• BioChip Journal
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• 제12권4호
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• pp.287-293
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• 2018

Bacillus cereus can cause blood infections (i.e., sepsis). Its early detection is very important for treating patients. However, an antibody with high binding affinity to B. cereus is not currently available. Bacteriophage cell wall-binding domain (CBD) has strong and specific binding affinity to B. cereus. Here, we report the improvement in the sensitivity of an ATP bioluminescence assay for B. cereus detection using CBD-conjugated magnetic nanoparticles (CBD-MNPs). The assay was able to detect as few as 10 colony forming units (CFU) per mL and $10^3CFU\;per\;mL$ in buffer and blood. CBD-MNPs did not show any cross-reactivity with other microorganisms. These results demonstrate the feasibility of the ATP assay for the detection of B. cereus.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.81.2-82
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• 2003

Plants have evolved along with pathogens, and they have developed sophisticated defense systems against specific microorganisms to survive. G-protons are considered one of the upstream signaling components working as a key for the defense signal transduction pathway. For activation and inactivation of G-protein, GTP-biding proteins are involved. GTP -binding proteins are found in all organisms. Small GTP-binding proteins, having masses of 21 to 30kD, belong to a superfamily, often named the Ras supefamily because the founding members are encoded by human Ras genes initially discovered as cellular homologs of the viral ras oncogene. Members of this supefamily share several common structural features, including several guanine nucleotide binding domains and an effector binding domain. However, exhibiting a remarkable diversity in both structure and function. They are important molecular switches that cycle between the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. In addition, most GTP-binding proteins cycle between membrane-bound and cytosolic forms. such as the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture secondary wall formation, meristem signaling, and defense against pathogens. But their molecular mechanisms and functions are not well known. We isolated a RacB homolog from rice to study its role of defense against pathogens. We introduced the constitutively active and the dominant negative forms of the GTP-hinging protein OsRacB into the wild type rice. The dominant negative foms are using two forms (full-sequence and specific RNA interference with RacB). Employing southern, and protein analysis, we examine to different things between the wild type and the transformed plant. And analyzing biolistic bombardment of onion epidermal cell with GFP-RacB fusion protein revealed association with the nucle.

• BMB Reports
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• 제29권2호
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• BMB Reports
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• 제44권4호
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• pp.250-255
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• 2011

In multicellular organisms, including humans, understanding expression specificity at the tissue level is essential for interpreting protein function, such as tissue differentiation. We developed a prediction approach via generated sequence features from overrepresented patterns in housekeeping (HK) and tissue-specific (TS) genes to classify TS expression in humans. Using TS domains and transcriptional factor binding sites (TFBSs), sequence characteristics were used as indices of expressed tissues in a Random Forest algorithm by scoring exclusive patterns considering the biological intuition; TFBSs regulate gene expression, and the domains reflect the functional specificity of a TS gene. Our proposed approach displayed better performance than previous attempts and was validated using computational and experimental methods.

• Parasites, Hosts and Diseases
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• 제60권2호
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• pp.143-147
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• 2022

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

• Fisheries and Aquatic Sciences
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• 제3권1호
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• pp.52-63
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• 2000

Non-specific reaction has been a problem in doing, especially, research and diagnosis for infectious agents. Avidin-biotin-peroxidase complex (ABC) techniques has widely been used to amplify a reaction. Photobacterium damse1a subsp. piscicdia (formerly Pasteurella piscicida) exhibited a capacity to bind with streptavidin non-specifically. The band, estimated 26 K Da in Western blotted paper, was blocked with biotin but incompletely. In an attempt to explore an involvement of the non-specific substance in attaching piscine cells, cell attachment test performed using anti- Ph. d. subsp piscicida sera raised mouse and rabbit exhibited slightly blocking effects for Mediterranean (1736) and significantly for Japanese (Sp 92144) isolate. Biotin decreased the attachment ability significantly for Sp92144 but it was not effective to 1736. Both isolates showed greatly enhanced attachment ability with poly-L-lysin. The non-specific binding substance was contained in bacterial extracellular products (ECPs). The substance was able to purified with 2-imminobiotin affinity column, the purified substance appeared to have 4 bands in silver staining, and had a carbohydrate branch. This purified substance showed cytotoxic effects selectively between 5 piscine cell lines. Moreover, it stimulated rainbow trout macrophage in terms of reduction of cytochrome cas well as yeast phagocytosis, significantly.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1134-1145
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• 2022

