This report describes the different responses to dapsone treatment in two cases of sterile nodular panniculitis (SNP). Two dogs were presented with ulcerative skin lesions, painful and erythematous papules, and nodules. History and physical examination revealed systemic signs such as pyrexia, lethargy, depression, and anorexia, in addition to ulcerated and ruptured nodules on the skin. The dermatological diagnostics included clear taping, trichogram, skin scraping, impression smears, fungal and bacterial cultures, and histopathology and special stainings of multiple punch biopsies obtained from the skin lesions. Based on the clinical and histopathologic findings, the absence of microbiological infection, and the positive response to immunosuppressive therapy, both the dogs were diagnosed with SNP. Although both dogs had been treated with various immunosuppressive drugs including prednisolone, cyclosporine, azathioprine, and triamcinolone, therapy was switched to dapsone due to recurrent dermatological signs and presumed steroidinduced hepatotoxicity. The clinical responses to dapsone were opposite in the two cases. In the first case, combination therapy with prednisolone and cyclosporine was effective in attenuating ulcerative lesions, while dapsone alone did not control the clinical signs. In contrast, in the second case, the therapeutic response to the common immunomodulatory drugs such as prednisolone, triamcinolone, and azathioprine was inadequate. Interestingly, dapsone alone was effective in controlling the clinical signs without causing undue side effects. Although the usefulness of dapsone for the treatment of canine SNP is unknown, it may be considered in mild to moderate cases of SNP when the use of steroids is not recommended due to its low efficacy or side effects.

물벼룩 급성 독성 평가를 위한 비타민A 강화벼의 분자생물학적 특성을 분석한 결과, Southern blot에서 베타-카로틴 생합성을 위한 Psy와 CrtI 유전자들이 one-copy로 도입됨을 확인하였으며, 선발마커인 Bar 유전자의 단백질 검출 immunostrip 분석에서도 비타민A 강화벼에서만 검출되었다. 비타민A 강화벼의 목적하는 최종 산물인 베타-카로틴 함량도 낙동벼에 비해 8.9배 증가됨을 확인하였다. 비타민A 강화벼와 낙동벼의 농업환경 생물지표종인 물벼룩(Daphniamagna)에 대한 급성독성시험을 실시한 결과, 비타민A 강화벼의 48시간-EC50은 3,311.40 mg/L(95% 신뢰한계 : 2,901.39 ~ 3,779.23 mg/L), 무영 향농도(NOEC)는 1,800 mg/L였고, 낙동벼는 48시간-EC50은 3,655.23 mg/L(95% 신뢰한계 : 3,156.71 ~ 4,232.86 mg/L), 무영향농도는 1,800 mg/L였다. 따라서 Psy와 CrtI 유전자가 형질전환된 비타민A 강화벼 및 낙동벼가 환경 지표생물종인 물벼룩에 미치는 영향 평가 결과 상대적 동등성을 보였으며, 이는 Psy와 CrtI 유전자의 단백질 노출이 물벼룩에 부정적인 영향을 미치지 않은 것으로 판단된다.

Anthocyanins, providing the bright red-orange to blue-violet colors, flavonoid-derived pigments with strong antioxidant activity that have benefits for human health. We isolated RsMYB1, which encodes an R2R3 MYB transcription factor (TF), from red radish plants (Raphanus sativus L.) that accumulate high levels of anthocyanins. RsMYB1 shows higher expression in red radish than in common white radish, in both leaves and roots, at different growth stages. regulatory genes. Transient expression of RsMYB1 in tobacco showed that RsMYB1 is a positive regulator of anthocyanin production. Also, the synergistic effect of RsMYB1 with B-Peru was larger than the effect of Arabidopsis plants stably expressing RsMYB1 produced red pigmentation throughout the plant, accompanied by up-regulation of the six structural and two regulatory genes for anthocyanin production. This broad transcriptional activation of anthocyanin biosynthetic machinery in Arabidopsis included up-regulation of TRANSPARENT TESTA 8, which encodes a bHLH-type TF. These results suggest that overexpression of RsMYB1 promotes anthocyanin production by triggering the expression of endogenous bHLH genes as potential binding partners for RsMYB1. In addition, RsMYB1-overexpressing Arabidopsis plants had a higher antioxidant capacity than did non-transgenic control plants. Taken together, RsMYB1 is an actively positive regulator for anthocyanins biosynthesis in radish plants and it might be one of the best targets for anthocyanin production by single gene manipulation being applicable in diverse plant species.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium -mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium-mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

