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.

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral and antimicrobial responses. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity. The transiently expressed CpBV-IkB3 inhibited expression of hemolin, but did not those of lysozyme and cecropin in P. xylostella, while both lysozyme and cecropin were inhibited in the treated Spodoptera exigua. When the recombinant AcNPV was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene.

Antimicrobial peptides represent an essential alternative first line of defense. Naturally occurring molecules associated with the innate immune system in disease-bearing vectors such as mosquito, tick could be the target for searching more potent and effective agents to combat against the pathogens resistant to conventionally used antibiotics. Recently, we explored expression of a defensinlike peptide, longicin from the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analog (P4) displayed antimicrobial/fungicidal/parasiticidal activity and, therefore, proposed to be a chemotherapeutic compounds against tick-borne disease organisms. Structural characterization of antimicrobial peptides is very important to understand the peptide activity. In addition, harmful side effects such as lysis of red blood cells or cytotoxicity towards mammalian host cells commonly associated with antimicrobial peptides as potential therapeutic agent should also be elucidated. In this study, we analyzed some structural features using bioinformatics tool, CD Spectroscopy, and also determined cytolytic activity of P4 peptide. According to the chemicophysical characteristics, the P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of β-sheet which was also observed in modelled tertiary structure. CD spectroscopy results also revealed the existence of a β-sheet and changes of helical content in the presence of membrane-mimic condition. These structural observations on P4 suggest that the antimicrobial activity could be due to the well developed β-sheet. In addition, sequence homology search showed that antimicrobial molecule identified in other ticks and in organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved antimicrobial domain. The peptide P4 also showed less cytolytic activity against various cell lines or erythrocytes of various species. The data presented here strongly suggests that the peptide P4 could be developed as future therapeutic agent against tick-borne microorganisms.

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.

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.

Avian Influenza (AI) is an avian disease that break out by AI virus (AIV). As Hemagglutinin (HA) that is a main antigen surface protein of influenza virus that causes these influenza infection changes continuously, HA escapes bond of guided antibody by host"s immunity system. Specification region (HA91-261) of HA molecule that reconize receptor (sialic acid) of host cell is well-preserved without changing relatively, but is gotten buried on inside of trimer structure in natural conditions, is reported that development of effective vaccine or cure for HA protein is difficult because is not exposed to immunity system. Aptamer is relatively small strand DNA or RNA, and has high affinity to specific proteins. In this study, new aptamer DNAs were screened which can specifically bind to the receptor binding region of HA H9 protein.

Atrial natriuretic peptide (ANP), a circulating peptide hormone with 28 amino acids secreted from cardiac atria, plays an important role in the maintenance of fluid and electrolytes homeostasis. The actions of ANP appear to be mediated by specific receptors on target cells. However, the specific antagonist for ANP receptors is not yet to be defined. The present study was undertaken to evaluate the antagonistic effects of water extract obtained from the gardenia (Gardenia jasminoides Ellis) on the natriuretic peptide receptor system in the kidney. In the kidney of Sprague Dawley rats, specific 125I-ANP binding sites were localized in glomerulus, inner medulla, intrarenal artery, and vasa recta bundle by using quantitative in vitro receptor autoradiography. These specific bindings were competitively displaced in a dose dependent manner by water extract obtained from the gardenia. Moreover, productions of 3',5'-cyclic guanosine monophosphate (cGMP) via activation of particulate guanylyl cyclase (GC) by ANP were increased in receptor proteins from the glomerulus and inner medulla of rat kidney. These cGMP productions were inhibited in a dose dependent manner by water extract obtained from the gardenia. The inhibitory effect of water extract obtained from the gardenia on activation of GC was quantitatively more potent rather than on 125I-ANP bindings to these renal structures. From affinity cross-linking study on the specific receptor proteins, the water extract obtained from the gardenia inhibited the 125I-ANP labeling of GC-coupled natriuretic peptide receptor from renal glomerulus of rat. Intravenous infusion of ANP elicits the potent diuresis, increment of urine osmolariry, and excretions of urinary chloride, creatinine and PAH in New Zealand White rabbit. These renal effects of ANP were inhibited by the intravenous pre-treatment of water extract obtained from the gardenia without changes in systemic hemodynamics including mean arterial blood pressure and heart rate. These results indicate that the water extract obtained from the gardenia may could be block the GC-coupled natriuretic peptide receptor, and suggest that the extract may contain a natural antagonistic molecule regulating the renal natriuretic peptide system.

The present study was undertaken to evaluate the antagonistic effects of water extracts of Chinese gooseberry (Actinidia chinensis P.) root on the natriuretic peptide receptor system in the rat kidney. By using in vitro receptor autoradiography, specific 125I-atrial natriuretic peptide (125I-ANP) binding sites were localized in glomerulus, inner medulla, intrarenal artery, vasa recta bundle, and renal pelvis of Sprague Dawley rat kidney. These specific bindings were competitively displaced by water extracts of Chinese gooseberry root in a dose dependent manner. Also, productions of 3',5'-cyclic guanosine monophosphate (cGMP) by activation of particulate guanylyl cyclase (GC) were stimulated by ANP in the glomerular and inner medullary membranes from rat kidney. These cGMP productions were inhibited by water extracts of Chinese gooseberry root in a dose dependent manner. The inhibitory effect of water extracts of Chinese gooseberry root on activation of GC was more potent rather than on 125I-ANP bindings to these renal structures. From affinity cross-linking study, the water extracts of Chinese gooseberry root inhibited the 125I-ANP labeling of GC-coupled natriuretic peptide receptor from renal glomerulus. Intravenous infusion of ANP elicits a potent diuresis, and urinary sodium and chloride excretions in New Zealand White rabbit. The pre-treatment of intravenous water extracts of Chinese gooseberry root infusion decreased competitively various renal effects of ANP with out changes in systemic hemodynamics. These results indicate that the water extracts of Chinese gooseberry root specifically inhibits the GC-coupled natriuretic peptide receptor subtypes (NPR-A), and suggest that the water extracts of Chinese gooseberry root may contain an antagonistic molecule regulating the biological functions of ANP system in the kidney and other organs.