• Journal of Microbiology and Biotechnology
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• 제6권4호
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• pp.295-298
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• 1996

Under glutamine-limited condition, $2\times10^6$ (viable cells/ml) of maximum cell density and 13.5 ($\mu g$/ml) of tissue-type Plasminogen Activators (tPA) production were maintained by spike feeding fresh medium in fed-batch cultivation of human recombinant melanoma cells. It showed that tPA production was much seriously affected than cell growth according to initial glutamine concentrations. Above 3.4 (mmol/I) of glutamine concentration both cell growth and tPA production were not much affected by increasing initial glutamine concentration. Glutamine depleted situation was occurred at latter periods of batch and fed-batch cultivations below 5.4 (mmole/I) of initial glutamine concentration. It also showed that maximum glutamine consumption and ammonia evolution rates were closely related to initial glutamine concentrations. Maximum specific tPA production rate was estimated as $8.1\times19^{-6}$ ($\mu g$/cells/h) at 3.4(mmol/I) of glutamine concentration, which is higher than that from other batch and fed-batch processes.

• 한국미생물·생명공학회지
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• 제14권5호
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• pp.427-432
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• 1986

The regulation of three ammonia assimilatory enzymes, GDH (glutamate dehydrogenase), GS (glutamine synthetase) and GOGAT (glutamate synthase), has been examined in C. glutamicum. Three kinds of arginine auxotrophs blocked in each step of arginine biosynthetic pathway from glutamate were selected as arg 5, arg 6, arg 8. Histidine and tryptophan auxotrophs were also selected because histidine and tryptophan repressed GS biosynthesis in E. coli. These strains were cultured on the media containing nitrogen-excess and limited conditions, to compare the specific activities of ${\alpha}$-ketoglutarate dehydrogenase(${\alpha}-KGDH$), GDH, GS, GOGAT from the cell-free extracts. These results showed that enzyme levels of ${\alpha}-KGDH$ and GDH from 3 kinds of arginine auxotrophs, histidine and tryptophan auxotrophs in nitrogen-excess condition and those of GS and GOGAT in nitrogen limited condition were increased compared with opposite condition. The tryptophan and histidine auxotrophs showed higher level of glutamate and glutamine than parental strains and other mutants. it is assumed that the higher levels of ${\alpha-KGDH}$ and GDH from mutants in nitrogen-excess condition promoted the accumulation of glutamate and glutamine in fermentation broth. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with histidine, tryptophan, glycine, alanine, serine and GMP implied that a system of feedback inhibition were effective. The GDH, GS and GOGAT biosynthesis in culture broth was markedly repressed by the nature and kinds of available nitrogen sources such as tryptophan, proline, glycine, alanine, serine and tyrosine.

• 미생물학회지
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• 제24권2호
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• pp.113-118
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• 1986

Optimum temperature and pH of glutamine synthetase activity (E.C. 3.6.1.2.) of R. sphaeroides D-230 was $35^{\circ}C$ and 6.8, respectively. The adenylated state of GS in R. sphaeroides D-230 was stabilized by addition of 0.2mg/ml of cethyltrimethylammoniumbromide. Valine, histidine, proline, isoleucine, and lysine were good nitrogen source for the growth of R. sphaeroides D-230. The growth of R. sphaeroides D-230 in $N_2,\;NaNO_3\;or\;NH_4Cl$ as sole nitrogen source was lower than in any otherculture conditions. GS activity was inhibited, more or less, by various amino acid. THe relative inhibition rate of the enzyme by added 7mM arginine, $NH_4Cl,\;N_2,\;and\;NaNO_3$ was 63.8%, 26.79%, 6.24%, and 10.64%, drespectively. THe hydrogen evolution of R. sphaeroides D-230 grown in N-limited media was inhibited by 0.1mM MSX, irreversible GS inhibitor. GS activity was completely inhibited by 1.0mM MSX but ammonia released maximally at the same concentration of MSX. Ammonia release by added MSX was increased up to 1.0mM MS, but decreased above 1.0mM MSX. It is probably due to inhibition of nitrogenase actixity by MSX. Nitrogenase activity was not inhibited at low concentration of MSX. These results suggests that the inhibition of nitrogenase activity by ammonia is mediated by products of ammonia assimilation rather than by ammonia itself.

• Asian Pacific Journal of Cancer Prevention
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• 제16권11호
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• pp.4769-4775
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• 2015

Background: Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and thirdly leading cause of cancer-related death worldwide. The estimated risk of hepatocellular carcinoma is 15 to 20 times as high among persons infected with HCV as it is among those who are not infected, with most of the excess risk limited to those with advanced hepatic fibrosis or cirrhosis. Glypican3 (GPC3) plays a key role in relation to signaling with growth factors, regulating the proliferative activity of cancer cells. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia in the mammalian liver. GS was suggested as a specific marker for tracing cell lineage relationships during hepatocarcinogenesis. In normal liver, GS expression is seen in pericentral hepatocytes, but not by midzonal or periportal hepatocytes. In HCC, strong and diffuse GS expression in seen in tumor cells. Results: Glypican3 immunopositvity was highly specific and sensitive indicator for hepatocellular carcinoma as well as glutamine synthetase which was found to be a sensitive and specific indicator for development of hepatocellular carcinoma when compared to cirrhosis, liver cell dyspalsia and metastatic carcinomas. Statistical analysis revealed a significant association between GPC3 and GS with tumor size (P=0.003, p=0.006, respectively). Diffuse staining significantly associated with large tumor size while, focal and mixed staining was detected more with small tumor size. Studying the relation with tumor grade also revealed significant association between diffuse GPC3 and GS staining with high tumor grade. Diffuse staining was detected in 91.7% and 100% respectively of poorly differentiated specimens and only in 33.3% and 22.2% of well differentiated specimens. Conclusions: While using GPC3 and GS to screen for premalignant hepatic lesions remains controversial, our data suggest that GPC3 and GS may be a reliable diagnostic immunomarkers to distinguish HCC from benign hepatocellular lesions. However, negative immunostaining should not exclude the diagnosis of HCC.

• 한국초지조사료학회지
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• 제29권2호
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• pp.103-110
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• 2009

유채(Brassica napus L.)에서 황 결핍이 $NO_3^-$ 흡수와 동화에 대한 영향을 알아보고자 $SO_4^{2-}$ 농도를 세가지 수준(1 mM $SO_4^{2-}$, 대조구; 0.1mM $SO_4^{2-}$, 결핍; 0 mM $SO_4^{2-}$, 무공급)으로 25시간 처리한 후 $NO_3^-$ 흡수량, 식물조직내의 nitrate reductase (NR) 및 glutamine syntheetase (GS) 활성을 분석하였다. 25시간 처리과정에서 황결핍 조건하에서의 $NO_3^-$의 흡수는 대조구와 큰 차이를 나타내지 않은 반면, 황 무공급구에서는 $NO_3^-$의 흡수는 유의적으로 감소하였다. NR 활성은 잎과 뿌리 조직에서 공히 $SO_4^{2-}$ 농도가 감소됨에 따라 감소하였다. 특히 어린잎과 중간 잎의 황 결핍 처리구에서 각각 35% 및 황 무공급구에서 70%의 뚜렸한 감소를 보였다. 뿌리에서는 오직 황 무공급구에서 유의적인 감소 (-29%)를 보였다. $SO_4^{2-}$ 농도에 따른 잎 조직에서 GS 활성은 잎의 성숙 정도에 따라 차이를 보였으며, 전반적으로 황 결핍과 황 무공급구에서 감소하는 경향을 보였다. 뿌리조직에서 GS 활성은 황 무공급구에서만 유의적인 감소를 보였다. 이러한 결과들은 황 공급이 제한을 받는 조건에서 질산염의 흡수가 감소되며, 식물조직체내의 질산염환원과 아미노산 합성관련 효소의 활성이 제한을 받았다는 것을 보여준다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권10호
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• pp.1470-1477
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• 2006

