시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제 대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었 다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자 의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추 정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시 간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대 해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과 는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

In insect exoskeleton/cuticle, structural cuticular proteins (CPs) and the polysaccharide chitin are the major components of the procuticle. CPs are cross-linked by quinones or quinone methides produced by the laccase2 (Lac2)- mediated oxidation of N-acylcatechols. We reported that two major CPs, TcCPR27 and TcCPR18, belong to the CPR family that contain the RR-2 consensus motif (Rebers & Riddiford), are essential for formation and stabilization of the rigid cuticle of Tribolium castaneum adults. In this study, we characterized and investigated functions of the third most abundant protein, TcCP30, in extracts of elytra. TcCP30 cDNA encodes a protein with 171 amino acid residues containing a putative signal peptide. Unlike TcCPR27 and TcCPR18, TcCP30 mature protein lacks an RR motif, with a very unique amino composition, 36% Glu, 21% His, 20% Arg and 16% Gly. TcCP30 gene is highly expressed right before and after eclosion (in 5 d-old pupae and 0 d-old adults). Immunohistochemical studies revealed that TcCP30 protein was present in rigid cuticle such as elytra and ventral abdomen but not soft cuticle such as hindwings and dorsal abdomen of adult T. castaneum. Injection of dsRNA for TcCP30 into late instar larvae had no affect on larval and pupal growth and development. However, the subsequent pupal-adult molt, more than 50% adults were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults exhibited wrinkled, warped and split elytra. TcCP30-deficient adults could not fold their hindwings properly because probably due to the malformed elytra. These results indicate that TcCP30 is critical for formation of rigid adult cuticle as well as development and growth of T. castaneum.

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus. Our previous study indicated that overexpression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. This study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to analyze its inhibitory activity against cysteine protease and physiological role in the parasitism of an endoparsitoid wasp, Cotesia plutellae. The open reading frame (ORF) of CpBV-CST1 encodes a polypeptide of 138 amino acids (15 kDa). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was overexpressed by 0.5 mM IPTG for 4 h. In biological activity assay, partially purified GST-fused rCpBV-CST1 showed inhibitory activity against papain. It also inhibited larval development of P. xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 plays a role in retardation of larval development of P. xylostella during parasitism.

Phospholipase A2 (PLA2) catalyze the committed step for eicosanoid biosynthesis and releases arachidonic acid (AA), which is oxygenated into eicosanoids that mediate immune responses in insects. Thus, any inhibition of PLA2 activity would lead to a significant immuno suppression due to lack of eicosanoids. Among more than 15 families of PLA2s, group Ⅳ cytosolic PLA2 (cPLA2) has been mainly associated with the production of eicosanoids associated with immune responses. However, no cPLA2 has been reported in all invertebrates including insects. AcPLA2 candidate gene (SecPLA2) has been identified from a hemocyte transcriptome of the beet armyworm, Spodoptera exigua. RNA interference of SecPLA2 expression significantly reduced cellular immune responses of hemocytes. When the SecPLA2 was expressed and purified, the recombinant SecPLA2 catalyzed a substrate, phosphoatidyl choline, atsn-2 position. Its catalytic activity was sensitive to pH, temperature, and calciumlevel. Furthermore, there combinant SecPLA2 was specifically sensitive to a cPLA2-specificinhibitor, methyl arachidonyl fluorophosphonate.

Mycoplasma (M.) hyopneumoniae is the causative agent of swine enzootic pneumonia, a disease that is prevalent in every country where pigs are raised. In this study, we aimed to develop a sensitive and specific PCR assay to detect M. hyopneumoniae in pigs. The suitability of this PCR assay for the detection of mycoplasmal infection was also tested using clinical lung samples from slaughtered pigs. We de- veloped a probe and M. hyopneumoniae-specific primer pairs, MhyoP-F and MhyoP-R, for the new PCR assay based on regions in the Mycoplasma protein P97 gene that are unique to M. hyopneumoniae. The developed PCR as- say was very specific and sensitive for the detection of M. hyopneumoniae. The assay was able to detect the equivalent of 10 pg of target template DNA, which indicates that the assay was very sensitive. In addition, the M. hyopneumoniae PCR assay detected only M. hyopneumoniae and no other Mycoplasma spp. or bacterial species of another genera. Further, the newly developed PCR assay effectively detected M. hyopneumoniae infection in pigs. We suggest that this PCR assay using M. hyopneumoniae-specific primer pairs, MhyoP-F and MhyoP-R, will be useful and effective for monitoring M. hyopneumoniae infection in pigs.