이차원 전기영동 분석을 이용하여 국내 밀 32 품종의 HMW-GS 단백질 발현의 정성 및 정량적인 분석을 통해 품 HMW-GS 발현 정도를 평가하여 국내 밀 품종 육성의 초 자료로 활용하고자 수행하였다. 평균 HMW-GS 스팟 수는 11.78개였으며, Glu-A1 1.31개, Glu-B1 5.53개, 그리고 Glu-D1에서 4.94 개였다. Glu-B1과 Glu-D1에서는 subunit에 따른 단백질 스팟 수가 차이가 없기 때문에, Glu-A1에서는 1과 2* subunit을 지닌 품종이 null allele 품종에 비하여 단백질 스팟 수가 많았다. 단백질 스팟 수는 조경밀이 18개로가장 많았으며, 다홍밀은 7개로 제일 적었다. 단백질의 상대적인 발현량을 조사한 결과 평균 0.44로 대비 품종인 Chinese Spring에 비하여(1.0) 낮았고, 고분밀이 1.11로 가장 높았으며, 은파밀이 0.24로 가장 낮았다. 단백질 스팟수와 발현량을 이용한 유연관계 분석 결과, 국내 밀 품종을 6개 그룹으로 분류할 수 있었다.

국내 밀 품종 조경, 금강 그리고 중국 밀 품종인 Chinese spring의 genomic DNA를 주형으로 LMW-GS 특이 프라이머세트를 이용하여 3개의 새로운 LMW-GS i 타입 유전자를분리하였고 이들의 분리된 유전자는 각 각 조경 II-2, CSIII-5 그리고 금강 6-12로 명명하였다. 이들의 유추 아미노산을 분석한 결과 20개의 시그널 펩타이드, 이소루신으로 시작하는 N-말단 부분 그리고 글루타민이 많은 반복도메인 그리고 C-말단 부분으로 구성되어 있으며 조경 II-2와 CS III-5는 전형적인 LMW-GS i-type 유전자처럼 C-말단에 8개의 시스테인 잔기가 있었다. 금강 6-12는 특이하게도 하나 더 많은 9개의 시스테인 잔기가 존재하였는데 이 여분의 시스테인 잔기는7번째 시스테인 잔기의 11잔기 앞에 존재하며 TAT(타이로신)이 TGT(시스테인)로 바뀐 결과이다. LMW-i 타입 글루테닌 유전자들 간의 SNP와 InDel을 확인하기 위해서 본 연구에서 클로닝 된 조경 II-2, CS III-5 그리고 이전에 본 그룹에서 확인된 조경 HQ619933와 기존 문헌에 나와 있는 6배 체 밀 유래의 10개의 LMW-GS i 타입 유전자들과 다중염기서열 분석을 실시하였고, 이들 사이에서 15개의 SNP와 1개의 insertion이 확인되었다. 밀 품종 조경의 Glu-A3 단백질을 동정하기 위해 글루테닌을 추출 이차원전기영동을 하고 Glu-A3c 위치의 스팟을 절취하여 in-gel digestion한 후 LC-ESI MS/MS 분석을 수행한 결과 조경의 i 타입 LMW-GS 유전자 좌는 Glu-A3c로 확인되었다. LMW-i 타입 글루테닌 유전자들의 연관 관계를 분석하기 위해 본 연구 그룹에서 클로닝 한 조경 II-2, CS III-5, 금강 6-12 그리고 조경 HQ6199333와 Genebank DB의 35개의 LMW-i 타입 글루테닌 유전자의 유추 아미노산 서열을 이용하여 Phylogenic tree를 완성하였다. 이들 39개의 계통도 분석 결과 이배체 밀과 4배체 밀의 LMi 타입 글루테닌이 육배체 밀의 LMW-i 타입 글루테닌과 크게 나눠지는 것을 확인하였으며, 육배체 밀의 LMW-i 타입 글루테닌들은 Glu-A3a부터 GluA-3g까지 7개 subgroup으로 나눠지는 것을 확인하였다. 금강 6-12는 GluA-3a와 GluA-3c 사이에 존재하였고 조경 II-2와 CS III-5는 GluA-3d와 일본 연질 밀인 농림 61의 AB062878과 같은 subgroup에 존재하였고 조경 HQ6199333은 Glu-A3c subgroup에 위치하였다. LMW-i 타입 글루테닌 유전자들의 유추 아미노산 다중서열분석결과 반복 도메인은 length polymorphism은 179～149개 정도의 long 타입과 91, 51, 10, 2개의 short 타입으로 나눠지고 이것은 long 타입과 short 타입 LMW-i 타입 글루테닌 유전자를 구분 할 수 있는 마커의 근거가 된다.