One hundred and sixty crossbred pigs ($6.62{\pm}0.36kg$) weaned at day $18{\pm}1$ were used to investigate the effects of lactitol and tributyrin on performance, small intestinal morphology and enzyme activity. The pigs were assigned to one of five dietary groups (4 pens/diet with 8 pigs/pen) and were fed the negative control diet or the negative control diet supplemented with 10 g/kg glutamine (as a positive control), or 3 g/kg lactitol (${\beta}$-D-galactopyranosyl-($1{\rightarrow}4$)-D-sorbitol), or 5 g/kg tributyrin (butanoic acid 1,2,3-propanetriyl ester), or 3 g/kg lactitol+5 g/kg tributyrin. Body weight and feed intake were measured weekly during the 4-week study. On day 7, four pigs per dietary treatment were sacrificed to examine small intestinal morphology and enzyme activity. The results showed that: (1) Compared with the negative control diet, the positive control diet improved weight gain and feed efficiency during weeks 1-2 and over the entire study (p<0.05), and also decreased duodenal and ileal crypt depth (p<0.05), but did not alter intestinal enzyme activity (p>0.05). Lactitol improved feed efficiency during weeks 3-4 and over the entire study (p<0.05), but did not improve weight gain and feed intake, intestinal morphology or enzyme activity (p>0.05). Tributyrin improved weight gain and reduced feed/gain during weeks 3-4 and over the entire study. Tributyrin significantly decreased crypt depth in the duodenum and ileum, and increased duodenal lactase and ileal maltase activity (p<0.05). Lactitol+tributyrin increased weight gain during weeks 3-4 and over the entire study, and improved feed efficiency during weeks 1-2 and 3-4 and over the entire study (p<0.05). Lactitol+tributyrin increased the jejunal villus height, and decreased the duodenal and ileal crypt depth (p<0.05). Lactitol+tributyrin also increased jejunal lactase and sucrase activity (p<0.05). (2) Compared with the positive control, tributyrin improved weight gain and reduced feed/gain during weeks 3-4 (p<0.05), decreased the ileal crypt depth, and improved the duodenal lactase and sucrase activity (p<0.05). Lactitol+ tributyrin improved weight gain during weeks 3-4, improved feed efficiency during weeks 3-4 and over the entire study, increased the ileal villus height, and increased jejunal lactase, sucrase and maltase activity (p<0.05). These results showed that tributyrin improved performance, intestinal morphology and enzyme activity, while the effect of lactitol was very limited. These results also showed that, compared with glutamine, tributyrin was more effective in improving intestinal morphology and enzyme activity, and tributyrin exerted a superior effect in improving performance as weaning progressed. These observations suggest that, as a chemical for repairing intestinal atrophy, glutamine and tributyrin should be used in the first and second periods of the starter phase, respectively.

• KSBB Journal
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• 제27권3호
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• pp.186-194
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• 2012

Various human host cell lines, which are more effective than the other original human cell lines, have been developed and used. Highly efficient human cell line can be obtained from the fusion between human embryonic kidney 293 (HEK293) and human Burkitt's lymphoma cells (Namalwa). Fused cell line has the advantages of both cell lines such as the high transfection efficacy of HEK293 cells and the constitutive expression of Epstein-Barr virus (EBV) genome which is related with high expression of target protein and anti-apoptotic growth of Namalwa cells. In this study, characterization of two original cell lines was performed by using design of experiment (DOE) considering cell maintenance, media development, optimization of culture condition, and scale-up. The formation of aggregates was apparent with high glutamine concentration at more than 6 mM. Supplementation of hydrolysates showed positive effects on the growth performances of HEK293 cells. On the contrary, Namalwa cells showed negative results. It was confirmed that Namalwa cells were more sensitive to lower temperature at $35^{\circ}C$ and hyperosmotic condition over 260 mOsm/kg. In addition, both cell lines showed limited growth in 3-L bioreactor due to shear stress.