• International Journal of Oral Biology
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• 제43권4호
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• pp.201-207
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• 2018

Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.

• 한국식품영양과학회지
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• 제14권2호
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• pp.108-116
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• 1985

This study was designed to investigate the nutritional anemic state and hematological findings of children, aged 11 and 12 years, living in Haenam Koon (living at home) and in Mokpo City (living in an orphan asylum) located in Chonnam. The mean red blood cell number of male was higher than female in both groups of living in the rural area and in the city orphan asylum (p<0.01), but the white blood cell count was not significantly different. The levels of average hemoglobin, hematocrit and mean corpuscular hemoglobin concentration of the children in both groups were similar, and 19.8% of children living at home in the rural area and 32.1% of children living in the city orphan asylum were anemic. The mean levels of serum total protein, albumin and A/G ratio in children of both group were not statistically different, and 18.4% of children living at home in the rural area and 13.2% of children living in the city orphan asylum were insufficient in the serum total protein value. The average serum cholesterol level of children living at home in the rural area was higher than that of children living in the city orphan asylum(p<0.01), and that of female living at home was higher than that of male (p<0.05). The mean levels of serum iron, total iron binding capacity and transferrin saturation of children living at home in the rural area were significantly higher than those of children living in the city orphan asylum (p<0.01).

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.556-561
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• 2010

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

• KSBB Journal
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• 제31권2호
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• pp.105-112
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• 2016

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at $30^{\circ}C$, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at $OD_{600}$ (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

• Journal of Microbiology
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• 제44권1호
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• pp.54-63
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• 2006

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the $^{32}P-labeled$ partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5' -untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

• Journal of Plant Biotechnology
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• 제29권4호
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• pp.229-233
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• 2002

식물의 항산화력을 증진하여 식물병원균 저항성 작물을 개발하고자, 철분 결합 단백질인 대두의 ferritin 유전자를 CaMV 35S와 hsr203J promoter에 연결하여 감자에 형질전환하였다. PCR및 Northern분석에 의한 형질전환 감자에 ferritin 유전자가 존재하는 것과 이들 유전자 식물체내에서 발현되는 것을 확인하였다. ferritin 유전자를 담배 유래 병원균 특이 발현promoter인 hsr203J와 연결하여 획득된 형질전환 감자 식물체는 감자역병균 접종 후 24시간대에서 전사체 발현량이 가장 많았으며 그 후 줄어드는 경향을 나타내었다. 유전자 도입이 확인된 형질전환체 감자괴경의 철분 함량은 CaMV 35S와 ferritin 유전자 도입 형질전환체가 2.5배, hsr203J promoter와 ferritin 유전자 도입 형질전환체가 1.5배 각각 증가하는 것으로 나타냈다. 또한 이들 형질전환체는 감자 무름병균에 대한 저항성 증진효과를 관찰할 수 있었다.

• Molecules and Cells
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• 제26권6호
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• pp.536-547
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• 2008

Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.

• 생명과학회지
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• 제26권6호
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• pp.698-704
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• 2016

세포내소기관과 단백질 복합체의 운반은 kinesin superfamily proteins (KIFs)에 의해 매개된다. 처음으로 밝혀진 kinesin인 kinesin 1은 motor단백질로서 세포 내에서 미세소관을 따라 이동하며, 다양한 세포내 소기관이나 단백질복합체를 운반한다. Kinesin 1은 장쇄(KHC, 또한 KIF5s로도 통칭) 2분자와 단쇄(KLCs) 2분자로 구성된 4합체(tetramer) 구조를 가진다. KIF5s의 운반체 결합영역을 포함하는 말단영역은 다수의 운반체와 결합하지만, 결합운반체에 관하여 아직 충분히 밝혀지지 않았다. 본 연구에서 KIF5A의 결합 단백질을 동정하기 위하여 효모 two-hybrid screening을 수행하였고 철 저장 및 해독 기능을 하는 단백질인 ferritin heavy chain (Frt-h)을 찾아내었다. Frt-h은 KIF5A의 아미노산 800번과 940번 사이의 부위와 결합하며, 다른 KIF5s와도 결합함을 효모 two-hybrid assay로 확인하였다. 또한 Frt-h의 coiled-coil 도메인이 KIF5A와의 결합에 필수영역임을 밝혔다. 한편, ferritin light chain (Frt-l) 또한 KIF5s와 결합함을 효모 two-hybrid assay로 확인하였다. 이러한 단백질간의 결합을 glutathione S-transferase (GST) pull-down assay를 통하여 검증하였다. 추가적으로 생쥐의 뇌 파쇄액을 항 KHC 항체로 면역침강을 행한 결과, KLC1뿐만 아니라 Frt-h와 Frt-l도 같이 침강하였다. 이러한 결과들은 세포 내에서 kinesin 1이 ferritin 복합체를 운반함을 시사한다.

