Phospholipase A2 (PLA2) hydrolyzes phospholipids at sn-2 position to release free fatty acids. PLA2 consists of a superfamily mainly categorized as secretory PLA2 (sPLA2), cellular Ca 2+ -dependent PLA2 (cPLA2), and cellular Ca 2+ -independent PLA2 (iPLA2). We are the first to report iPLA2 in insect. Here an objective of our study is to purify a recombinant iPLA2 protein (SeiPLA2) of Spodoptera exigua using bacterial expression system. An open reading frame of SeiPLA2 was cloned into an expression vector and then transformed into Escherichia coli BL21. Over-expression with IPTG yielded recombinant SeiPLA2 (rSeiPLA2), which was then purified by an affinity chromatography using Ni-NTA column. The purified rSeiPLA2 gave significant PLA2 activity using a pyrene substrate. Its activity was inhibited by an iPLA2 specific inhibitor (BEL), but not by sPLA2 inhibitor (BPB) or cPLA2 inhibitor (MAFP

In the present study, we investigated the role of peripheral ionotropic receptors in artemin-induced thermal hyperalgesia in the orofacial area. Male Sprague-Dawley rats weighting 230 to 280 g were used in the study. Under anesthesia, a polyethylene tube was implanted in the subcutaneous area of the vibrissa pad, which enabled drug-injection. After subcutaneous injection of artemin, changes in air-puff thresholds and head withdrawal latency time were evaluated. Subcutaneous injection of artemin (0.5 or 1 μg) produced significant thermal hyperalgesia in a dose-dependent manner. However, subcutaneous injection of artemin showed no effect on air-puff thresholds. IRTX (4 μg), a TRPV1 receptor antagonist, D-AP5 (40 or 80 μg), an NMDA receptor antagonist, or NBQX (20 or 40 μg), an AMPA receptor antagonist, was injected subcutaneously 10 min prior to the artemin injection. Pretreatment with IRTX and D-AP5 significantly inhibited the artemin-induced thermal hyperalgesia. In contrast, pretreatment with both doses of NBQX showed no effect on artemin-induced thermal hyperalgesia. Moreover, pretreatment with H-89, a PKA inhibitor, and chelerythrine, a PKC inhibitor, decreased the artemin-induced thermal hyperalgesia. These results suggested that artemin-induced thermal hyperalgesia is mediated by the sensitized peripheral TRPV1 and NMDA receptor via activation of protein kinases.

We report the use of carrot, a new and inexpensive biomaterial source, for preparing high quality carbon dots (CDs) instead of semi-conductive quantum dots for bioimaging application. The as-derived CDs possessing down and up-conversion photoluminescence features were obtained from carrot juice by commonly used hydrothermal treatment. The corresponding physiochemical and optical properties were investigated by electron microscopy, fluorescent spectrometry, and other spectroscopic methods. The surfaces of obtained CDs were highly covered with hydroxyl groups and nitrogen groups without further modification. The quantum yield of as-obtained CDs was as high as 5.16%. The cell viability of HaCaT cells against a purified CD aqueous solution was higher than 85% even at higher concentration (700 μg mL−1) after 24 h incubation. Finally, CD cultured cells exhibited distinguished blue, green, and red colors, respectively, during in vitro imaging when excited by three wavelength lasers under a confocal microscope. Offering excellent optical properties, biocompatibility, low cytotoxicity, and good cellular imaging capability, the carrot juice derived CDs are a promising candidate for biomedical applications.

최근 조직공학 기술의 발달로 재해와 질병으로 인한 손상된 조직과 장기의 대체연구들 이 진행되고 있다. 장기 대체 연구의 핵심 요소 중 하나는 재건된 조직이나 장기가 혈관 망을 형성하여 host tissue로부터 양분과 산소의 전달이다. 본 연구는 조직공학 기법을 이용하여 장기 재건에 필수적인 혈관 재건을 위해 혈관을 구성하는 주세포인 endothelial cell을 체외에서 배양하는 것이다. Endothelial cell(EC)배양을 위해서는 세포지지체인 세 포외 기질(External cellular metrics, ECM)을 필요로 하기 때문에 ECM중에 대표적인 collagen과 gelatin을 사용하여 지지체에 따른 체외배양능을 비교하였다. 실험 동물로는 돼지 대동맥을 채취하여, 대동맥 속에 collagenase type I을 주입하고, 혈관의 입·출구를 봉합한 상태로 10분 간 37℃에서 처리하였다. 관류된 용액은 10% FBS가 함유된 기본배 양액(EGM-2 media)을 사용하여 2번 수세한 후 회수된 세포를 각각의 ECM이 처리된 dish위에서 배양 하였다. EC세포인지를 확인하기 위해서 EC표지 인자인 CD31과 vWF 항체의 발현을 flow cytometry로 확인 하였고, 회수된 세포에서 두 단백질이 모두 발현 되었다. ECM에 따른 EC의 세포 형태를 비교하였을 때 형태학적 차이는 없었다. Basement Membrane Extract위에서 calcein-AM으로 염색된 EC는 ECM의 종류와 상관 없이 2-6시간 사이에 Tube Formation을 보였다. 또한 endothelial cell의 표지 마크인 CD31, Flk1, vWF의 mRNA 발현양과 IHC에 의한 단백질 발현을 조사한 결과 collagen 지지체 위에서 배양된 endothelial cells에서 발현양이 더 높았다. 결론적으로 두 가지 ECM에서 모두 성공적으로 endothelial cell의 배양이 가능하지만 collagen위에서 배양된 endothelial cell이 더 우수한 maintenance능력을 가짐을 확인 할 수 있었다.

