• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.749-755
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• 2005

The ccpA gene encoding catabolite control protein A (CcpA) of Leuconostoc mesenteroides SYl, a strain isolated from kimchi, was cloned, sequenced, analyzed for transcript, and overexpressed in Escherichia coli. The ccpA ORF (open reading frame) is 1,011 bp in size, which can encode a protein of 336 amino acid residues with a molecular mass of 36,739 Da. The transcription start site was mapped at a position 49 nucleotides upstream of the start codon, and promoter sequences were also identified. The putative cre site overlapped with the -35 promoter sequence. The deduced amino acid sequence of the CcpA contained the helix-turn-helix motif found in many DNA-binding regulatory proteins. CcpA from 1. mesenteroides SY1 had $54.6\%$ identity with CcpA from Lactobacillus casei. The Northern blot experiment showed that ccpA was transcribed as a single 1.1 kb transcript, and transcription was repressed when grown on media containing glucose. CcpA was overproduced in E. coli BL21(DE3) cells using the pET expression vector, and purified to an apparent homogeneity. Gel Mobility Shift Assay with purified CcpA and a DNA fragment containing the ere sequence of the $\alpha$-galactosidase gene (aga) from L. mesenteroides SY1 revealed that CcpA bound specifically to the cre site of aga.

• Journal of Microbiology
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• 제42권4호
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

• Mass Spectrometry Letters
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• 제5권3호
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• pp.63-69
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• 2014

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

• Journal of Nutrition and Health
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• 제35권9호
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• pp.912-918
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• 2002

In nature, two different types of fructose polymers (fructan) are generally found in dietary fibers; these are the fructose homopolymers levan, which is of high molecular weight and is $\beta$-(2,6)-linked, and inulin, which is of low molecular weight and is $\beta$-(2,1)-linked. The effects of levan and inulin on the intestinal physiology of rats were compared. Sprague Dawley rats were fed one of three diets for 3 weeks: a control diet, a basal diet containing 7% of levan, and a basal diet containing 7% of inulin. Cecal enlargement, together with the lowering of cecal pH, occurred in rats fed on the levan and inulin diets (p < 0.05). The levan and inulin diets resulted in a two-fold increase in the amount of short-chain fatty acids in the cecum, when compared to the control diet. The number of total microbes and of lactic acid-producing bacteria in the feces were higher in rats fed the fructan diets than those in rats fed control diet (p < 0.05). The levan diet also significantly increased the cecal $\alpha$-galactosidase activity by 3.8-fold, when compared to the control diet, indicating that levan stimulated the growth of Bifidobacteria in the cecum. These results show that the intake of levan and inulin stimulated the growth of lactic acid-producing bacteria in the cecum and thereby improved intestinal conditions in rats. (Korean J Nutrition 35(9) : 912~918,2002)

• 한국가금학회지
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• 제29권1호
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• pp.19-23
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• 2002

본 연구의 목적은 산란계 사료내 복합효소제의 첨가가 난각특성 및 영양소 소화율에 미치는 영향을 조사하기 위하여 실시하였다. 사양시험은 47주령 ISA Brown산란계 144수를 공시하였으며, 처리구로는 옥수수-대두박 위주의 사료(CON; 기초사료), 기초사료에 복합효소제를 0.7% 첨가한 구(ME0.1; 기초사료 + 0.1%복합효소제), 기초사료에 복합효소제를 0.2% 첨가한 구(ME0.2; 기초사료 + 0.2% 복합효소제)로 3개 처리로 구성되었다. 총 28일간의 사양시험 기간 동안, 산란을, 난중, 난각강도 그리고 난각두께에 있어서 처리구간에 유의적인 차이는 보이지 않았다. 난황색에 있어서는 복합효소제를 첨가함에 따라 유의적으로 증가하였다. 난황계수에 있어서도 복합효소제의 첨가수준이 증가함에 따라 유의적으로 높아졌다. 건물 소화율에서 처리구간에 유의적인 차이는 없었으나, 질소 소화율에 있어서는 복합효소제의 첨가수준이 증가함에 따라 대조구와 비교하여 유의적으로 높았다. 결론적으로, 산란계 사료내 복합효소제의 첨가가 난황색, 난황계수 그리고 질소 소화율을 향상시키는 것으로 사료된다

• Molecules and Cells
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• 제28권5호
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• pp.489-494
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• 2009

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and $p21^{Cip1/WAF1}$ in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated ${\beta}$-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or $CKII{\alpha}$ siRNA, but this response was almost abolished in p53- or $p21^{Cip1/WAF1}$-null cells. Increased cellular levels of p53 and $p21^{Cip1/WAF1}$ protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and $p21^{Cip1/WAF1}$ expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the $p53-p21^{Cip1/WAF1}$ pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

• 한국축산식품학회:학술대회논문집
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• 한국축산식품학회 2004년도 정기총회 및 제33차 춘계 학술대회
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• pp.89-119
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• 2004

This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.