• 한국식품영양과학회지
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• 제33권10호
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• pp.1606-1610
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• 2004

본 실험은 자돈식이내 $\delta$-아미노레불린산의 첨가가 이유자돈의 성장능력 및 혈액성상에 미치는 영향을 조사하기 위하여 실시하였다. 사양시험은 3원 교잡종 자돈 75두(평균체중 7.21$\pm$0.02 kg)를 공시하였다. 시 험 설계는 1) NC(negative control; 무항생제 기초식이), 2) PC(positive control; NC diet+0.1% Apramycin+0.1% Oxytetracycline),3) ALA0.1 (NC diet+0.1% $\delta$-아미노레불린산), 4) ALA0.2(NC diet+0.2% $\delta$-아미노레불린산),5) ALA + AB (PC diet+0.2% $\delta$-아미노레불린산)의 5개 처리를 하였다. 총 사양시험기간동안, 일당증체 량은 ALA+AB 처리구가 NC 처리구와 비교하여 유의적으로 높게 평가되었으나(p<0.05), PC, ALA0.1 그리고 ALA0.2처리구와는 통계적인 차이는 보이지 않았다. 일당식 이섭취량과 식이효율에 있어서는 처리구간의 유의적 차이가 없었다. 시험 개시 후 20일령 에 측정한 영양소 소화율에 있어서는 건물과 질소 소화율에 있어서 ALA+AB처리구가 NC 처리구 및 ALA0.1 처리구와 비교하여 높게 평가되었다(p<0.05). 또한, 혈청내 total protein의 농도에 있어서는 ALA+AB처리구가 NC 처리구 및 PC 처리구와 비교하여 높게 평가되었다(p<0.05). 혈청내 iron의 농도는 $\delta$-아미노레불린산을 첨가한 처리구가 NC 및 PC 처리구에 비하여 유의적으로 높게 평가되었다(p<0.05). 혈청내 TIBC에 있어서는 ALA+AB 처리구가 NC, PC 그리고 ALA0.1 처리구와 비교하여 통계적으로 높게 평가되었다(p<0.05). 혈액내 Hb 및 HCT의 농도는 ALA0.2 처리구와 ALA+AB 처리구가 NC 처리구와 PC 처리구와 비교하여 유의적으로 높게 평가되었다(p<0.05). 혈액내 RBC와 WBC의 농도는 ALA0.2 처리구와 ALA +AB 처리구가 NC 처리구와 비교하여 유의적으로 높게 평가되었다(P<0.05). 혈액내 lymphocyte는 $\delta$-아미노레불린산 처리구가 NC 처리구와 비교하여 유의적으로 증가하였다(p<0,05). 결론적으로, 자돈식 이내 $\delta$-아미노레불린산의 첨가는 자돈의 성장 및 소화율을 향상시키며, 혈액 내 total protein, iron, hemoglobin, lymphocyte의 수준을 증가시켰다. 또한, $\delta$-아미노레불린산과 항생제의 혼합급여는 자돈의 성장 능력에 있어 상승효과를 갖는 것으로 사료된다.

• Journal of Plant Biotechnology
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• 제34권2호
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• pp.145-152
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• 2007

Lactoferrin is an 80-kDa iron-binding glycoprotein known to exert many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Rice can be a useful target for edible food plants to introduce human lactoferrin, because it has lower allergenicity and is likely to be safer than microorganisms or transgenic animals. A cDNA fragment encoding human lactoferrin (HLF) driven by the maize polyubiquitin promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was introduced into rice (Oryza sativa L. cv. Dong Jin) using the Agrobacterium -mediated transformation system. Putative transformants were initially selected on the medium containing bialaphos. The stable integration of the bar and HLF genes into transgenic rice plants was further confirmed through polymerase chain reaction (PCR) and Southern blot analyses. The expression of the full length HLF protein from various tissues such as grains and young leaves of transgenic rice was verified by Western blot analysis. Analysis of progeny also demonstrated that introduced genes were stably inherited to the next generation at the Mendelian fashion.