An FDA approved drug for the treatment of type II diabetes, Troglitazone (TRO), a peroxisome proliferator–activated receptor gamma agonist, is withdrawn due to severe idiosyncratic hepatotoxicity. In the search for new applications of TRO, we investigated the cellular effects of TRO on YD15 tongue carcinoma cells. TRO suppressed the growth of YD15 cells in the MTT assay. The inhibition of cell growth was accompanied by the induction of cell cycle arrest at G0/G1 and apoptosis, which are confirmed by flow cytometry and western blotting. TRO also suppressed the expression of cell cycle proteins such as cyclin D1, cdk2, cdk4, cyclin B1, cdk1(or cdc2), cyclin E1 and cyclin A. The inhibition of cell cycle proteins was coincident with the up-regulation of p21CIP1/WAF1 and p27KIP1. In addition, TRO induces the activation of caspase-3 and caspase-7, as well as the cleavage of PARP. Further, TRO suppressed the expressions of Bcl-2 without affecting the expressions of Bad and Bax. Overall, our data supports that TRO induces cell cycle arrest and apoptosis on YD15 cells.

아이코사노이드는 탄소수 20 개의 다가불포화지방산 산화물로 구성된다. 이들 다가불포화지방산의 생합성 전구물질을 탐지하기 위해 파 밤나방(Spodoptera exigua)의 서로 다른 조직으로부터 지방산을 분리하여 GC/MS로 조성을 분석하였다. 파밤나방 5령 유충에서 소화관, 지방체, 혈구 및 체벽을 분리하고, 각 조직에서 지질을 추출하여 각각 중성지질, 당지질 및 인지질로 분리하였다. 대부분의 조직은 palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) 그리고 linolenic acid (18:3)를 주요 지방산으로 함유하였다. 그러나 이들 지방산의 조성은 조직과 지질 종류에 따라 상이하였다. 지방체와 혈구세포는 이들 주요 지방산 이외에 myristic acid (14:0)와 3 종류의 미동정 지방산들이 추가로 검출되었다. 서로 다른 지질 종류 가운데 인지질은 중성지질이나 당지질에 비해 상대적으로 높은 linolenic acid를 지닌 반면 포화지방산의 함유량은 낮았다. 전체 불포화지방산의 조성도 조직과 지질 종류에 따라 상이하였다. 인지질은 지방체, 혈구 및 소화관에서 높은 불포화지방산 함유 량을 나타냈다. 세포성 인지질분해효소인 calcium-independent phospholipase A2 (iPLA2)는 지방산 조성을 조절하는 데 역할을 담당하였다. 이 유전자의 RNA 간섭은 중성지질과 인지질에서 지방산 조성의 변화를 유발하였다. 본 연구는 아이코사노이드 생합성의 전구물질로 여겨지는 아라키도닉산을 검출하지 못했다. 이는 곤충에 있어서 아이코사노이드는 포유동물과는 다른 새로운 생합성 과정을 통해 형성되는 것으로 추정된다.

Repetitive applications of drugs to tumor tissues and animals induced resistance and/or tolerances which caused severe problem in agriculture and medicine. However, we still do not clearly understand the molecular and cellular mechanisms underlying development of resistance and tolerance to chemicals. Drosophila is one of the most widely used model for studying fundamental phenomena in sciences using its available genetic and genomic resources. To investigate unknown molecular and cellular basis of drug resistance development, we applied Drosophila with two different concentrations of a chemical after treating them with Ethyl methanesulfonate mutatgenesis. We found that flies treating with two different concentration of chemicals showed different susceptibility to a chemical. We have established two different lines showing different susceptibility to a chemical. We will use these lines to compare any differences in mRNA expression profiling and enzyme activities. (This work was supported by project title: Investigation on cross drug resistance mechanisms using Drosophila as a model (PJ010821032016) from Rural Development Administration).