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 dental infections have been increased due to the widespread overuse and exposure to antibiotics. One of the well-known pathogens is S. aureus The pathogenic properties of S. aureus is associated with the virulence gene. The objective of this study is to investigate the distribution of virulence gene of S. aureus which may contribute to the prevention and treatment of bacterial infections. 54 strains of S. aureus were separated from saliva taken from 129 outpatients diagnosed with periodontitis from Jun. to Dec., 2010 in Seoul. 44 (46.6%) and 13 (31.7%) strains of them were obtained from 88 and 41 outpatients from the S and E dental clinics, respectively. Then, the distribution of virulence gene and genetic diversity were analyzed with the PCR technique. The result of the S. aureus isolates possessed coagulase gene and showed six polymorphism types 390~470 bp (1.8%), 550~633 bp (3.7%), 630~714 bp (9.3%), 715~795 bp (20.4%), 796~876 bp (40.7%) and 877~957 bp(22.2%) due to variable numbers of tandem repeats present within the gene. In this study it will be anticipated that this study can contribute to establish efficiently the countermeasure about the prevention and treatment of antibiotic bacterial infections.

Insect blood cells, hemocytes, inhibit spreading behavior upon bacterial challenge to perform cellular immune responses. Hemocyte spreading is accomplished by cytoskeleton rearrangement, which is activated by various immune mediators, such as biogenic monoamins, plasmatocyte-spreading peptide(psp), and eicosanoids. However, little is known how these, immune mediators. acitvate hemocyte spreading behavior. A small G protein, Rac1, gene was identified in hemocytes of Spodoptera exiqua. Its expressed in most developmental stages accept egg and expecially expresses in hemocytes and fat body of Larval stage. In response to bacterial challenge, its expression was segnificantly up-regulated. However, RNA inteference (RNAi) of Rac1 expression inhibited hemocyte spreading behavior. under RNAi condition of Rac1, octopamine and psp failed to activate hemocyte spreading behavior. Interestingly, as addition of prostaglandinE2 to the RNAiconditioned Larval rescued the mediation of octopamine and psp. These results indicate that Rac1 is required for mediation of octopamine and psp on hemocytespreading behavior and suggest that Rac1 may activate eicosanoid biosnthesis.

We prepared the polyclonal antibody anti-20α-hydroxysteroid dehydrogenase (anti-20α-HSD) against the recombinant full-length protein bovine 20α-HSD in Escherichia coli. The specificity of anti-20α-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine 20α-HSD and bovine placental tissues. According to western blot analysis, anti-20α-HSD specifically recognizes the 37-kDa protein bovine 20α-HSD. The protein is not present in untransfected CHO cells. Anti-20α-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine 20α-HSD protein in the cultured luteal cells during the estrous cycle later.

쌀 가공식품 부산물의 효소 처리로 인한 단백질과 식이섬유, 색도의 차이는 시료에 따라 각각 다르게 나타났다. Gravimetric method와 Kjeldahl method 간에 단백질 함량의 차이가 조금씩 있는 것으로 확인 되었지만, 전체적으로 두 값의 패턴이 같은 것으로 보아 이 두 가지 단백질 측정 방법간에 유의성이 존재한다고 판단하였고, 두 값의 비교를 통해 단백질 함량의 변화를 더욱 자세히 비교 할 수 있었다. 주박의 경우 다른 효소보다 Termamyl이 단백질 함량을 높이는데 영향이 큰 것으로 나타났고, 효소를 혼합하였을 때 그 시너지 효과가 두드러졌다. 특히 세가지 효소를 모두 혼합하였을 경우 가장 좋은 시너지 효과를 나타냈다. 그러나 식이섬유의 경우 Termamyl 단일효소를 처리했을 경우가 식이섬유 함량을 높이는 데 가장 효과적인 것을 확인 할 수 있었으며, 탈지미강의 경우 역시 Termamyl이 가장 효율 적으로 나타났고 주박과 비슷한 패턴을 보여, 역시 혼합효소로 시너지 효과를 확인 할 수 있었다. 그러나 식이섬유 함량의 경우, Termamyl 처리를 했을 때 약한 증가 경향을 보인 반면, 다른 효소 처리로 인한 식이섬유 함량은 감소하는 경향을 나타냈다. 시럽박의 경우 초기 단백질 함량이 높아서 단백질 함량이 크게 증가하지 않았다. 최적의 효소라고 평가할 만한 효소를 찾을 수 없었고, 시너지 효과 역시 나타나지 않았다. 그러나 식이섬유의 경우 효소 처리로 인해 상당량이 증가하는 것으로 나타났다. 특히 Viscozyme 처리로 인한 식이섬유 함량은 큰 증가를 보였으나, 시너지 효과는 크게 나타나지 않았다.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C.plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. A recent study on CpBV replication by analysis of ovary transcriptome of C.Plutellae suggests several candidate coat protein genes. This study was conducted to confirm the coat protein genes by analyzing coat proteins of CpBV viral particles by a tandem mass MALDI-TOF. Immunoprecipitation of ovary protein extract with a polyclonal CpBV antibody captured three proteins named as p35, p60, and p70. More number of coat proteins were resolved in a protein extract directly from viral particles. All candidate coat proteins are analyzed in amino acid sequences by MALDI-TOF. A comprehensive analysis of viral proteomics and ovary transcriptome determined novel viral coat proteins from CpBV

