Prevotella intermedia에서의 Hemin 결합 단백질 유전자의 분리 및 염기서열 분석

김신,김성조 大韓小兒齒科學會 2006 大韓小兒齒科學會誌 Vol.33 No.2

본 연구는 치주질환 주요 병인균주 중의 하나인 P.intermedia를 대상으로 하여, 이 균주에서의 hemin 결합 단백질 유전자를 분리하고 염기서열을 결정하기 위하여 수행되었다. 본 연구에서는 약 5,000개의 recombinant colony들을 스크리닝하여 hemin 결합 단백질 유전자를 포함하고 있는 것으로 여겨지는 1개의 클론(pHem1)을 확인하였다. Restriction enzyme mapping 결과 pHem1의 insert DNA의 크기는 약 2.5kb이었으며, P. intermedia chromosomal DNA 내에는 hemin 결합 단백질 유전자가 single copy로 존재하였고, transcript의 크기는 약 1.8kb이었다. 분리한 pHem1 유전자는 1개의 ORF를 가지고 있으며, ORF의 크기는 2,550bp로 약 850개의 아미노산 폴리펩타이드로 구성되어 있다. 또한, pHem1 유전자는 이미 밝혀진 다른 유전자들과 상동성을 보이지 않았다. 본 연구는 P.intermedia에서의 porphyrin 생리 및 hemin 획득기전을 분자생물학적으로 규명하는데 있어 중요한 의의가 있으리라 사료된다. 향후 pHem1 유전자의 특성 분석 등이 수행되어야 할 것으로 여겨진다. Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain DH5^(α), and screened for recombinant clones using hemin-binding activity by plating onto hemin-contraining agar. Approximately 5,000 recombinant E.coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phenotype. The 2.5 kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHⅠ, BgIⅡ, EcoRⅠ, HIndⅢ and PstⅠ-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity. It need further investigation to clarify the mechanisms of heme uptake in P. intermedia.

Characterization of hemin-binding of oral streptococci

Eun Jeong Kim,Si Young Lee 조선대학교 치의학연구원 2022 Oral Biology Research (Oral Biol Res) Vol.46 No.3

Certain pathogenic bacteria obtain iron for growth through heme or heme compounds. Streptococci are gram-positive bacteria that reside in the mouth and require iron for growth. However, the iron accumulation mechanism in streptococci remains unknown. Therefore, this study investigated the hemin-binding properties of streptococci to confirm the potential of oral streptococci to obtain iron through heme compounds. The hemin-binding ability of streptococci was evaluated by incubation with hemin and subsequent measurement of the optical density of the supernatant to determine the amount of hemin bound by the bacteria using the hemin concentration standard curve. The hemin-binding of streptococci was proportional to the concentration of bacteria (Streptococcus gordonii) and hemin added. However, when a high hemin concentration of up to 60 μg/mL was added, the amount of hemin bound by S. gordonii remained almost unchanged. The incubation temperature had no significant effect on hemin-binding, but pretreatment with trypsin and protease decreased hemin-binding. Additionally, we experimentally confirmed that S. gordonii could use hemin for growth. This study demonstrated that streptococci possess hemin-binding sites and can acquire iron through hemin.

Characterization of the hemin-binding property of Porphyromonas endodontalis

( Eun Jeong Kim ),( Si Young Lee ) 조선대학교 치의학연구원(구 조선대학교 구강생물학연구소) 2021 Oral Biology Research (Oral Biol Res) Vol.45 No.2

Porphyromonas endodontalis, found in the root canal of teeth, requires iron for growth. However, the mechanism of iron uptake in P. endodontalis remains unclear. The ability of bacteria to utilize heme compounds to acquire iron for growth has been reported in some pathogenic bacteria. In the present study, we analyzed the ability of P. endodontalis to obtain iron from heme compounds. Further, we investigated the hemin-binding characteristics of P. endodontalis and the relationship between hemin binding and Congo red binding. To confirm the bacterial growth in hemin-supplemented medium, iron was removed from the medium with an ironchelator, and hemin was supplemented to an iron-free medium. The hemin-binding characteristics of P. endodontalis were analyzed by incubating bacteria with hemin and measuring the optical density of the supernatant obtained via centrifugation, using hemin concentration standard curve. Although growth of P. endodontalis was not observed in the iron-depleted medium, it was observed in a hemin-supplemented medium. Further, hemin binding was dependent on the concentrations of hemin and bacteria. Hemin binding proceeded quickly in P. endodontalis, and the incubation temperature had no effect on this binding. Similar to hemin binding, Congo red binding of P. endodontalis was dependent on Congo red and bacterial concentrations. In addition, Congo red binding of P. endodontalis was inhibited by hemin prebinding. Hemin-agarose beads and SDS-PAGE were used to identify a 40-kDa protein that could be involved in hemin binding. The results showed that P. endodontalis could bind to and use hemin to obtain the iron required for growth.