Exposure to several common acting through oxidation stress environmental toxins has been shown to be associated with Parkinson’s disease (PD). One recently identified inherited Parkinson’s disease (PD) gene, DJ-1, may have a role in protection from oxidative stress, thus potentially linking a genetic cause with critical environmental risk factors [1]. In the present study, initially we assessed the antioxidant activity of Silk Worm Powder (SWP) in selected chemical systems and further explored the efficacy of SWP in Drosophila lacking DJ-1 function (This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project title: Elucidation the health improvement effects of boiled silk worm larvae, Project No: PJ01082801) Rural Development Administration).

Malignant gliomas and glioblastomas are the most common type of primary brain tumors. The treatment of malignant glioma involves surgery, radiation, and chemotherapy. These therapies have not been successful in curing malignant glioma and typically associated with dismal prognosis. Therefore, we can investigate thymidine kinase activity and cytotoxic effect after transfer suicide gene in U-251 glioma cells. We assessed expression patterns of green fluorescence protein(GFP) after infected with adenovirus in U-251 cells. After infection of HSV-tk in U-251 cells, we observed thymidine kinase activity with [3H]-penciclovir and cytotoxic effect by treated with ganciclovir. We could observe that expression level of GFP was increased according to infected concentrations in U-251 cells. GFP was not expressed in 1moi and 10moi, and slightly expressed in 30moi. Expression level of GFP was largely increased in 50moi and almost cells expressed GFP in 100moi. GFP expression has shown clear image in 100moi compared with other concentrations. We also investigated thymidine kinase activity using [3H]-penciclovir after infection of suicide gene HSV-tk into U-251 cells. Thymidine kinase activity increased in 10moi concentration compared with empty adenovirus infection. We could find that thymidine kinase activity was elevated proportional to HSV-tk infection amount in 30moi and 50moi. For evaluation of cellular cytotoxic effect of HSV-tk, we treated ganciclovir to U-251 cells and assessed cytotoxicity by using MTT assay. We could identifiy that cytotoicity appeared in very low concentration of HSV-tk compared with cancer cells originated with other organs. Cytotoxic effect was shown about 15% of U-251 cells of total cells in 5moi. By infection 10moi of HSV-tk, cytotoxic effect was intensively increased and about 60% of U-251 cells became extinct. About 70% cells exhibited cytotoxic effect in 30moi and more than 80% cells also appeared cytotoxic effect by infection of HSV-tk in 50moi, 100moi, and 200moi. Therefore, we could confirm to gene expression in U-251 cells was increased proportional to infected gene concentrations. Also we could find that thymidine kinase activity elevated with according to infected concentration and cellular cytotoxic effect was shown in very low concentration and higher cytotoxic effect also appeared by infection of suicide gene HSV-tk into U-251 glioma cells. These results suggest that gene therapy with suicide gene will be successful in curing brain tumors containing malignant glioma and glioblastoma.

The PHBV nonofibrous membrane fabricated by electron-spinning method for tissue engineered bone regeneration scaffold was evaulated in terms of cellular prolieration and cryopreservation efficiency. The rat calvarial periosteum derived primary cells were cultured with PHBV nanofibrous membranes and analyzed the cellular proliferation and differention fashion and cryopreservation potential by in vitro MTT assay as well as ALP staining and Alizarine red staining with or without cryopreservation for 2 weeks. The rat calvarial periosteum derived primary cells cultured with PHBV nonofibrous membrane showed favorable proliferation and alkaline phosphatase activity with numerous mineral nodule formation regardless of cryopreservation, even though its efficiency was slightly decreased in cryopreserved condition. These findings suggest that PHBV nanofibrous membrane can be applicable as an efficient cell engineered membrane for guided bone regeneration or scaffold for tissue engineered bone regeneration.