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993⁺. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

• 한국어병학회지
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• 제14권1호
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• pp.31-36
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• 2001

해수에 적응된 뱀장어, Anguilla japonica의 장 세포막으로부터 새로운 clonidine의 결합부위가 있음이 밝혀졌다. Clonidine의 특이적인 결합부위는 적어도 2개의 부위 (high affinity $K_d=1.4{\pm}0.3$ nMn= 5, low affinity $K_d=175{\pm}34$ nM)를 가지고 있었다. 2nM [$^3H$]clonidine의 특이적인 결합은 $20^{\circ}C$, pH 7.5에서 최적 결합능을 가지고 있었고, 라벨되지 않은 clonidine에 의해 가역적인 반응을 보였다. 이러한 결합은 adrenaline, yohimbine과 rauwolscine에 의한 저해능은 약하였다. 그리고 대부분의 결합부위는 $\alpha_2$-adrenoceptor와는 상이하였다. Clonidine의 특이적인 결합은 다양한 imidazoline/guanidinium약물에 의해 억제되었다. Competition 실험의 결과, 2nM[$^3H$]clonidine의 치환 rank order는 다음과 같다. guanabenz > cirazoline = naphazoline=UK14,304= ST587 $\geq$ clonidine $\geq$ idazoxan = RX821002 = tolazoline > ST93 = oxymetazoline = amiloride = ST91 > yohimbine = efaroxan = rauwolscine $\geq$ adrenaline = ST567 = histamine = agmatine. 이러한 순서는 포유류에 분류된 imidazoline receptorl($I_1$), imidazoline receptor 2($I_2$) 및 imidazoline/guanidinium receptive sites(IGRS)형태와는 틀리기 때문에 새로운 imidazoline receptor라고 생각된다. 지금까지 보고된 포유류의 세포와 조직에서 IGRS의 생리학적인 역할은 명확하지 않지만, 해수뱀장어 장은 IGRS의 세포에 있어서의 역할 그리고 IGRS의 내인성(內因性) ligand가 무엇인가에 대한 좋은 모델이 될 수 있다고 생각되어진다.

• 한국지하수토양환경학회지:지하수토양환경
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• 제9권3호
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• pp.12-19
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• 2004

정제된 Aldrich 휴믹산(PAHA)과 한외 여과된 다양한 크기의 PAHA 성분들(PAHA UF fractions)을 이용하여 여러 고리 방향족 탄화수소(PAH)와의 결합 메커니즘을 조사하였다. 유기탄소 결합계수($K_oc$)는 전통적인 Stern-Volmer 형광 소광법과 변형 형광 소광법 두 가지를 이용하여 구하였다. 구해진 Pyrene $K_oc$ 값은 PAHA 농도와 자유 용존 pyrene 농도에 의존하였다. 이러한 비선형 흡착결합 양상은 두 물질간의 흡착성 고유상호작용이 존재한다는 것을 암시하였다. 상대적으로 분자크기가 작은 naphthalene은 중간 크기의 PAHA UF fractions과의 결합에서 높은 $KK_oc$ 값을 보여준 반면 분자 크기의 큰 PAH,즉 pyrene의 경우에는 PAHA UF fractions 크기가 크면 클수록 더 결합이 잘 되었다. 이러한 두 PAH 물질간의 불일치한 크기별 결합양상은 현재까지 제시된 고유 결합 메커니즘들 중의 하나 인 크기별 배제(size exclusion) 효과로 잘 설명되었다. 다양한 pH 조건하에서의 PAH $K_oc$ 실험에서는 일반적으로 pH가 낮아질수록 휴믹산의 산성작용기가 중화되고 그에 따라 휴믹산내의 소수성 영역이 커짐으로 인해 pyrene과 휴믹산과의 결합정도는 커졌다. 그러나 큰 사이즈의 PAHA UF fraction(>100K Da)을 사용한 실험에서는 낮은 pH가 아닌 특정 pH 범위에서 또 하나의 높은 pyrene $KK_oc$ 값을 보여줌으로서 크기별 배제 효과가 존재함을 뚜렷이 보여주었다. 이러한 크기별 배제 효과는 휴믹산의 홀(hole)구조가 PAH 크기에 적합하게 구성되어 있는 조건(휴믹성분 크기 혹은 pH)에서만 작용하는 것으로 보인다.