• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.

• 한국식품위생안전성학회지
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• 제26권4호
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• pp.349-354
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• 2011

시중에서 판매된 일반우유와 포름알데히드를 처리한 참치어유 배합사료를 먹여 DHA를 강화한 DHA 강화우유내의 포름알데히드 함량을 분석함으로써 포름알데히드를 처리한 사료를 먹여 DHA를 강화한 우유의 안전성을 평가하고자 하였다. 시중에서 판매된 일반우유와 DHA 강화우유를 CM, DHA-K, DHA-B로 구분하였다. 이화학 검사를 통해 세 제품이 모두 기준치 이내의 정상 수준을 나타내는 신선한 제품임을 확인하였고, 전반적인 시료간의 유의적 차이는 거의 없었으나, DHA-K를 다른 두 시료와 비교하면 지방함량은 두 시료의 지방함량의 절반 정도인 1.85%이었으며, 단백질은 약 1% 높게 나타났다. DHA 강화우유에 함유된 DHA의 함량을 분석해본 결과 DHA-B가 DHA-K보다 약 2배이상 높게 나타났으며, 이러한 차이는 연령별 섭취량 기준과 비슷한 양상을 나타내는 것으로 보인다. HPLC를 이용하여 우유 내 포름알데히드를 분석한 결과, 일반우유와 포름알데히드를 처리한 참치어유배합사료를 먹인 DHA 강화우유 모두 WHO에서 정한 신선유 중 포름알데히드 함량 기준치인 0.013~0.057 ppm사이에 존재했으며, 또한 세 시료간에 유의적 차이가 없는 것으로 보아 DHA를 강화하기 위해 포름알데히드를 첨가한 참치어유 배합사료를 소에게 먹이더라도 포름알데히드가 우유로 전이되지는 않는 것으로 보이며, 포름알데히드를 처리한 사료를 먹인 DHA 강화우유의 포름알데히드에 대한 안전성은 일반우유 수준이라고 판단된다.

• 생명과학회지
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• 제26권3호
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• pp.376-386
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• 2016

에폽토시스에 의한 세포자멸사는 암세포에 대한 항암제 효능의 핵심적 기전이다. 항암제의 대표적인 두 종류로 알려진 DNA-손상 약제(DNA-damaging agents, DDAs)와 미세소관-손상 약제(microtubule-damaging agents, MDAs)가 암세포에 야기하는 초기 항암신호전달 기전은 다르지만, 최종적으로는 대부분 미토콘드리아 의존-에폽토시스를 통해 암세포를 사멸시킨다. 한편, DDAs에 의한 에폽토시스 유도에는 wild-type 종양억제 단백질 p53의 역할이 매우 중요하다. 그러나 인체 암의 약 50% 이상이 p53유전자의 돌연변이 때문에 종양억제 단백질로서의 p53 기능이 불활성화 되어 있다. 따라서 p53과 무관하게 에폽토시스를 유도할 수 있는 MDAs를 이용한 항암치료는 돌연변이 p53을 지닌 암세포에 대해 유리한 화학요법으로 이해된다. 최근 본 연구진은 인체 급성 백혈병 세포주인 Jurkat T 세포를 모델로 하여, MDAs (nocodazole, 17-α-estradiol, 혹은 2-methoxyestradiol)의 항암작용과 관련된 세포주기 정지 및 에폽토시스 유도 기전을 구명하였다. 그 결과, Jurkat T 세포를 MDAs로 처리할 경우, 유사분열방추사의 결함에 의한 세포주기(전중기, prometaphase) 정지, 장시간에 걸친 Cdk1의 활성화, 활성화된 Cdk1에 의한 에폽토시스 조절인자들(Bcl-2, Bcl-xL, Mcl-1 및 Bim)의 인산화, 이에 따른 Bak 활성화, 미토콘드리아막 손상 및 카스파아제 연쇄 활성화에 의해 에폽토시스가 유도됨을 밝혔다. 또한 동일한 MDA 처리 조건하에서 Bcl-2 혹은 Bcl-xL의 과발현시켜 에폽토시스 진행을 차단할 경우, Jurkat T 세포는 약제처리 후에 전중기 정지된 4N 상태에 도달하지만, 이어서 유사분열 불이행(mitotic slippage) 및 내재복제(endoreduplication)가 진행되어 다배수체들(polyploids; 8N, 16N)을 생성하게 됨을 확인하였다. 이러한 결과는 MDAs처리에 따른 다배수체들의 생성을 차단하는 세포 내 기전으로서, 전중기 정지된 4N 세포의 에폽토시스에 의한 제거가 매우 중요함을 보여준다. 특히, 다배수체는 유전적으로 매우 불안정하여 암세포의 항암제 내성 획득 및 암 재발과 직접 연관되는 것으로 알려져 있으므로, 에폽토시스 기전에 결함이 있는 암세포를 대상으로 MDAs를 이용한 항암 화학요법을 시행할 경우에는 다배수체 세포의 생성을 차단하기 위한 새로운 수단이 반드시 병행되어야 할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제18권11호
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• pp.1600-1608
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• 2005

The study was carried out to evaluate the effect of exogenous bovine somatotropin on water metabolism in relation to mammary function in early lactation of crossbred Holstein cattle. Ten, 87.5% crossbred Holstein cattle were divided into two groups of 5 animals each. At day 60 of lactation, the control group was given placebo while animals in the experimental group were given recombinant bovine somatotropin (rbST) by subcutaneous injection with 500 mg of rbST (14-days prolonged-release rbST). In rbSTtreated animals, milk yield increased 19.8%, which coincided with a significant increase in water intake (p<0.01), while DM daily intake was not different when compared to the control animals. Water turnover rate as absolute values significantly increased (p<0.05), while the biological half-life of water did not change in rbST-treated animals. Total body water (TBW) and total body water space (TOH) as absolute values significantly increased (p<0.01) in rbST-treated animals, while it was decreased in the control animals. Absolute values of empty body water (EBW) markedly increased (p<0.05), which was associated with an increase in the extracellular fluid (ECF) volume. Absolute values of plasma volume and blood volume were also significantly increased (p<0.05) in rbST-treated animals. The increase in mammary blood flow in rbST-treated animals was proportionally higher than an increase in milk production. The plasma IGF-1 concentration was significantly increased (p<0.01) in rbST-treated animals when compared with those of control animals during the treatment period. Milk fat concentration increased during rbST treatment, while the concentrations of both protein and lactose in milk were not affected. The present results indicate that rbST exerts its effect on an increase in both TBW and EBW. An increased ECF compartment in rbST-treated animals might partly result from the decrease in fat mass during early lactation. The action of rbST on mammary blood flow might not be mediated solely by the action of IGF-1 for increase in blood flow to mammary gland. An elevation of body fluid during rbST treatment in early lactation may be partly a result of an increase in mammary blood flow in distribution of milk precursors to the gland.

• Molecules and Cells
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• 제38권6호
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• pp.528-534
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• 2015

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-$1{\alpha}$ stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-$1{\alpha}$ in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-$1{\alpha}$, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-$1{\alpha}$ and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-$1{\alpha}$ stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제35권6호
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• pp.397-402
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• 2009

The purpose of this study is to investigate the effects of alendronate and pamidronate on proliferation and the alkaline phosphatase activity of human bone marrow derived mesenchymal stem cells and to relate the results with bisphosphonate related osteonecrosis of the jaw(BRONJ). With the consent of patients with no systemic disease and undergoing iliac bone graft, cancellous bone was collected to obtain human bone marrow derived mesenchymal stem cells through cell culture. 96 well plate were prepared with a concentration of $10^4$cell/ well. Alendronate and pamidronate were added to each well with the concentration of $10^{-6}M$, $10^{-8}M$ and $10^{-10}M$, respectively. Then proliferation capacity of each well was evaluated with the cell counting kit. 24 well plates were prepared with a concentration of $10^5$cell/ml/well and with the bone supplement, alendronate and pamidronate were added with the concentration of $10^{-6}M$, $10^{-8}M$ and $10^{-10}M$, respectively on each plate. The plates were cultured for either 24 or 72 hours. Then the cells were sonicated to measure the alkaline phosphatase activity and protein assay was done to standardize the data for analysis. As the concentration of alendronate or pamidronate added to the culture increased, the proliferation capacity of the cells decreased. However, no statistical significance was found between the group with $10^{-10}M$ of bisphophonate and the control group. Pamidronate was not capable of increasing the alkaline phosphatase activity in all trials. However, alkaline phosphatase activity increased with 24 hours of $10^{-8}M$ of alendronate treatment and with 48 hours of $10^{-10}M$ of alendronate treatment. Cell toxicity increased as the bisphosphonate concentration increased. This seems to be associated with the long half life of bisphosphonate, resulting in high concentration of bisphosphonate in the jaw and thus displaying delayed healing after surgical procedures. Alendronate has shown to increase the alkaline phophatase activity of human bone marrow derived mesenchymal stem cells. However, this data is insufficient to conclude that alendronate facilitates the differentiation of human bone marrow derived mesenchymal stem cells. Further studies on DNA level and animal studies are required to support these results.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.69-73
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• 2005