Insect immunity is innate and consists of cellular and humoral immune responses. Cellular immune response usually requires hemocyte-spreading behavior, which is accompanied by cytoskeletal rearrangement. A glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), catalyzes an oxidation reaction of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate in the cytosol. Another function of GAPDH in mammalian cell is to bind C-terminal α-tubulin to facilitate cytoskeletal arrangement. An immunoprecipitation (IP) of viral protein, CpBV-CrV1, against hemocyte protein lysate revealed that CpBV-CrV1 binds to GAPDH, identified by MALDI-TOF analysis. RNA interference (RNAi) of GAPDH significantly suppressed cellular immune response, but neither RNAi of hexokinase nor aldolase suppressed the cellular immune response. A common molecular motif of CpBV-CrV1 and a-tubulin at C-terminal region supported the IP analysis. To test the role of α-tubulin motif in CpBV-CrV1, point mutations of CpBV-CrV1 were applied and resulted in loss of the biological activity of CpBV-CrV1. Furthermore, an immunofluorescence assay indicates CpBV-CrV1 colocalized with a-tubulin in hemocytes collected from Plutella xylostella parasitized by Cotesia plutellae possessing C. plutellae bracovirus (CpBV). This result suggests that GAPDH plays a critical role in hemocyte-spreading behavior during immune challenge, and it is a molecular target of the pathogenic virus.

Although it is believed that internal nutrient sensors play important roles in feeding behaviors, their molecular and neural mechanisms underlying of the modulation of physiological status and cell growth are poorly understood. Using a Ca2+ imaging experiments with heterologous expression systems, we show that one of the gustatory receptors in the western honey bee Apis mellifera is selectively tuned to amino acids. Remarkably, we report that this gustatory receptor of the honey bee is highly expressed in hypopharyngeal gland, which plays a role in caste differentiation as well as royal jelly production and secretion. Knocking down this gustatory receptor gene reduces cellular pathways responsible for nutritional sensing such as mTOR signals in hypopharageal gland. Furthermore, the interfering expression of this gustatory receptor gene not only alters morphological changes and developmental retardation of the hypopharyngeal gland, but it also blocks cellular growth signals to induce autophagy. This new report indicates that internal sensing and downstream signals detecting nutrients is essential for honey bee to maintain the cellular growth and development of internal organs essential for caste development and maintenance of social structure in the honey bee.

We present a case of cellular angiofibroma arising from right neck in a 46-year-old woman. Surgical excision was performed and the patient was disease free till now. Cellular angiofibroma usually arised in the inguinoscrotal of vulvovaginal regions. Only 3 cases of cellular angiofibroma involving maxillofacial region have been reported in the english written literature. Cellular angiofibromas are rare benign tumor characterized by bland spindle shaped cells arranged in a stroma with wispy collagen and numerous vessels. Immunohistochemical stains of the tumor cells showed the positivity for CD34, PR, EMA, but negativity for neurofilament, desmin and actin. The tumor should be differentiated from aggressive angiomyxoma and angiomyofibroblastoma, angiofibroma because of its clinical and histological similarity. We report a rare case of benign cellular angiofibroma involving right neck and study other published articles.

A poor prognosis of oral squamous cell carcinoma (SCC) is partly due to the invasiveness and metastasis of the tumor. One of key elements in tumor invasion and metastasis in the degradation of extracellular matrix is tissue inhibitors of metalloproteinases (TIMPs). This study was performed to determine the expression of TIMP-1 and TIMP-2 of oral SCCs with regard to the histologic invasiveness and differentiation in 5 normal oral mucosa and 36 oral SCCs. The histologic invasiveness of oral SCCs were classified into 4 grades. The differentiation of oral SCCs was divided into 3 grades. The StreptAvidin-Biotin immunohistochemical process, using TIMP-1 and TIMP-2 monoclonal antibodies, was performed to determine the expression of TIMP-1 and TIMP-2. The expression of TIMP-1 was positive in 5 of 17 oral SCCs with weak invasiveness and was positive in 8 of 19 oral SCCs with strong invasiveness. The TIMP-1 expression did not increase significantly with respect to the invasiveness of oral SCCs (P>0.05). The expression of TIMP-2 was strongly positive in 5 out of 17 SCCs with weak invasiveness and was strongly positive in 15 of 19 SCCs with strong invasiveness. The TIMP-2 expression increased significantly with respect to the invasiveness of oral SCCs; the stronger the expression, the stronger the invasiveness (P<0.05). The expression of TIMP-1 and TIMP-2 did not increase significantly with respect to the histologic differentiation. We concluded that with respect to the invasiveness, the TIMP-2 expression increases significantly in oral SCCs but the TIMP-1 expression does not; and that with respect to the histologic differentiation, their expressions do not increase significantly. These results suggested that TIMP-2 can be used as a tool to evaluate the invasiveness of oral SCCs.