Effect of hemin and ascorbic acid on colonic pre-neoplastic lesions induced by azoxymethane in mice

Jun Ho Kim,Tae Ryong Kim,Hyo Suk Choi,Sung June Kim,Jae Hwang Jeong,Sang Yoon Nam,Young Won Yun,Beom Jun Lee 한국예방수의학회 2017 예방수의학회지 Vol.41 No.4

Colorectal cancer (CRC) is the third most prevalent cancer in the world, and heme iron is known to promote the CRC in an animal model. This study was conducted to investigate the effects of ascorbic acid in the presence of hemin on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/disodium sulfate (DSS) in mice. After acclimation for 1 week, five-week old mice received three s.c. injections (0-2 weeks of the experiment) of AOM [10 mg/kg body weight (BW)] weekly and were treated with 2% DSS in drinking water for the next week to induce aberrant crypt foci (ACF). All animals were fed the AIN-76A purified rodent diet for experimental period of 6 weeks. Experimental groups were then divided into three groups: carboxymethylcellulose (CMC) alone (control), CMC + Hemin, CMC + Hemin + ascorbic acid (AA). The CMC was used as a solvent for hemin. The daily doses were 534 mg/kg BW hemin and 246 mg/kg BW ascorbic acid administered orally. After the colonic mucosa were stained with methylene blue, aberrant crypt foci (ACF), aberrant crypt (AC) and polyps were counted. Lipid peroxidation in liver was evaluated by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF, AC and large ACF (≥4 AC/ACF) per colon increased in the hemin group compared to the control group, while they decreased significantly in the hemin + ascorbic acid group compared to the control group or hemin group (p<0.01). The number of polyps/colon in the hemin + AA group was significantly decreased compared to the hemin group (p<0.05). In the liver, the TBARS value of the hemin group was significantly higher than that of the control group (p<0.01). Additionally, the TBARS value of the hemin + AA group decreased slightly compared to that of the hemin group. Taken together, these results suggest that hemin can promote colon carcinogenesis in a mouse model and that ascorbic acid has a protective effect against hemin-promoted colon carcinogenesis.

테레프탈알데하이드의 전자전달 강화효과에 따른 헴 단백질 모방 촉매의 성능 향상 및 이를 이용한 비분리막형 과산화수소 연료전지

전시은,안희연,정용진,Jeon, Sieun,An, Heeyeon,Chung, Yongjin 한국화학공학회 2022 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.60 No.4

Terephthalaldehyde (TPA) is introduced as a cross liker to enhance electron transfer of hemin-based cathodic catalyst consisting of polyethyleneimine (PEI), carbon nanotube (CNT) for hydrogen peroxide reduction reaction (HPRR). In the cyclic voltammetry (CV) test with 10 mM H₂O₂ in phosphate buffer solution (pH 7.4), the current density for HPRR of the suggested catalyst (CNT/PEI/hemin/PEI/TPA) shows 0.2813 mA cm^-2 (at 0.2 V vs. Ag/AgCl), which is 2.43 and 1.87 times of non-cross-linked (CNT/PEI/hemin/PEI) and conventional cross liker (glutaraldehyde, GA) used catalyst (CNT/PEI/hemin/PEI/GA), respectively. In the case of onset potential for HPRR, that of CNT/PEI/hemin/PEI/TPA is observed at 0.544 V, while those of CNT/PEI/hemin/PEI and CNT/PEI/hemin/PEI/GA are 0.511 and 0.471 V, respectively. These results indicate that TPA plays a role in facilitating electron transfer between the electrodes and substrates due to the π-conjugated cross-linking bonds, whereas conventional GA cross-linker increases the overpotential by interrupting electron and mass transfer. Electrochemical impedance spectroscopy (EIS) results also display the same tendency. The charge transfer resistance (R_ct) of CNT/PEI/hemin/PEI/TPA decreases about 6.2% from that of CNT/PEI/hemin/PEI, while CNT/PEI/hemin/PEI/GA shows the highest R_ct. The polarization curve using each catalyst also supports the superiority of TPA cross liker. The maximum power density of CNT/PEI/hemin/PEI/TPA (36.34±1.41 μWcm^-2) is significantly higher than those of CNT/PEI/hemin/PEI (27.87±0.95 μWcm^-2) and CNT/PEI/hemin/PEI/GA (25.57±1.32 μWcm^-2), demonstrating again that the cathode using TPA has the best performance in HPRR.

Porphyromonas gingivalis에서의 Hemin 결합 단백질 유전자의 특성 연구

김성조,Kim, Sung-Jo 대한치주과학회 1999 Journal of Periodontal & Implant Science Vol.29 No.3

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$ , and screened for recombinant clones with hemin-binding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa hemin-binding protein.