The hematopoietic growth factor erythropoietin (EPO) is required for the maintenance, proliferation, and differentiation of the stem cells that produce erythrocytes. To analyse the biological activity of the recombinant human EPO (rec-hEPO), we have cloned the EPO cDNA and genomic DNA and produced rec-hEPO in the CHO cell lines. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of rec-hEPO. MIT assay values were increased by survival of F36E cells at 24h or 72h. The hematocrit and RBC values were increased by subcutaneous injection of 20 IU (in mice) and 100IU(in rats) rec-hEPO. Hematocrit values remarkably increased at $13.2\%$ (in mice) and $12.2\%$ (in rats). The pharmacokinetic behavior with injection of 6 IU of rec-hEPO remained detectable after 24 h in all mice tested. The highest peat appeared at 2h after injection. The long half-life of rec-hEPO is likely to confer clinical advantages by allowing less frequent dosing in patients treated for anemia. These data demonstratethat ree-hEPO produced in this study has a potent activity in vivo and in vitro. The results also suggest that biological activity of ree-hEPO could be remarkably enhanced by genetic engineering that affects the potential activity, including mutants with added oligosaccharide chain and designed to produce EPO-EPO fusion protein.

• Journal of Nutrition and Health
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• 제42권5호
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• pp.434-441
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• 2009

본 연구는 J774 대식세포에서 FPN 유전자 발현 조절에 구리가 미치는 영향을 알아보기 위하여 수행되었으며 그 결과는 다음과 같다. J774 대식 세포에 구리를 처리하였을 때, iron exporter FPN의 mRNA 수준이 농도 의존적으로 증가하는 것으로 나타났다. 반면, iron importer DMT1의 mRNA 수준은 구리 처리에 의해 영향을 받지 않았다. Actinomycin D를 이용하여 mRNA 합성을 억제한 상태에서 FPN mRNA 분해 정도를 시간별로 추적한 결과, acitnomycin D 처리 후 9시간 경과시 FPN mRNA 수준이 처음 수준의 약 60% 정도로 감소하였다. 배양액에 구리를 첨가한 경우에도 FPN mRNA의 분해 정도는 아무것도 처리하지 않은 대조군과 유의적인 차이가 없었으며, 이로 볼 때 구리는 FPN mRNA의 안정성에 영향을 미치지 않는 것으로 생각된다. 한편, reporter assay 실험 결과 구리의 첨가는 FPN 프로모터 활성을 유의적으로 증가시키는 것으로 나타나, 구리가 FPN mRNA의 전사 과정을 직접적으로 촉진함을 알 수 있었다. 또한, FPN 5'-UTR에 위치하는 IRE (iron response element)의 존재 여부는 구리에 의한 FPN 전사 개시 활성에 영향을 주지 않는 것으로 나타났으며, 이로 볼 때 구리는 철분과는 독립적인 작용 기작에 의해 FPN 유전자 발현을 조절하는 것으로 사료된다. 이상의 결과를 종합해 볼 때, 구리는 대식 세포에서 전사개시 과정을 활성화함으로써 농도 의존적으로 FPN 유전자 발현을 촉진하는 것으로 생각되며, 이는 구리가 철분의 대사에 미치는 새로운 작용 기작을 제시한다. 앞으로, 구리와 철 분의 상호 작용이 FPN의 철분 및 다른 무기질 이온의 세포내 외 수송 (transport)에 어떤 영향을 미치는지에 대한 기능적 연구가 계속적으로 이루어져야 할 것으로 사료된다.