Rice seed storage proteins (SSPs) are accumulated in storage organelles of the endosperm during seed maturation. The SSPs from the rice seeds consist of glutelins as a major SSP, and prolamins and globulins comprise about the rest 20 % of the SSPs. To improve the nutritional quality of rice seeds or processing properties of rice flour, we are attempting to change the composition of the SSPs in rice seeds. For this purpose, we generated many transgenic rice plants, which show the altered levels of the SSPs, by using the RNA interference (RNAi). Accumulation of glutelins was 76% reduced in the GluA-RNAi lines. The Pro-RNAi lines revealed the reduced levels of prolamins to 36%. The protein level of globulins was 61% reduced in the Glb-RNAi lines. Interestingly, an obvious reduction of glutelins, prolamins, and globulins was not examined in the GluA:Pro:Glb-RNAi lines. This suggests that a reduction of a few SSPs could be compensated by the increases of other SSPs at the protein levels. We are also attempting to generate transgenic rice plants expressing both a high-molecular-weight (HMW) glutelin subunit and a low-molecular-weight (LMW) glutelin subunit. These manipulations of rice SSPs might be an important contribution on improving the functional properties of rice seeds.

To develop a strong root-specific gene expression system, six gene promoters were investigated by using transgenic Arabidopsis and a GUS:GFP reporter gene. These promoters were initially selected from Arabidopsis genes which are specifically expressed only in roots, based on the TIGR information. The GUS activity of these promoters was measured in several tissues of Arabidopsis by using both histochemical and fluorimetric GUS assays. The results showed that the activity of these promoters was strongly detected only roots. This was also confirmed by RT-PCR analysis. Therefore, these six promoters could be used for utilization of a root-specific expression of target genes.

Although it is known that the composition of high-molecular weight glutenin subunits (HMW-GSs) and low-molecular weight glutenin subunits (LMW-GSs) are important factor for bread, noodle and cookie, it is still not clear which HMW-GSs and LMW-GSs confer improved processing properties and how those HMW-GSs and LMW-GSs interact each other. In this study, to investigate distinctive glutenin proteins for characteristic processing properties for noodle, glutenins extracted from seeds of several Korean and Chinese wheat cultivars were focused in IPG gel strip and subjected to SDS-polyacrylamide gel electrophoresis. Differential protein expression level was analyzed using image master platinum 6. Then to characterize the HMW-GSs of Korean cultivar Uri, extracted glutenin proteins were separated by two dimensional gel electrophoresis. Nine spots digested with trypsin resulting peptide fragmentation were identified by LC ESI-MS/MS and MASCOT database. We also separated HMW-GSs from wheat cultivar Uri by fast protein liquid chromatography (FPLC) withResource Phe column using gradient buffer condition with 4M Urea and 0.45M ammonium sulfate.Each single band of 1Dx 2.2, 1Bx7 and double bands of 1Dy8 and 1By12 were separated.

β-carotene producing transformants were produced in the background of "Nagdong", a japonica rice cultivar. Introgression of the caroteniod locus into the elite cultivar "Ilpum". Was started to initiate a backcrossing program, we surveyed 220 SSR markers and found that 38% of them were polymorphic between the “Ilpum” as the recurrent parent and "Nagdong" transfomed as a recipient parent. First, backcross progenies have been produced and genotyped by the transgene specific PCR-based marker. Blast search indicated that the transgene PAC4-2 was located between SSR markers, RM5631 and RM5916. We produced 240 BC3F2 plants derived from thirteen BC3F1 plants containing the caroteniod gene. To develop near isogenic line, additional experiments including target and background selection are being performed using SSR markers. Traits of agronomic importance such as heading date, panicle number and culm length will also be evaluated. The results will be discussed.

There are many evidences that carotenoids may act as antioxidants and protect humans from serious disorders such as skin degeneration and aging, cardiovascular disease, certain types of cancer, and age-related diseases of the eye. Carrots (Daucus carota L.) are consumed as an important dietary source of b-carotene, a-carotene and lutein. Astaxanthin, a keto-carotenoid has been used to raise red color of fish body and to improve immune activity in fish-breeding industry. In this study, transgenic carrot plants were generated to overproduce carotenoids including astaxanthin, a non-natural ketocarotenoid in this plant, using an efficient storage root-expression system. Among the nineteen transgenic carrot plants, transformed by a storage root-specific (ibMads) or a storage root (ibAGP1) or the constitutive CaMV35S promoters with three genes involved in carotenoid synthesis [Psy (Phytoen synthase), Crtl (Lycopen-β-cyclase), CrtO (β-carotene ketolase)], transgenic plants with ibAGP1 promoter, an amyloplast targeting sequence (TP1) and a single CrtO gene gave high content of keto-carotenoids and b-carotene. For fish body coloration, carotenoid extract or astaxanthin significantly made the body color of red seabreams more reddish than those of normal diet-fish in the 3 weeks feeding. In addition, the serum lysozyme activity in carotene-treated fish was significantly higher than that in normal diet-fed fish (P<0.05) in the 6 weeks feeding. In these cases, neither carotenoid extract- nor astaxanthin-contained diet did influence on growth rate and food utilization in red seabreams. These results suggested that carotenoid extract prepared in the present study may be useful in the body coloration and the enhancement of nonspecific immune response of red seabreams. Meanwhile, b-carotene (50 mM) up-regulated peroxisome proliferator-activated receptor a expression (PPAR-a) by about two fold in CV-1 cells, while the carotenoid extracts and astaxanthin failed to affect on the expression. Carotenoid extracts (250 mg/ml) from wild type carrot or transgenic carrots showed moderate DPPH scavenging activity.

Carrots (Daucus carota L.) are consumed as an important dietary source of provitamin A including β-carotene, α-carotene and lutein. An HPLC method was applied to determine the content of the carotenoid composition in carrot cutivars cultivated in Korea. HPLC analyses were carried out with five carrot cultivars (Socheon-5-chon, Hongsim-5-chon, Myeongju-5-chon, Seonhongbom-5-chon and Betarich) sown at April, 2007 and six cultivars (Yeoreum-5-chon, Hanyeoreum-5-chon, Sinheukjeon-5-chon, Bibariheukjeon, Manina and Betarich) sown at August of the same year. In general, the former varieties are not used for the sowing at summer because of their bolting (growth of floral axis). The former and the latter carrots were harvested after 110 and 96 days from seeding, respectively, and the carotenoids were extracted with acetone after freeze-drying. The amount of α-carotene (117.7∼205.3 μg/g・DW) was similar to that of β-carotene (113.1∼189.6 μg/g・ DW) for the carrot cultivars sown at spring, while the content (46.2∼71.1 μg/g・DW) was about a half of β-carotene content (92.5∼140.2 μg/g・DW) for the latter cultivars. In addition, the average content of lutein (25.2 μg/g・DW) in the former cultivars was eight times higher than that in the latter cultivars (3.1 μg/g・DW). Among the spring cultivation types, Socheon-5-chon and Myeongju-5-chon showed higher amount of α-carotene and β-carotene, while the higher amount was determined in Yeoreum-5-chon and Sinheukjeon-5-chon among the autumn cultivation types. Validation of the HPLC-DAD method showed good linearity (r2 > 0.997) of the three compounds analyzed in a wide concentration range (0.025∼20 μg/ml). The R.S.D. values for intra-day and inter-day precision were less than 19.2% and the mean recovery of each compound was 85.4∼104.7%.