Activation of transient receptor potential vanilloid 1 (TRPV1), a calcium permeable channel expressed in primary sensory neurons, induces the release of glutamate from their central and peripheral afferents during normal acute and pathological pain. However, little information is available regarding the glutamate release mechanism associated with TRPV1 activation in primary sensory neurons. To address this issue, we investigated the expression of vesicular glutamate transporter (VGLUT) in TRPV1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) under normal and complete Freund’s adjuvant (CFA)-induced inflammatory pain conditions using behavioral testing as well as double immunofluorescence staining with antisera against TRPV1 and VGLUT1 or VGLUT2. TRPV1 was primarily expressed in small and medium-sized TG neurons. TRPV1+ neurons constituted approximately 27% of all TG neurons. Among all TRPV1+ neurons, the proportion of TRPV1+ neurons coexpressing VGLUT1 (VGLUT1+/ TRPV1+ neurons) and VGLUT2 (VGLUT2+/TRPV1+ neurons) was 0.4% ± 0.2% and 22.4% ± 2.8%, respectively. The proportion of TRPV1+ and VGLUT2+ neurons was higher in the CFA group than in the control group (TRPV1+ neurons: 31.5% ± 2.5% vs. 26.5% ± 1.2%, VGLUT2+ neurons: 31.8% ± 1.1% vs. 24.6% ± 1.5%, p < 0.05), whereas the proportion of VGLUT1+, VGLUT1+/TRPV1+, and VGLUT2+/TRPV1+ neurons did not differ significantly between the CFA and control groups. These findings together suggest that VGLUT2, a major isoform of VGLUTs, is involved in TRPV1 activation-associated glutamate release during normal acute and inflammatory pain.

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

The virus-like particle (VLP) is similar to a pathogenic virus and has a high immunogenicity. However, in the selection of various structural proteins to form VLP, all expression systems commonly consume most of the time and suffer from various difficulties. Therefore, there is a need for a system that can rapidly determine the structural proteins required for effective VLP production. This study aims to construct a transient expression platform using insect cells to solve this problem. Plasmid-based expression vectors and baculovirus-inducible expression vectors were constructed. The vectors were evaluated for overexpression using EGFP. We also confirmed the formation of virus like particles through overexpression of virus structural proteins.

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic Ca2+ ([Ca2+]i), transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular Ca2+ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent Ca2+ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced [Ca2+]i increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

Bromoviridae과 Cucumovirus속에 속하는 대표 바이러스인 오이모자이크바이러스 (Cucumber mosaic virus: CMV)는 많은 경제적으로 중요한 원예작물 및 관상식물들에 심한 손실을 초래하는 바이러스이다. 다중염기서열 비교는 현재까지 서브그룹1에 속하는 모든 CMV 계통들에서 3'말단부의 보전적 염기서열들이 존재하고 있음을 보여주었다. 이런 관찰에 기초하여, 우리는 CMV RNA3와 상동성을 가지는 162 bp 상보적 DNA를 포함하며 CMV감염에 대하여 식물 유래 RNA간섭 현상을 유도할 수 있는 도치된 반복 구조를 가지는 머리핀RNA (pIR-CMVNCR)를 발현시킬 수 있는 벡터를 제작하였다. 아그로박테리움을 이용하여 IR-CMVNCR의 일시 발현은 CMV 감염을 저해하였으며, 아그로박테리움이 접종된 식물의 상엽에서는 CMV 병징이 발현되지 않았다. 또한 RT-PCR결과는 아그로박테리움이 접종된 식물의 접종엽 및 상엽에서 CMV 서브유전자 4를 포함하는 CMV RNA들이 모두 검출이 되지 않았다. CMV 유래 작은 저해 RNA들의 축적이 관찰되었으며, 이의 결과의 의미는 아그로박테리움에 의해 IRCMVNCR을 일시 발현시킨 야생담배 (Nicotiana benthamiana)의 접종엽에서 RNA 간섭 현상이 유도되어 CMV 감염을 억제시키는 것으로 판명되었다.