• 대한한의학회지
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• 제24권2호
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• pp.166-178
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• 2003

Objectives : Nitric oxide (NO) plays an important role in normal and pathophysiological cells as a messenger molecule, neurotransmitter, microbiological agent, or dilator of blood vessels and arteriosclerosis, respectively. This study was undertaken to understand the mechanism of NO production and effect of Hyeolbuchukeo-tang (Xiefuzhuyu-tang) on NO production in cultured vascular smooth muscle cell (VSMC). Methods and Results : VSMC was isolated from aorta and cultured. Cultured primary cells were identified as VSMC with anti--smooth muscle actin antibody. A large amount of NO was produced in cultured VSMC treated with $IFN-{\gamma}$ plus TNF in a time- and dose-dependent manner. $TNF-{\alpha}$ was a more efficient stimulator than $IFN-{\gamma}$ in NO production of cultured VSMC. iNOS protein wasdetected within 3 hrs and it increased up to 12 hrs in a time-dependent manner. However, accumulated NO in cytokine-treated VSMC was not detected within 3 hrs. NO production in cytokine-treated VSMC showed the dose- and time-dependent manner, and increased up to 48 hrs. The activated VSMC produced a large amount of NO (about 60 uM). Hyeolbuchukeo-tang (Xiefuzhuyu-tang) alone did not induceNO production, but it potentiated the effect of $TNF-{\alpha}$ on NO production and increased NO production by about 20%. Hyeolbuchukeo-tang (Xiefuzhuyu-tang) did not affect the transcriptional activity of iNOS gene, but increased the accumulation of iNOS. These results indicate that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) could modulate the translational level of iNOS. PKC did not modulate NO production, but calcium ionophore A23187 decreased NO production. However, Hyeolbuchukeo-tang (Xiefuzhuyu-tang) elevated the decreased NO production in A23187-treated VSMC by modulating the stability of iNOS transcripts. Half-life of the synthesized transcripts appeared to have about 6 hrs. PDTC, an $NF-{\kappa}B$ inhibitor, blocked the accumulation of iNOS mRNA, indicating that $NF-{\kappa}B$ served as an important modulator in the transcriptional regulation of iNOS. As Hyeolbuchukeo-tang (Xiefuzhuyu-tang) potentiated the effect of the $TNF-{\alpha}$ on NO production but had no additional effect on PDTC-modulated NO production, it is suggested that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) enhances the $TNF-{\alpha}-mediated$ NO production of VSMC by modulating the iNOS activity and the stability of iNOS transcripts in activated VSMC having the elevated intracellular calcium ion. Conclusions : This study suggests that Hyeolbuchukeo-tang (Xiefuzhuyu-tang) has a potential capacity for preventing and treating diseases of the circulation system, including arteriosclerosis.

• 미생물학회지
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• 제51권1호
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• pp.68-74
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• 2015

Thermococcus pacificus P-4의 유전체 서열 분석을 통하여 예측되는 GH1 ${\beta}$-glucosidase를 암호화하는 유전자를 동정하였다. 그 유전자는 487 아미노산들을 암호화하는 1,464 bp 나타내었으며, 그 아미노산 서열은 Pyrococcus furiosus ${\beta}$-glucosidase와 77% 상동성을 나타내었다. 그 유전자는 Escherichia coli 시스템 내에서 복제 및 발현하였다. 재조합 된 단백질은 금속 친화크로마토그래피를 통하여 정제하고 특성을 분석하였다. 정제된 단백질(Tpa-Glu)은 pH 7.5와 $75^{\circ}C$에서 최적활성을 나타내었으며, 열안정성은 $90^{\circ}C$에서 약 6시간의 반감기를 보였다. Tpa-Glu는 pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, 그리고 pNP-${\beta}$-xylopyranoside 순으로 우수한 $k_{cat}/K_m$ 값을 나타내었다. 또한, Tpa-Glu는 ${\beta}$-1,3-linked polysaccharide (laminarin) 그리고 ${\beta}$-1,3-와 ${\beta}$-1,4-linked oligosaccharides에 대하여 exo-hydrolyzing 활성을 보였다. 본 연구는 초고온 고세균으로터 ${\beta}$-glucosidase가 exohydrolyzing 활성을 처음 확인한 것으로 이 효소는 laminarin의 당화공정에 ${\beta}$-1,3-endoglucanase와 함께 적용할 수 있을 것으로 기대된다.