쌀단백질을 이용한 고부가가치 천연 조미소재를 개발하기 위하여 쌀단백질을 프로테아제로 효소분해한 배지에서 Saccharomyces cerevisiae를 배양하여 제조한 효모 추출물(Yx)에, 효소분해 후 남은 쌀단백질 잔사를 Bacillus licheniformis 혹은 Bacillus subtilis로 발효하여 얻은 발효물(Rfl, Rfs)을 각각 첨가하였다. 쌀단백질 잔사의 발효물이 첨가된 효모추출물(YxRfl, YxRfs)의 전체적 선호도는 첨가전의 효모추출물(Yx)에 비하여 높았으며, 특히 쌀단백질 발효물의 보충에 의해서 감칠맛과 같은 풍미가 증가함을 미각센서 분석 및 관능검사에 의해서 확인할 수 있었다. 쌀단백질 잔사발효물에 의한 감칠맛의 상승은 감칠맛을 내는 아미노산 이 외에도 쌀단백질의 발효에 따라 유리된 다양한 펩타이드 분획의 영향이 있었을 것으로 예상된다. 이와 같이 감칠맛 아미노산 및 펩타이드가 함유된 쌀단백질 발효물이 보충된 효모추출물은 감칠맛과 풍미의 상승작용으로 전체적인 기호도가 높아짐에 따라 고부가가치 천연조미소재의 제조에 응용될 수 있을 것으로 기대되었다.

Betulinic acid (BA), a naturally occurring triterpene found in the bark of the white birch tree, has been investigated to induce apoptosis in various cancer cells and animal models. However, there is no report of the chemopreventive effect of BA in cervical cancer cells. Using KB human cervical cancer cells as a model, we currently show that BA decreases cell viability and induces apoptotic cell death. The mechanism of the BA-induced anti-growth response in KB cells is due to the down-regulation of specificity protein 1 (Sp1) and its downstream targets, myeloid cell leukemia-1(Mcl-1) and survivin. Thus, BA acts as a novel chemopreventive agent through the regulation of Sp1that is highly expressed in tumors.

Venom allergen-like protein 2 (Vap2) was characterized from the pinewood nematode, Bursaphelenchus xylophilus, which is a destructive pathogen in several countries including Japan, China and Korea. Among three vaps of B. xylophilus (Bx-vap)reported in GenBank, Bx-vap2 showed the highest transcript level in both pine-grown propagative stage (PGPS) and media-grown propagative stage (MGPS). Bx-vap2 also was revealed that its transcript level over 10-fold increased in PGPS. In addition, western blot using BxVap2-polyclonal antibody showed that expression level of BxVap2 was significantly increased in PGPS. In immunohistochemistry, moreover, strong signals were detected around putative subventral gland in PGPS, whereas weak signals were observed in MGPS. These experimental results suppose pathogenic function of BxVap2 and migration assay using Bx-vap2 knock-down worms by RNA interference supports this postulation.

In the previous molecular cloning study from human salivary gland cDNA l ibrary a novel clone (C77-091) was known as a candidate gene for antimicrobial protein by GenBank database search and RNA in situ hybridization. This study is aimed to identify the molecular characteristics of C77-091 protein, which showed an antimicrobial activity on E.coli, thereby named as salivary antimicrobial protein (SAMP). SAMP consisted of a typical hydrophobic amino acid rich domain in the N-terminus, a cluster of basic amino acids, carbohydrate attachment site, a possible transglutaminase catalyzed cross-linking site, and multiple consensus sequences of phosphorylation site in the C-terminus. Western blot analysis of human organs and tissue with the monospecific antibody to the synthetic SAMP peptide showed strong interacting protein from the extracts from submandibular gland and parotid saliva but absent in the mixed saliva, and the immunohistochemical staining detected a strong positive regions in the secretory granules in the luminal cytoplasm of interlobular ductal cells of salivary gland. The SAMP was also distributed in the human sebaceous gland and prostate. These data suggest that C77-091 named SAMP gene is a novel antimicrobial protein in human salivary gland, which may play a role for the innate immunity by protecting and stabilizing the mucosal epithelium to maintain homeostasis of oral mucosa.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

Luteal cells produce progesterone that supports pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. In the present study, the corpus luteum (CL) in early pregnancy established from luteal phase and pregnant phase was analyzed. The first study determined progesterone changes in the bovine CL at day 19 (early maternal recognition period) and day 90 in mid-pregnancy and compared them to the CL from day 12 of the estrous cycle. CL alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing CL from luteal phase to those from pregnant phase counterparts, significant changes in expression level were found in 23 proteins. Of these proteins 17 were not expressed in pregnant phase CL but expressed in luteal phase counterpart, whereas, the expression of the other 6 proteins was limited only in pregnant phase CL. Among these proteins, vimentin is considered to be involved in regulation of post-implantation development. In particular, vimentin may be used as marker for CL development during pregnancy because the expression level changed considerably in pregnant phase CL tissue compared with its luteal phase counterpart. Data from 2-DE suggest that protein expression was disorientated in mid pregnancy from luteal phase, but these changes was regulated with progression of pregnancy. These findings demonstrate CL development during mid-pregnancy from luteal phase and suggest that alternations of specific CL protein expression may be involved in maintenance of pregnancy.