• Asian-Australasian Journal of Animal Sciences
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• 제17권4호
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• pp.533-537
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• 2004

The experiment was conducted to test the hypothesis that supplementing diets of lactating first parity sows with a mixture of carbohydrases (CS) improves lactation performance and second parity reproductive performance. The CS used in this study contained 7 units/g of $\alpha$-1,6-galactosidase, 22 units/g of $\beta$-1,4-mannanase, $\beta$-1,4-mannosidase and trace amounts of other enzymes. Twenty primiparous sows (Newsham Hybrid) were allotted to either the control group (no CS supplement) or the CS group (0.1% CS supplement) and fed the experimental diets during 21 d lactation period. Sows and nursing pigs were weighed at birth and weekly until weaning. Days of weaning-to-estrus were recorded. Sows had free access to feed and water. Feed intake of sows was measured daily. During the second parity gestation and lactation, all the sows were fed the same gestation and lactation diets and their reproductive performance was measured. During the second parity, there were 14 sows (7 sows per group) remained productive. For the first lactation, maternal body weight loss of the CS group was smaller (p<0.05) than that of the control group. There was no difference in litter weight gain between two groups. Voluntary feed intake of sows did not differ between the two groups. Days of weaning-to-estrus of the CS group were smaller (p<0.05) than those of the control group. In the second parity, there was no difference in the reproductive performance between the two groups. In conclusion, supplementing CS in the diet of lactating sows during the first parity decreased body weight loss and days of weaning-to-estrus of sows. However, these effects of the CS supplementation in the first parity were not successfully carried over to the second parity.

• International Journal of Oral Biology
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• 제32권1호
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• 대한소아치과학회지
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• 제28권1호
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• pp.129-141
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• 2001

우식치아의 교합면과 정상치아의 교합면에서 균을 채취한 결과, 우식치아의 교합면에서는 $3.43\times10^5$ CFU, 정상치아의 교합면에서는$3.43\times10^3$ CFU가 MSB 배지상에서 검출되었다. API test를 이용하여 당발효 및 생화학적 성상을 관찰한 결과, 우식치면에서 분리된 세균은 20종 모두 S. mutans였으나, 건강한 치면에서는 2개의 세균만 S. mutans로 동정되었다. 우식치면과 정상치면에서 분리된 균주 S. mutans SM1과 S. mutans SM2는 $\alpha-galactosidase$ 활성을 제외하곤 당발효 및 생화학적 성상이 모두 일치하였다. 증식에 있어서, 두 균주 모두 pH 5.5에서 증식이 가장 활발하였고, 자당의 농도는 SM1은 20%일 때 SM2는 5%일 때 최대 증식을 보였다. SM1은 배지의 $CaCl_2$농도가 16mM, KCl농도가 160mM, $MgCl_2$농도가 6.4mM 이었을 때 증식이 가장 활발하였고, SM2는 $CaCl_2$ 농도가 16mM, KCl 농도가 40mM, $MgCl_2$ 농도가 6.4mM 이었을때 증식이 가장 활발하였다. Sodium bicarbonate 완충액과 Sodium phosphate 완충액의 경우, SM1과 SM2 모두 1mM에서 증식이 활발하였다. Tris 완충액의 경우, SM1은 1mM에서, SM2는 10mM에서 증식이 활발하였다. Potassium phosphate 완충액의 경우, SM2는 농도가 증가함에 따라 증식이 억제된 반면, SM1은 100mM 까지는 농도가 증가할수록 증식이 활발하였다. SM1과 SM2의 염색체를 추출한 후 Primer gtfB-F961과 gtfC-R5574를 사용하여 gtf 유전자를 PCR 한결과, 4.6kb의 단일 band를 얻었고 이 band를 분리하여 제한효소로 처리한 결과, EcoR I 처치시 S. mutans GS-5, SM1과 SM2모두 약 0.8kb와 3.8kb의 DNA절편을 보여 같은 양상을 나타냈고, Hind III 처치시 GS-5와 SM1은 잘리지 않았고, SM2의 경우 2.4kb, 1.8kb, 400bp의 3조각의 절편으로 나뉘어 SM1과 SM2의 gtf 유전자의 상사성이 관찰되었다. BamH I처치시 SM1과 SM2는 4조각의 절편으로 절단되어 같은 양상을 보였고, GS톤의 경우는 3조각의 절편으로 절단되었다. Kpn I, Sma I, Xho I 그리고 Pst I에는 절단되지 않았다.