Dental professionals are repeatedly exposed to many microorganisms present in both blood and saliva. Thus, dental professionals are at a greater risk of acquiring and spreading infections, and the implementation of infections control guidelines is necessary. Cellular phones have become a necessary device for communicating in hospitals. Cellular phones contaminated with bacteria may serve as a fomite in the transmission of pathogens by the hands of medical personnel. Nevertheless, studies about rate and levels of bacterial contamination of cellular phones have been extremely limited with regards to dental personnel. The purpose of this study was to identify bacterial flora on the cellular phones of dentists by a molecular biological method using the 16S rRNA cloning and sequencing method. We acquired total 200 clones from dentists’ cell phones and identified the bacterial species. Pseudomonas (34.6%), Lactobacillus (18.5%), Azomonas (11.5%), and Janthinobacterium (6%) were the dominant genera on dentists’ cell phones. The oral bacteria identified were Anaerococcus lactolyticus, Gibbsiella dentisursi, Lactobacills leiae, Streptococcus mitis, Streptococcus oligofermentans, and Streptococcus sanguinis. Pathogenic bacteria and opportunistic pathogens such as Carnobacterium funditum, Raoultella planticola, Shigella flexneri, Lactobacillus iners, Staphylococcus aureus, and Staphylococcus epidermidis were also identified.

곤충병원성세균 Xenorhabdus nematophila (Xn)의 일부 대사물질은 대상 곤충의 phospholipase A2 (PLA2)를 억제하여 아이코사노이드 생합성 활성을 저해시킨다. 그러나 이들 세균 대사물질이 억제하는 곤충의 PLA2에 대해서는 알려져 있지 않다. Xn의 배양액에서 화학구조가 동정된 8 가지 대사물질들은 두 종의 나비목 배추좀나방(Plutella xylostella)과 파밤나방(Spodoptera exigua)의 유충에 대하여 살충 활성을 보유했다.특별히 이들 물질은 모두 Bacillus thuringiensis (비티)의 살충력을 크게 향상시켰다. 파밤나방의 세포성 인지질 분해효소(SecPLA2)를 클로닝하고 대장균에서 과발현시켰다. 분리된 SecPLA2를 지방체에서 얻은 인지질과 반응시켰을 때 여러 다가불포화지방산을 해리시켰다. 이 효소활성이 Xn 유래 대사물질들에 의해 뚜렷이 억제되었다. 또한 SecPLA2에 대한 억제효과와 비티 살충력 상승효과 사이에 정의 상관관계를 보였다. 본 연구는 SecPLA2가 Xn 대사물질의 억제 대상 분자 종말점 가운데 하나라고 제시하고 있다.

Suicide gene transfer has been study extensively for therapies in various human diseases. We can evaluate cellular activity of thymidine kinase and cytotoxic effect in colon cancer cells after suicide gene transfer. We observed cellular expression of green fluorescence protein after transfer with adenovirus into colon adenocarcinoma HCT-15 cells. After transfer HSVtk, we also estimated thymidine kinase activity using [3H]-penciclovir and cellular cytotoxicity by MTT assay. After transfer green fluorescence protein into HCT-15 cells, we could observed fluorescence expression in 10 moi concentration. Expression level of green fluorescence protein markedly increased in 30 moi and most of HCT-15 cells expressed green fluorescence protein in 100 moi. By infection with HSVtk in HCT-15 cells and HT-29 cells, thymidine kinase activity in HCT-15 cells was about two fold higher than that HT-29 cells. Thymidine kinase activity at 1 moi concentration makes no difference with 0 moi in both cells. At 10 moi concentration, thymidine kinase activity increased about three fold compared with 1moi in HCT-15 cells, but not observed high increase in HT-29 cells. Thymidine kinase activity at 100 moi showed about three fold increase in HCT-15 cells and one and a half fold in HT-29 cells compared with 10 moi. By treatment of HSVtk at various mois and ganciclovir to HCT-15 cells, we could find that increased cytotoxic effect according to HSVtk concentration. Cellular cytotoxic effect was slightly appeared at 5 moi concentration and intensively increased at 30 moi concentration, dead colon cancer cells were reached about 30% of total colon cancer cells. Cellular cytotoxic effect was consistently increased until 50 moi, and about 50% of cells at 100 moi and less then 50% of HCT-15 cells at 200 moi were survived. Finally, we can identify that suicide gene transfer into HCT-15 cells is performed according to concentration of suicide gene and thymidine kinase activity also increase with HSVtk concentration in both HCT-15 cells and HT-29 cells. Additionally, we also find that suicide gene therapy by HSVtk with ganciclovir intensively increase cellular cytotoxicity in colon cancer cells. Therefore, our findings suggest that suicide gene therapy by HSVtk can affect cytotoxicy for colon cancer cells and eventually seems to influence therapeutic efficacy.

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in Protaetia brevitarsis seulensis. we identified the professional phagocytes, granulocytes (GRs), which mediate encapsulation and phagocytosis of pathogens. The GRs were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo.