• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.61-74
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• 1996

Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.231-243
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• 1998

In the present study, we provide evidence that ginsenoside $Rh_2$ (G-$Rh_2$) as well as staurosporine induces apoptosis of human hepatoma SK-HEP-1 cells by caspase 3-mediated processing of $p21^{WAFI/CIPI}$ in the early stage of apoptosls. Immunoblottings showed that G-$Rh_2$ as well as statrosporine induced the processing of caspase-3 to an active form, pl7. In stable Bcl-2 transfectants however, G-$Rh_2$ induced DNA fragmentation, while staurosporine did not. In the early stage of apoptosis, $p21^{WAFI/CIPI}$ was detected to undergo proteolytic processing specifically conducted by caspase-3. $p21^{WAFI/CIPI}$ translated in vitro was cleaved into a p14 fragment, when incubated with cell extracts obtained from either G-$Rh_2$- or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-cho, a specific caspase-3 inhibitor, but not by Ac-YVkD-cho, a specific caspase-l inhibitor. Similarly, $p21^{WAFI/CIPI}$ was efficiently leaved by recombinant caspase-3 overexpressed in E. coli. Moreover, the endogenous $p21^{WAFI/CIPI}$ of untreated-cell extracts was also cleaved by recombinant caspase-3. Mutation analysis allowed identification of two caspase-3 cleavage sites, $DHVD^{112}$/L and $SMTD^{149}$/F, which are located within, or near the interaction domains for cyclins, Cdks, and PCNA. Taken together, these results show that G-$Rh_2$ as well as staurosporine increases caspase-3 activity, which in turn directly cleaves $p21^{WAFI/CIPI}$ resulting in elevation of Cdk kinase activity in the early stages of apoptosis. We propose that proteolytic cleavage of $p21^{WAFI/CIPI}$ is a functionally relevant event that allows unleashing the cyclin/Cdk activity from the inhibitor seen in the earlier stage of apoptosis, the event of which may be associated with the triggering mechanism for the execution of apoptosis.

• Korean Journal of Environmental Agriculture
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• v.19 no.2
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• pp.147-153
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• 2000

Cigarette smoking deals a harmful effect directly to smokers and even to non-smokers through environmental tobacco smoke. The major damaging component in cigarette smoke is nicotine which converts to various carcinogens. Among the carcinogenic metabolites, nitrosamine-4-(methylnitrosamino)-1- (3-pyridyl)-1- butanone (NNK) is responsible for many types of lung cancers. Recent studies report that activation of NNK is markedly inhibited in the presence of cotinine, a safer metabolite from nicotine. It is well known that tea extract have potentials to prevent cancers. This study aims to correlate green tea's potential for cancer prevention with an accelerated formation of cotinine. In the presence of tea extract, a nicotine to cotinine conversion was studied in established cell lines and xenopus oocytes. Among three lines of cell used, PLC/PRF5 and 293 cells showed a fast turnover from nicotine to cotinine while HepG2 cell line showed a marginal difference between groups treated and non-treated with tea extract. A microinjection procedure using Xenopus oocyte was utilized to probe for the effect of tea extract in accelerating nicotine conversion to cotinine. According to this procedure, tea extract's unusual potential for converting nicotine to cotinine is also substantiated. Overall, this present study indicated that tea extract have an unusual effect on conversion of nicotine to cotinine in cells.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.71-80
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• 2005

Effects of low temperature ($8^{\circ}C$) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber; Cucumis sativus L.) and a chilling-resistant (figleaf gourd; Cucurbita ficifolia Bouche) crop have been measured at the levels of whole root systems (root hydraulic conductivity, $Lp_r$) and of individual cortical cells (cell hydraulic conductivity, Lp). In figleaf gourd, there was a reduction only in hydrostatic $Lp_r$ but not in osmotic $Lp_r$ suggesting that the activity of water channels was not much affected by low root temperature (LRT)treatment in this species. Changes in cell Lp in response to chilling and recovery were similar asroot level, although they were more intense at the root level. Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (both at the root and cell level) often resulted in Lp and $Lp_r$ values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger forosmotic (representing the cell-to-cell passage of water) than for hydrostatic $Lp_r$. After a short term (1 d) exposure to $8\;^{\circ}C$ followed by 1 d at $20\;^{\circ}C$, hydrostatic $Lp_r$ of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. On the contrary, osmotic $Lp_r$ and cell Lp in both species remained high by a factor of 3 as compared to the control, possibly due to an increased activity of water channels. After pre-conditioning of roots at LRT, increased hydraulic conductivitywas completely inhibited by $HgCl_2$ at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins and alterations of root anatomy determine the water uptake in both species. To better understand the aquaporin function in plants under various stress conditions, we examined the transgenic Arabidopsisand tobacco plants that constitutively overexpress ArabidopsisPIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates were found between the transgenic and wild-type plants under favorable growth conditions. By contrast, overexpression of PIP1;4 or PIP2;5 had a negative effect on seed germination and seedling growth under drought stress, whereas it had a positive effect under cold stress and no effect under salt stress. Measurement of water transport by cell pressure probe revealed that these observed phenotypes under different stress conditions were closely correlated with the ability of water transport by each aquaporin in the transgenic plants. Together, our results demonstrate that PIP-type aquaporins play roles in seed germination, seedling growth, and stress response of Arabidopsis and tobacco plants under various stress conditions, and emphasize the importance of a single aquaporin-mediated water transport in these cellular processes.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.45-52
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• 2009

In order to understand the molecular interactions that occur between rice and the rice blast fungus during infection, we previously identified a number of rice blast fungal elicitor-responsive genes from rice (Oryza sativa cv. Milyang 117). Here, we report the cloning and characterization of the rice fungal elicitor-inducible gene Oshin1 (GenBank Accession Number AF039532). Sequence analysis revealed that the Oshin1 cDNA is 1067 bp long and contains an open reading frame encoding 205 amino acid residues. The Oshin1 gene shows considerable sequence similarity to the tobacco hin1 and hin2 genes. The predicted Oshin1 protein has a cysteine-rich domain at the N-terminus and is rich in leucine, serine, and alanine residues. Southern blot analysis suggests that Oshin1 gene is a member of a small gene family in the rice genome. To examine the expression of Oshin1, Northern blot analysis was conducted. Expression of the Oshin1 transcript is rapidly induced in suspension-cultured rice cells treated with fungal elicitor, salicylic acid or hydrogen peroxide. In addition, Oshin1 transcript levels are rapidly increased by treatment with $Ca^{2+}$/A23187. The expression of Oshin1 was also elevated in 3-week old leaf tissues upon ethephon application or fungal elicitor treatment. Our results suggest that the Oshin1 gene is involved in plant defense responses to environmental stresses.

• Current Optics and Photonics
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• v.3 no.2
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• pp.150-153
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• 2019

Larynx cancer is a potentially terminal and severe type of neck and head cancer in which malignant cells start to grow and spread upwards in the larynx, or voice box. Smoking tobacco, drinking hot beverages and drinking alcohol are the main risk factors for these tumors. In this study, we aimed to develop a precise, accurate and rapid chemometrics assisted Raman spectroscopy method for diagnosis of larynx cancer in deparaffinized tissue samples. In the proposed method, samples were deparaffinized and 20 microns of each tissue were located on a coverslip. Both healthy (n = 13) and cancerous tissues (n = 13) were exposed to a Raman laser (785 nm) and excitations were recorded between wavenumbers of $50{\sim}1500cm^{-1}$. An Orthogonal Partial Least Square algorithm was applied to evaluate the Raman spectrum obtained. Sensitivity and specificity of the proposed method is high enough with the aid of Principal Component Analysis (PCA) to test the whole model. Healthy and cancerous tissues were accurately and precisely clustered. A rapid, easy and precise diagnosis algorithm was developed for larynx cancer. By this method, some useful data about differences in biomolecules of each group (phospholipids, amides, tyrosine, phenylalanine collagen etc.) was also obtained from the spectra. It is claimed that the optimized method has a great potential for clustering and separating tumor tissues from healthy ones. This novel, rapid, precise and objective diagnosis method may be an alternative for the conventional methods in literature for diagnosis of larynx cancer.

• Journal of Periodontal and Implant Science
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• v.31 no.3
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• pp.597-610
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• 2001

Normal gingival fibroblasts functioning is fundamental for the maintenance of periodontal connective tissue as well as wound healing. Nicotine have been found to affect DNA synthesis and cell proliferation, which appear to depend on the type of cells. This in vitro study was done to determine the effects of nicotine, a major component of tobacco, on cell proliferation, viability, activity, cell cycle distribution, and expression of cell cycle regulatory proteins in human gingival fibroblasts. Nicotine has been tested for 2 days or 4 days in 5 different concentrations; $0.1{\mu}g/ml$; $1{\mu}g/ml$; $10{\mu}g/ml$; $100{\mu}g/ml$; $1000{\mu}g/ml$. To assess cell proliferation and viability, viable and non-viable cells were counted by hemocytometer; to evaluate cellular activity, MTT assay was employed; to analyze cell cycle distribution, fluorescent propidium iodide-DNA complex were measured using fluorocytometer; to determine the expression of cell cycle regulatory proteins, western blot analysis was performed. After 2 days and 4 days incubation respectively, at concentrations of $1{\mu}g/ml$ - $1000{\mu}g/ml$, nicotine significantly inhibited proliferation comparing to non-supplemented controls. The cell viability was significantly decreased after 2 days and 4 days at concentrations of $1{\mu}g/ml$ - $1000{\mu}g/ml$ and at $10{\mu}g/ml$ - $1000{\mu}g/ml$ respectively. After 2 days and 4 days, the cellular activity was significantly decreased at concentrations of $10{\mu}g/ml$ - $1000{\mu}g/ml$. Treatment with $100{\mu}g/ml$ nicotine for 48 hours caused an increase in the proportion of G1-phase cells (from 46.41% to 53.46%) and a decrease in the proportion of S-phase cells (from 17.80% to 14.27%). The levels of cyclin $D_1$ and CDK 4 proteins in nicotine-treated fibroblasts were lower than that of controls, whereas the levels of p16 and pRB were higher than that of controls. These results suggest that the decrease of cell proliferation and lengthened Gap phases (G1) by nicotine may due to the increased expression of p16 and pRB as well as decreased expression of cyclin $D_1$ and CDK 4 in human gingival fibroblasts.

• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.300-311
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• 1998

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most notorious toxic environmental pollutants, induces various toxic effects in many organs including testes and is regarded as an endocrine disruptor. Korean ginseng, on the other hand, has been well known for its preventive effects on lox- ins, diabetes melltus and hyperlipidemia. We investigated, histopathologically, the effect of Korean Red ginseng water extract (KR-WE) on guinea pig testes damaged by TCDD. Ninety guinea pigs were divided into 6 groups: normal control (NC) group received vehicle and saline; TCDD,1191kg b.w., was administered intraperitoneally to the single dose TCDD-treated (77) group; 100 mghg b.w.16 and 200mg1kg b.w./d KR-WE were injected intraperitoneally to the preventive groups (PIOO and P2OO, respectively) for 28 days from 1 week before TCDD injection, and to the therapeutic groups (CIOO and C2OO, respectively) for 14 days since 1 week after TCDD administration. Increment of body weight was retarded to a larger extent by TCDD. Moreover, body weight of the 77 group decreased significantly 7 days after TCDD exposure, while that of preventive groups kept increasing. Decrease in body weight was not observed in KR-WE-treated groups. Weight decrease in testes caused by TCDD was remarkably protected by KR-WE. Testicles in 77 group displayed decreased tubular size and maturation arrest at the primary or secondary spermatocyte stage. On the other hand, maturation arrest in germ cells by TCDD was improved in KR-WE treated groups. Almost complete protection of the testes was observed in PIOO and P2OO groups. In addition, the therapeutic effect was noticed in C 100 and C2OO groups. These results provided strong evidence that Korean Red ginseng might be a useful agent for the prevention and treatment of testicular damage induced by environmental pollutants.

• Journal of Ginseng Research
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• v.23 no.3 s.55
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• pp.123-129
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• 1999

Under the light condition of 25,000 Lux (12 hrs dark and light cycle) with scrapping treatment of aerial mycelia of Cylindrocarpon destructans on potato dextrose agar (PDA), V-8 juice agar, and ginseng extract agar, production of the macroconidia was increased to $3.7\~8.1$ fold over them produced in the dark. They were also produced $7.7\~18.0$ times more in the liquid cultures under the light condition than under the dark as well. PDA and V-8 juice agar among the tested were the best for the macroconidium production. On PDA, 1,585 $macroconidia/mm^2$ were produced under the light of 25,000 Lux with scrapping treatment of aerial mycelia of C. destructans, which is 3.2 and 1.4 times more than those produced under 3,000 and 10,000 Lux, respectively. Meanwhile, $20\~99$ macroconidia/$mm^2$ were produced by the non-scrapping under the light condition between 3,000 Lux and 25,000 Lux. The macroconidia were, however, lysed at $6\~7$ days after being incubated under the above range of the light. They were consisted of $1\~3$ cells in a macroconidium while $69.4\~100\%$ of them were the two-celled and the number did not seem to be affected by either the scrapping or the light. Production of chlamydospore converted from mycelia of C. destructans seemed to be promoted by the light and the scrapping as well. The 1,285 chlamydospres/$mm^2$ were produced with the light (25,000 Lux), which is 2.8 and 1.2 times more than those with 3,000 and 10,000 Lux, respectively. Scrapping the aerial mycelia of the cultures increased the chlamydospore formation to 1.9, 2.5 and 1.4 times more than the non-scrapping under the light intensity of 3,000 Lux, 10,000 Lux, and 25,000 Lux, respectively. On PDA, 1 to 8 chlamydospore(s) per catena were formed by all treatments tested and $34.2\~58.9\%$ of them was a single chlamydospore, However, the numbers was affected by neither the light ($3,000\~25,000$ Lux) nor the scrapping the aerial mycelia.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.209-215
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• 2005

Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.