Ischemia, the leading cause of strokes, is known to be deeply related to synaptic plasticity and apoptosis in tissue damage due to ischemic conditions or trauma. The purpose of this study was to research the effects of NEES(needle electrode electrical stimulation) in brain cells of ischemiainduced rat, more specifically the effects of Poly[ADP-ribose] polymerase(PARP) on the corpus striatum. Ischemia was induced in SD mice by occluding the common carotid artery for 5 minutes, after which blood was re-perfused. NEES was applied to acupuncture points, at 12, 24, and 48 hours post-ischemia on the joksamri, and at 24 hours postischemia on the hapgok. Protein expression was investigated through PARP antibody immuno-reactive cells in the cerebral nerve cells and western blotting. The number of PARP reactive cells in the corpus striatum 24 hours post-ischemia was significantly(p<.05) smaller in the NEES group compared to the global ischemia(GI) group. PARP expression 24 hours post-ischemia was very significantly smaller in the NEES group compared to the GI group. Results show that ischemia increases PARP expression and stimulates necrosis, making it a leading cause of death of nerve cells. NEES can decrease protein expression related to cell death, protecting neurons and preventing neuronal apoptosis.

An efficient transient expression system has been developed and characterized for the production of foreign genes in seedlings. The seedlings can be easily produced from commercial seeds used for vegetable sprouts. In principal, a chemical abrasive was employed to generate wounds in seedlings prior to vacuum-infiltration with Agrobacterium tumefaciens bearing the target gene. This optimized chemical wounding-assisted agro-infiltration process resulted in up to 15-fold increase in β -glucuronidase (GUS) enzyme activity. This procedure has been used efficiently to express hepatitis B surface antigen (HBsAg) protein in a transient mode. Therefore, seedlings with proper wounds can be suggested as a convenient tool for the production of useful recombinant proteins.

Ceriporiopsis subvermispora is a unique white rot fungus degrading plant cell wall lignin without severe loss of cellulose. Recombinant plasmids containing homologous gene expression signals fused to the coding sequence of Escherichia coli hph which encodes for hygromycin phosphotransferase were introduced in protoplasts of wild type C. subvermispora using PEG/CaCl2 protocol. A number of hygromycin B resistant strains were isolated on a screening plate containing 100㎍/ml of hygromycin B: whereas, no colonies were observed when protoplasts were treated with no DNA as a negative control. It was demonstrated that most of the isolates lost their drug resistance during successive cultivations in the presence of the same concentration of hygromycin B, but some of the isolates showed stable drug resistance after five times repeated screening. They did not lose the drug resistance even after the cultivation in the absence of hygromycin B and incorporation of the hph sequence was confirmed by specific amplification of the target sequence in PCR experiments and Southern hybridization analysis. The stable transformation system will make it possible to do molecular genetic analysis, as well as breeding of genetically modified strains, in C. subvermispora. Moreover, it was demonstrated that recombinant constructs with truncated promoter showed reduced number of the drug resistant isolates on the first screening plate, in response with the length of the remaining promoter sequence. These findings indicated that unstable drug resistance observed in these isolates should originate from transient expression of the introducing marker genes, and that a promoter assay system has been developed for the first time in basidiomycete. This system is practically not affected by the positional effect of the integrated recombinant gene in the host chromosome.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp encoding two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28~50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. ORF301 and ORF302 were transiently expressed in hemocyte, fat body, gut, and epidermis of P. xylostella. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to non parasitized larvae of P. xylostella, in which the two genes were expressed at least for four days post-injection. The P. xylostella larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population, suppression of immune associated genes including cecropin, pro-phenoloxidase (PO) and serpin1, and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression by the viral segmenttreated larvae, but not by ORF302 specific dsRNA. The larval injected with CpBV-S3 exhibited an enhanced susceptibility to baculovirus infection. These results indicate that ORF301 of CpBV-S3, which containing BEN domain, suppresses both cellular and humoral immune responses in P. xylostella.

Perennial ryegrass (Lolium perenne L.) is one of the most important grass species in the world's temperate zones. It is used as high-quality forage in pastures and for recreational use as turf in golf courses, lawns and parks. Genetic improvement of perennial ryegrass is difficult due to its self-incompatibility. Consequently, progress by conventional breeding can be slow. Genetic transformation is an alternative that permits direct introduction of useful genes into a plant's genome and is becoming a powerful tool to complement conventional breeding. To improve environmental stress tolerance and quality of perennial ryegrass by introducing better and useful genes into the genome, we have developed a rapid and efficient transformation system using Agrobacterium-mediated gene transfer system.

