Screening for antimicrobial peptide genes in the immune-induced Antheraea yamamai larvae led to the identification of a novel antifungal moricin-like peptide (MLP10) gene. The complete MLP10 cDNA is comprised of 403 bp with 174 bp open reading frame encoding a 58 amino acid precursor that contains a putative 23-residue signal peptide, a 2-residue propeptide and a 33-residue mature peptide. The deduced amino acid sequence of MLP10 has 26∼52% identity to those of moricin-related peptides from lepidopteran insects. The MLP10 was highly expressed in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The resulting expressed KSI-MLP10 fusion protein was in a insoluble form. Recombinant MLP10 was released by cleavage of the fusion protein with cyanogen bromide (CNBr). Subsequently, we purified pure active MLP10 by FPLC chromatography, and 5.2mg of MLP10 was obtained from 1L culture medium. The purified MLP10 was prevented the growth of candida albicans at 6.25 uM, and was also active against gram negative and gram positive bacteria. This potent antimicrobial activity suggests that MLP10 may play a role in the immune response of A. yamamai.

Insulin in vertebrates plays a crucial role in maintaining homeostasis of blood sugar level. Insulin-like peptide (ILP) has been identified in insects, such as Drosophila melanogaster and Aedes aegypti. Plasma sugars and polyols of the diamondback moth, Plutella xylostella were separated by a Bio-LC. Among seven peaks, trehalose was the most predominant blood sugar and maintained at approximately 3.5 mM in the larval plasma. However, the feeding activity affected the plasma trehalose level, in which starvation significantly up-regulated the trehalose level. Analysis of ILP expression upon feeding indicated that feeding stimulated the gene expression of ILP. Interestingly, an injection of a vertebrate insulin significantly suppressed the hypertrehalosemia induced by starvation. These results suggest that ILP is a endocrine signal to down-regulate the plasma trehalose level in P. xylostella.

Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

Antimicrobial peptides (AMPs) are an important component of innate defense mechanisms with broad-spectrum activities against various pathogenic microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and viruses. Antibiotic resistance has become a pervasive and global health burden, resulting in the immediate need to develop a new class of antibiotic substances. We screened a 16-mer random peptide library using the yeast two-hybrid system with Beclin 1 as bait and found that two 16-mer peptides (named P4 and P30) appeared to interact with Beclin1 in the β-gal assay. The two candidate cDNAs were introduced into the yeast secretory system of Pichia pastoris and their expression induced in the presence of methanol. Spectrophotometric analysis and Disc clear zone assay using the supernatant of the yeast growth media showed that both of the two peptides had strong activities against Staphylococcus aureus, MRSA (methicillin resistance Staphylococcus aureus), MRSA2242, and MRSA-2250, but no effect on commensal Lactobacillus strains. PCR analysis of the genomic DNA of transformed Pichia pastoris using AOX1 primers revealed that the two cDNAs were integrated into the genome at the AOX1 locus. Our result suggests that these peptides could be developed as a useful alternative to classic chemical antibiotics.

The antibiotic peptide PAJE (RWKIFKKPFKISIHL-NH2), designed incorporating the N-terminal α-helical segments of papiliocin and jelleine, is a 15-residue hybrid peptide that has a broad spectrum of activity against Gram-negative, positive bacteria and fungi. In this study, we successfully expressed bioactive PAJE in Escherichia coli cells that are highly sensitive to this peptide. For the efficient production of peptide, we synthesized gene encoding PAJE, and fused the sequence in-frame to ketosteroid isomerase (KSI) gene to construct an expression vector pET29b-PAJE-KSI, which was then used to transform E. coli BL21 (DE3). The fusion protein PAJE-KSI was expressed as inclusion body at high level (more than 30% of the total proteins). Recombinant PAJE was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr). Subsequently, we purified the recombinant PAJE by FPLC chromatography. The purified PAJE displayed considerably antibacterial activity identical to that previously reported for chemically synthesized PAJE. The results indicated that successful expression of PAJE in E. coli cells and efficient procedure for purification may lead to a cost-effective platform for the mass production of PAJE.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Previously, we performed de novo RNA sequencing of Scolopendra subpinipes mutilans using high-throughput sequencing technology and identified several AMP candidates. Among them, a synthetic peptide (CP112) was designed based on the physicochemical properties of antimicrobial peptide such as length, charge, isoelectric point. Here, we have assessed the antimicrobial activities of CP112 against various microbes and the antioxidative effects. The results showed that CP112 had antimicrobial activities in radial diffusion assay and colony count assay. In addition, we found that CP112 bound to the surface of microorganisms via a specific interaction with lipoteichoic acid, lipopolysaccharide and peptidoglycan, which is one of bacteria cell wall components. Furthermore, CP112 has shown significant DPPH radical scavenging activity. Taken together, the results would be provided the basis for developing of peptide antibiotics and antioxidants.

Previously, we have performed de novo RNA sequencing of Scolpendra subpinipes mutilans using next generation sequencing technology and identified several AMP candidates. Among them, a synthetic peptide (scolopendrasin I) was designed based on SVM algorithm. In this study, we reported that the synthetic peptide scolopendrasin I had an antimicrobial and anticancer activity. As a result, scolopendrasin I showed antibacterial activities against Gram positive and Gram negative bacteria strains in radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin I bound to the surface of bacteria via a specific interaction with lipoteichoic acid and lipopolysaccharide, which is one of bacteria cell membrane components. In addition, we found that scolopendrasin I had anticancer activities in the human leukemic T lymphocyte cell line Jurkat using MTS assay. In conclusion, our results suggested that scolopendrasin I could be useful for developing peptide antibiotics and anticancer agents.

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74 nm and 1.4 nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.

In this research we extracted water-soluble collagen peptide from flatfish skin and compared it with commercially available collagen peptide extracted from Tilapia scale currently placed on the market in the aspect of physiochemical property. The physical property and nutritional components of FSCP appeared almost similarly to those of TSCP, and also in calorie, FSCP marking 3.82 Kcal showed no differences from TSCP marking 3.84 Kcal. As for forming amino acids, in aspartic acid, serine, histidine, tyrosine, methionine, FSCP had higher content than TSCP, but in OH-proline, proline and alanine FSCP had lower content than TSCP. Especially the content of essential amino acids of FSCP marked 22.74% with a higher content compared with 13.64% of TSCP. In the distribution of molecular weight FSCP with 1,000 Da showed omparatively low compared with TSCP, and in emulsion property and stability FSCP and TSCP showed similar excellent trend.

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.

Immune defense is indispensible for insect survival. However, uncontrolled and excessive immune responses would be highly detrimental and energy-consuming processes. An insect cytokine, plasmatocyte-spreading peptide (PSP), induces hemocyte-spreading behavior as well as activating phenoloxidase (PO) in the beet armyworm, Spodoptera exigua. A hemocyte transcriptome of S. exigua contains a partial sequence of a putative PSP-binding protein (SePSP-BP). SePSP-BP was expressed in all developmental stages especially in hemocytes and fat body. A quantitative RT-PCR showed that the bacterial infection significantly up-regulated the expression level of SePSP-BP. A double-stranded RNA specific to SePSP-BP (dsRNASePSP-BP) was injected and suppressed SePSP-BP expression even in response to bacterial challenge. The larvae treated with dsRNASePSP-BPsuffered high mortality to infection of nonpathogenic bacteria and prolonged high PO activity after the immune challenge. These results suggest that SePSP-BP may play a role in suppressing immune responses as a negative controller

The purpose of this study was to investigate anti-oxidant effects of loach muscle-derived peptides in vitro and in vivo. Loach muscle peptides were prepared by 4 different methods: boiling (B), enzymatic hydrolysis (E), boiling and enzymatic hydrolysis (BE), alkaline and enzymatic hydrolysis (AE). Two different in vitro analyses, DPPH radical scavenging activity and xanthine-xanthine oxidase-induced superoxide radical scavenging activity, were performed. All the four preparations showed concentration-dependent DPPH radical scavenging activity. However, superoxide radical scavenging activity was found only with AE and E preparations. To evaluate in vivo effects, mice were fed with 10% AE-containing diets for 4 weeks before hepatotoxicity induction with CCl4. In serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) levels, total antioxidant levels, and relative hepatic weight ratio, no evidence for anti-oxidant effects was found with AE indicating the absence of anti-oxidant effect in the in vivo mouse experiment. It needs to be clarified why anti-oxidant activity of loach protein hydrolysates was not evident in vivo. Furthermore, these results suggest that in vivo evaluation is crucial in demonstrating anti-oxidant activities.

Antimicrobial peptides are widely found in living organisms and are known to play a critical role in innate immunity. Numerous antimicrobial peptides from diverse species appear to be effective against pathogenic microorganisms of bacteria, fungi, protozoa, and viruses. Because antibiotic resistance is a global health issue in the fight against pathogenic microorganisms, there has been an urgent need for development of new antibiotic substances. In the current study, we performed yeast two hybrid screening using Beclin1 bait in order to find new peptide antibiotics from a random peptide library. Two candidate peptides from the screening were expressed in a yeast secretory system of Pichia pastoris and tested for any antimicrobial activity against Staphylococcus aureus, MRSA, MRSA2242, MRSA2250, Lactobacillus casei, and Lactobacillus acidophilus. Disc clear zone assay and spectrophotometric analysis revealed that the two peptides exert a decent activity against the pathogenic bacteria, in contrast to minimal effect on the commensal Lactobacillus strains. Taken together, this study presents novel peptides with antibacterial activity against the pathogenic forms of Staphylococcus aureus and suggests the possibility that these peptides, upon further characterization, may be developed as clinically useful antibiotics.

Insect blood cells (hemocytes) play a key role in defense against parasites and other pathogenic organisms that infect insects. Cellular immune responses exhibited by hemocytes are acute and effective to initially suppress pathogenic processes. Subsequently humoral immune responses executed by antimicrobial peptides completely cleared the pathogens with help of hemocytes. Two immune mediators, plasmatocyte-spreading peptide (PSP) and eicosanoid, are known to mediate cellular immune responses by activating hemocyte behavior. This study was focused on how these two immune mediators work together to express hemocyte spreading behavior. Both PSP and prostaglandins stimulate hemocyte spreading in dose-dependent manners in the beet armyworm, Spodoptera exigua. Interstingly, inhibition of eicosanoid biosynthesis inhibited PSP activity on mediating the hemocyte-spreading behavior. However, the addition of eicosanoid biosynthesis precursor, arachidonic acid, rescued the hemocytespreading activity. Inhibition of PSP or its receptor by each RNA interference are now under investigation to test whether PSP triggers eicosanoid signaling. These results suggest that there is a cross-talk between PSP and eicosanoid to express hemocyte-spreading behavior in response to bacterial challenge

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.

Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, post-mating behaviors are triggered by sex peptide (SP), a key modulatory substance produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors by acting on the sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the central nervous system (CNS). Interestingly, however, SPR is also expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. Based on these observations, we speculated that SPR may have additional ligands that are only distantly related to SP, if at all. If so, then this also raises questions on the evolution of SP-SPR signaling. To begin to address these questions, we set out to identify additional ligands for SPR. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes and Aplysia SPRs in vitro, yet are unable to trigger post-mating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for post-mating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors. Therefore, we propose that SPR has a different ancestral function, with a role in post-mating behavior arising only recently in Drosophila evolution, concomitant with the emergence of its novel SP ligand.

The present study was carried out to establish an animal model, displaying long-term learning and memory dysfunction, since single intracerebroventricular (icv) injection of amyloid β peptide (Aβ) causes a short-term memory impairment. Male ICR mice were fed a high-cholesterol diet (HCD) containing 3% cholesterol, 1% corn oil and 0.5% cholic acid, and 1 week later, icv injected with Aβ_1-42 (5 μg/head). Learning/ memory function was assessed via passive avoidance performances 1 day and 2, 4, and 6 weeks after Aβ_1-42 injection, in addition to blood biochemical analyses for lipid profiles and hepatic function. Total cholesterol, lowdensity lipoproteins and hepatic dysfunction parameters markedly increased, while high-density lipoproteins were reduced following HCD feeding. Whereas single injection of Aβ induced temporary memory loss 1 day after administration, exhibiting full recovery after 2 weeks, Aβ treatment in combination with HCD feeding lasted the learning/memory impairment up to 6 weeks. Therefore, it is suggested that hypercholesterolemia augments Aβ-induced memory loss, and that Aβ injection plus HCD feeding could be a long-term memorydeficit model suitable for long-term treatment with drugs or stem cells.

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.