• Biomolecules & Therapeutics
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• v.5 no.1
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• pp.53-58
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• 1997

$[Lys^7$]dermorphin, abbreviated K7DA, which has structural features similar to a metabolically stable $\mu$-opioid peptide agonist $[D-Arg^2, Lys^4$]dermorphin analogue (DALDA), but is intrinsically more potent with respect to binding to the $\mu$-opioid peptide receptor. The present studies report on attempts to enhance brain uptake of systemically administered K7DA by conjugation to a complex of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the blood-brain barrier (BBB). SA-OX26 conjugate mediates BBB transport of biotinylated therapeutics. The K7DA is monobiotinylated at the $\varepsilon$-amino group of the $[Lys^7$] residue with cleavable linker using NHS-SS-biotin. The brain uptake of $^{125}I$ labeled biotinylated K7DA ($^{125}I$-bio-SSa-K7DA) was very small and rapidly metabolized after intravenous injection. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the $^{125}I$-bio-55-K7DA bound to the SA-OX26 conjugate $^{125}I$-bio-SS-K7DA/SA-OX26) was 0.14$\pm$0.01, a level that is 2-fold greater than the brain uptake of morphine. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC and intravenous injection of labeled opioid chimeric peptides. The disulfide linker is stable in plasma in vivo but is cleaved in rat brain in vivo. In conclusion, these studies show that delivery of these potential opioid peptides to the brain may be improved by coupling them to vector-mediated BBB drug delivery system.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• Journal of Dairy Science and Biotechnology
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• v.33 no.4
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• pp.271-275
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• 2015

Colloidal semiconductor CdSe-ZnS core-shell nanocrystal quantum dot (Qdot) are luminescent inorganic fluorophores that show potential to overcome some of the functional limitations encountered with organic dyes in fluorescence labeling applications. Salmonella Enteritidis has emerged as a major cause of human salmonellosis worldwide since the 1980s. A rapid, specific, and sensitive method for the detection of Salmonella Enteritidis was developed using Qdot as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-Salmonella Enteritidis antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-Salmonella antibodies were added to form sandwich immune complexes. After magnetic separation, the immune complexes were labeled with Qdot via biotin-streptavidin conjugation, and fluorescence measurement was carried out using a fluorescence measurement system. The detection limit of the Qdot method was a Salmonella Enteritidis concentration of $10^3$ colony-forming units (CFU)/mL, whereas the conventional fluorescein isothiocyanate-based method required over $10^5CFU/mL$. The total detection time was within 2 h. In addition to the potential for general nanotechnology development, these results suggest a new rapid detection method of various pathogenic bacteria from a complex food matrix.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1030-1038
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• 2007

Results on localization of growth hormone receptor (GHR), interaction of growth hormone (GH) with receptor in buffalo adipose tissue and identification of activated signaling molecules in the action of GH are presented. Bovine GH (bGH) was labeled with fluorescein or biotin. Fluorescein-labelled bGH was used for localization of GHRs in buffalo adipocytes. The receptors were present on the cell surface. The affinity of binding of GH to its receptor was determined by designing an experiment in which buffalo adipose tissue explants, biotinylated GH and streptavidin-peroxidase conjugate were employed. The affinity constant was calculated to be $2{\times}10^8M^{-1}$. The receptor density on adipose tissue was found to be 1 femto mole per mg of tissue. Signalling molecules generated in the action of GH were tentatively identified by employing Western blot and enhanced chemiluminescence techniques using anti-phosphotyrosine antibody. Based on molecular weights of proteins reactive to anti-phosphotyrosine antibody, three signaling molecules viz. insulin receptor substrate, Janus activated kinase (Jak) and mitogen activated protein were tentatively identified. These signaling molecules appeared in a time (incubation time of explants with growth hormone) dependent way. The activation of Jak2 was confirmed by employing anti-Jak2 antibody in a Western blot. The activation of Jak2 occurred during 5 min incubation of buffalo adipose tissue explants with GH and incubation for an additional period, viz. 30 min. or 60 min., resulted in a drastic reduction in activation. The results suggest that Jak2 activation is an early event in the action of GH in buffalo adipose tissue.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2681-2685
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• 2012