외래단백질의 생산을 위하여 백합화분을 이용한 일시발현체계를 개발하였다. 충분히 성숙단계에 도달한 백합의 수술로부터 백합화분을 수집한 후 이들은 40-100 μm 크기의 aluminum oxide 입자와 함께 강하게 교반시켰다. 결과적으로 표면에 상해를 지닌 화분립에 대하여 erythropoietin(EPO) 유전자 DNA를 포함한 식물발현벡터(pBI-EPO)를 지니는 Agrobacterium tumefaciens LBA4404 세포들을 섞어 진공침윤을 수행하였다. Southern blot 및 cDNA blot hybridization의 분석 결과 EPO DNA의 화분 genomic DNA 삽입과 EPO mRNA의 생성이 각각 성공적으로 확인되었으며 이러한 결과는 Agrobacterium을 이용한 형질전환화분이 신속하고 편리한 단백질공장으로서 이용될 수 있음을 시사하고 있다.

The diamondback moth, Plutella xylostella parasitized by its endoparasitoid Cotesia plutellae undergoes various physiological alterations which involves immunosuppression and developmental arrest. Its symbiotic virus, C. plutellae bracovirus (CpBV) is highly essential for their successful parasitization which possesses more than 136 genes encoded. CpBV15βunique in CpBV genome is expressed at low levels in early and at higher levels during late parasitization period. This gene product alters the hemocyte-spreading behavior through inhibition of protein synthesis under in vitro conditions. In the current study, we investigated its specific involvement in physiological functions in the host by transient expression and RNA interference techniques. The open reading frame of CpBV15βwas cloned into a eukaryotic expression vector and this recombinant CpBV15β was transfected into healthy non-parasitized 3rd instar P. xylostella by microinjection. CpBV15βwas expressed as early as 24 h and was consistent up to 72 h. Due to the expression of this gene, the hemolymph storage protein levels were significantly reduced and the ability of the hemocytes to adhere and spread on extracellular matrix was altered or reduced, wherein CpBV15βwas detectable in the cytoplasm of hemocytes based on indirect immunofluorescence assay. To confirm the role of CpBV15β, its double stranded RNA could efficiently recover the functional efficacy of hemocytes towards non-self and synthesis of storage proteins. Thus these results clearly demonstrate the role of CpBV15βin altering the host physiology by involving in cellular immune response and host protein synthesis.

蓮(Nelumbo mucifera Gaertn)은 그 아름다운 꽃으로 유명하지만 또한 연 식물체의 거의 모든 부위는 식용 및 약용으로 쓰일 수 있다. 본 연구에서는 蓮根을 대상으로 Agrobacterium 세포감염 방법으로 유전적인 형질전환을 시도하였다. 즉, 蓮根을 1 mm 정도의 두께로 얇게 썰어서 절편으로 준비한 후 이에 대해 agroinfiltration 및 동시배양(3일, 22℃, 암 조건)을 수행하였다. 이러한 일반적인 agroinfiltra

Transient expression profiles for several chimeric β-glucuronidase (GUS) gene constructs were determined in microspore-derived embryos of wheat following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by six different promoters [Cauliflower Mosaic Virus 35S (CaMV35S), Nopaline synthase (NOS), Mannopine synthase (MAS), Chlorella Mosaic Virus Adenin methyltransferase (AMT), maize Ubiquitin 1 (UBI1), and enhanced 35S (E35S)]. The total numbers of GUS blue spot were determined manually under a dissecting microscope after histochemical staining for GUS. Results suggest that the E35S promoter is the most active and UBI1 promoter is the second active in embryos or embryogenic calli derived from wheat microspore. In addition, by flurometric assay on GUS, the E35S promoter was the best. Therefore, both UBI1 and E35S promoter are suitable for constitutive expression of the gene of interest in microspore-derived embryos of wheat. This information describing promoter functionality in wheat will be important when designing gene constructs for traits modification and when choosing appropriate cultivars for improvement through gene transfer experiments.