Porphyromonas gingivalis, Prevotella intermedia, 그리고 Prevotella nigrescens에서의 hemin 결합 단백질에 대한 연구

김성조,Kim, Sung-Jo 대한치주과학회 2006 Journal of Periodontal & Implant Science Vol.36 No.1

The results of this study confirm that the availability of hemin influences the expression of selected membrane proteins of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is hemin regulated was identified and purified in P. gingivalis. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when P. gingivalis cells were grown under hemin-limited conditions. A 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin binding protein from P. intermedia and P. nigrescens, respectively. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein from P. gingivalis revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins. N-terminal amino acid sequence of a 50 kDa putative hemin binding protein from P. intermedia was identical with Enolase from Streptococcus intermedia. Work is in progress to further characterize the molecular structure of these proteins.

Epigallocatechin-3-gallate suppresses hemin-aggravated colon carcinogenesis through Nrf2-inhibited mitochondrial reactive oxygen species accumulation

석주형,Dae Hyun Kim,Hye Jih Kim,Hang Hyo Jo,김은영,Jae-Hwang Jeong,Young Seok Park,Sang Hun Lee,Dae Joong Kim,Sang Yoon Nam,Beom Jun Lee,Hyun Jik Lee 대한수의학회 2022 Journal of Veterinary Science Vol.23 No.5

Background: Previous studies have presented evidence to support the significant association between red meat intake and colon cancer, suggesting that heme iron plays a key role in colon carcinogenesis. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, exhibits anti-oxidative and anti-cancer effects. However, the effect of EGCG on red meat-associated colon carcinogenesis is not well understood. Objectives: We aimed to investigate the regulatory effects of hemin and EGCG on colon carcinogenesis and the underlying mechanism of action. Methods: Hemin and EGCG were treated in Caco2 cells to perform the water-soluble tetrazolium salt-1 assay, lactate dehydrogenase release assay, reactive oxygen species (ROS) detection assay, real-time quantitative polymerase chain reaction and western blot. We investigated the regulatory effects of hemin and EGCG on an azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colon carcinogenesis mouse model. Results: In Caco2 cells, hemin increased cell proliferation and the expression of cell cycle regulatory proteins, and ROS levels. EGCG suppressed hemin-induced cell proliferation and cell cycle regulatory protein expression as well as mitochondrial ROS accumulation. Hemin increased nuclear factor erythroid-2-related factor 2 (Nrf2) expression, but decreased Keap1 expression. EGCG enhanced hemin-induced Nrf2 and antioxidant gene expression. Nrf2 inhibitor reversed EGCG reduced cell proliferation and cell cycle regulatory protein expression. In AOM/DSS mice, hemin treatment induced hyperplastic changes in colon tissues, inhibited by EGCG supplementation. EGCG reduced the hemin-induced numbers of total aberrant crypts and malondialdehyde concentration in the AOM/DSS model. Conclusions: We demonstrated that EGCG reduced hemin-induced proliferation and colon carcinogenesis through Nrf2-inhibited mitochondrial ROS accumulation.

Protective effect of selenium on hemin-aggravated experimental colon carcinogenesis in mice

Tae-Ryung Kim(Tae-Ryung Kim),Seongjoo Yoon(Seongjoo Yoon),Beom Jun Lee(Beom Jun Lee) 한국예방수의학회 2023 예방수의학회지 Vol.47 No.2

Selenium (Se) is known as an antioxidant mineral and heme iron is a major source for iron intake which can promote carcinogenesis in the body. This study was to investigate the effect of Se on heme-aggravated colon carcinogenesis in mice. Three experimental groups included control [normal diet + AOM (10 mg/kg body weight in saline)/DSS (2% in the drinking water)], [AOM/DSS + hemin (534 mg/kg body weight in CMC)], and [AOM/DSS + hemin + Se (2.82 mg/kg diet in CMC)] groups. Colonic mucosa were stained with 0.3% methylene blue and the colonic polyps, aberrant crypt (AC) and aberrant crypt foci (ACF) were counted. Lipid peroxidation in liver was evaluated by the thiobarbituric acid-reactive substances (TBARS) assay. The number of polyps in the hemin + Se group was 31.6% lower than that in the control group, and 41.4% lower than that in the hemin group. The number of AC in the hemin + Se group was 42.8% lower than that in the control group, and 49.1% lower than that in the hemin group. The number of ACF in the hemin + Se group was 49.0% lower than that in the control group, 45.7% than that in the hemin group. Hepatic TBARS level in the hemin + Se group was significantly low compared with the control group or the hemin group (p<0.05). These findings suggest that Se treatment may be protective against colon carcinogenesis promoted by a high heme-containing diet.

Hydroxyurea 처리시 적혈구계 전구세포에서의 감마글로빈 프로모터-269∼-240 부위의 역할

박주인,김태겸,김덕인,김인후,정진숙,한진영 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.1