is one dependent upon activity of caspase family protein, it was proved that COPRISIN medium cell kill process was one through a caspase-independent route such as AIF. Though it was found out that transcription of TNF-α and extracellular TNF-α secretion increased in blood cells stimulated by COPRISIN, it was also confirmed that TNF-α was a major medium factor in a COPRISIN induced cell kill process from the fact that a cell kill process by COPRISIN was not inhibited at all with TNF-α inhibiting antibody treatment. Above results revealed that COPRISIN, different from other tissue origin cells including kidney cells, can specifically induce apoptosis in immunocyte, which is caused by a caspase-independent cell signal transmission route.

Inflammatory bowel disease (IBD) is a group of chronic disorders of unknown etiology characterized by inflammation of the gastrointestinal tract. Recent data showed that the development of IBD is associated with the interplay of genetic, bacterial, and environmental factors and dysregulation of the intestinal immune system. We investigated how the gut cells were repaired after injury in Drosophila melanogaster. In this study we made D. melanogaster intestine damage model by oral feeding with variety IBD inducer such as pathogenic bacteria Serratia marcescens, Dextran Sulfate Sodium (DSS) and bleomycin, because its function is very similar with human, even though D. melanogaster has relatively simple organism. We repeated oral feeding with variety IBD inducer and got the survival rate and 50% lethal dose (LD50). After feeding with IBD inducer, we investigated the change of the intestinal stem cells, innate immune-related gene expression, and apoptosis in D. melanogaster gut. We examined the Delta, stem cell marker, staining image in the gut after feeding with DSS and S. marcescens with LD50 concentration. The Delta positive cells greatly increased in gut cells damaged by DSS or S. marcescens. This result supports the idea that intestinal gut stem cells are increased after gut cell damage and play very important role in damaged cell repair. Expression level of antimicrobial peptides was dramatically up-regulation after gut damage. As a result of TUNEL (terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling) assay, we confirmed that cell death by apoptosis was very increased in DSS feeding flies. Accordingly, we suggest that D. melanogaster is a proper IBD model organism to study how intestine damage can be repaired.

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3'rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease. There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects. RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant protein showed activity in the protease enzyme assay using gelatin as a substrate.

Attacin is an antibacterial protein that is secreted by fat body cells of insect larva in response to bacterial infection. A 949 bp cDNA encoding the antibacterial protein attacin was isolated by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from immunized Papilio xuthus larvae. The attacin cDNAs encoded 250 amino acid residues open reading frame with 60 residues prepropeptide. The deduced amino acid sequence of P. xuthus attacin showed significant identities with other Lepidopteran attacins. The attacin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The predicted mature attacin was expressed as soluble fusion protein efficiently in bacterial expression system. To increase productivity and solubility, attacin was translationally fused with thioredoxin (Trx) protein and expressed in E. coli cells that are highly sensitive to the mature attacin. The recombinant attacin exhibited antibacterial activity against Gram-negative bacteria.

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

To find some antibacterial peptides responsible for bacterial resistance, we performed differential hybridization with total cDNA probes which synthesized from normal and immunized larvae. Thirteen individual cDNA transcripts were expressed differentially in a total 1,862 random cDNA clones. One of upregulated genes is a novel member of the insect defensin-like peptide(Coprisin), a family of antibacterial peptide. Northern blot analysis showed that Coprisin was up-regulated at 4h and reached the highest point level at 16h after injection of E.coli. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with predicted molecular weight of 8.6 kDa and PI of 8.72. Comparison of the deduced amino acid mature portion of Coprisin with defensin-like peptide of other insect indicated that it has 79.1% and 67.4% identity with Anomala cuprea and Allomyrina dichotoma, respectively. To find antibacterial active region of Coprisin, we synthesized four peptides corresponding to amino acid residues 1V-43N-NH2(CopN1), 5-16(CopN2), 19-30(CopN3) and 31-43(CopN4) of coprisin having amidated amino acid residues at their Cterminal. A 12-mer amidated at its C-terminus, ACALHCIALRKK-NH2 (Ala19-Lys30-NH2) was synthesized based on the deduced amino acid sequence, assumed to be an active site sequence. This peptides showed antibacterial activity against E.coli, Staphylococcus aureus, MRSA, Psedomonas syringae, and Pectobacterium carotovorium. Modified 9-mer peptide, LRCIALRKK-NH2, showed strong antibacterial activity than mellitin peptide used as a positive control against gram-negative and gram-positive bacteria. This peptide showed no haemolytic activity and quite stable at 100℃ for several hours of incubation and in a wide pH range(pH2-12). Therefore, this peptide may be a good candidate for the development of new drug with potent antibacterial activity without cytotoxicity.