Objectives: This study focused on PTEN and Livin expression and associations with malignancy in human renal clear cell carcinomas (RCCC). Methods: PTEN and Livin expression was assessed in 100 RCCC tissue samples, 50 paracarcinoma cases, and 20 normal renal tissue samples using the immunohistochemical Streptavidin proxidase (SP) method. The relationships between binding and corresponding biological characteristics, such as histological grade, lymph node metastases, and clinical stages were analyzed. Results: Positive PTEN expression in RCCC was significantly lower than in renal tissue adjacent to carcinoma tissue and normal renal tissue (P<0.01). Livin expression in the renal tissue adjacent to the carcinoma and normal renal tissues exhibited only low levels, whereas overall Livin expression in RCCC was statistically significant (P<0.01). In RCCC, PTEN expression rate gradually decreased with an increase in clinical stage, whereas that of Livin increased to statistically significant levels (P<0.01), PTEN and Livin levels being negatively correlated (r=-0.395, P<0.01). Conclusions: PTEN and Livin are important in RCCC development. The two factors combined are expected to provide indices for estimating RCCC malignancy and progression levels, as well as references for RCCC diagnosis and treatment.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6905-6910
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• 2014

Background: The relation ofsurvivin gene expression to survival and surgical prognostic factors in the patients with endometrial carcinoma is unclear. Materials and Methods: In this study, 62 cases who were operated due to endometrial carcinoma were investigated between 2003 and 2011 in the the gynecological oncology clinic of Female Disease Training and Investigation Hospital of Etlik Zubeyde, Hanim, Turkey. Clinical and surgical prognostic factors were investigated by screening the records of these cases. With the standard streptavidin-biotin immune peroxidase method, cytoplasmic and nuclear expression of survivin was investigated in sections with specific antibodies (1:100, diagnostic Bio Systems, USA) primer. The aim was to elucidate any relation between survivin expression and defined prognostic factors and survival. Results: There was no statistically significant relationship between cytoplasmic and nuclear indexes identified for survivin and age, body mass index, the levels of preoperative hemoglobin, platelet and Ca 125, stage, grade, lymph node meastasis, the number of meta statical lymph nodes (total, paraaortic and pelvic), myometrial invasion, serosal invasion, adnexal involvement, the presence of acid in the first diagnosis, the involvement of omentum, the adjuvant treatment application of the cases, the presence of recurrence and rate of mortality (p>0.05). Statistical significance was noted for the presence of advanced stage lymph node metastasis (pelvic, paraaortic, pelvic and paraaortic), serosal involvement, positive cytology, lymph vascular space invasion, intra abdominal metastasis, and omentum involvement. When investigated the relation between cytoplasmic and nuclear survivin indexes and total survival, the result was not statistically significant (p>0.05). Conclusions: In our study, there was no statistically significant relationship between the rates of cytoplasmic and nuclear survivin expression with identified prognostic factors and total or non-disease survival.

• The Transactions of The Korean Institute of Electrical Engineers
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• v.58 no.1
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• pp.147-154
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• 2009

This paper presents a grating~integrated SPR (Surface Plasmon Resonance) sensor chip for simple and inexpensive biomolecule detection. The grating-integrated SPR sensor chip has two sensing channels having a nano grating for SPR coupling. An external mirror is used for multi channel SPR sensing. The present sensor chip replaces bulky and expensive optical components, such as fiber-optic switches or special shaped prisms, resulting in a simple and inexpensive wavelength modulated multi-channel SPR sensing system. We fabricate a SPR sensor chip integrated with 835 nm-pitch gratings by a micromolding technique to reduce the fabrication cost. In the experimental characterization, the refractive index sensitivity of each sensing channel is measured as $321.8{\pm}8.1nm$/RI and $514.3{\pm}8.lnm$/RI, respectively. 0.5uM of the target biomolecule (streptavidin) was detected by a $1.13{\pm}0.16nm$ shift of the SPR dip in the 10%-biotinylated sample channel, while the SPR dip in the reference channel for environmental perturbation monitoring remained at the same position. From the experimental results, multi-channel biomolecule detection capability of the present grating-integrated SPR sensor chip has been verified. On the basis of the preliminary experiments, we successfully measured the binding reaction rate for the $2\;nM{\sim}200\;nM$ monoclonal-antibiotin, thus verifying biomolecule concentration detectability of the present SPR sensor chip. The binding reaction rates measured from the present SPR sensor chip agredd well with those from a commercialized SPR sensor.

• BMB Reports
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• v.34 no.6
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.1
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• pp.35-41
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• 2008

This study was undertaken to investigate the distribution of major histocompatibility complex class II positive (MHC II+) (HLA-DP-like) cells in the cow uterus (cervix, corpus and cornu uteri) and to compare these cells between the estrus and diestrus phases of the estrous cycle. Twenty-nine multiparous cows were used. Tissue samples from the middle of the cervix, the corpus and the right cornu were taken immediately after slaughter at the estrus or diestrus phase. Streptavidin-biotin peroxidase complex staining was used to detect MHC II+ cells. The number of MHC II+ cells per unit area of tissue was counted using image analysis software under a light microscope. Numerous MHC II+ cells were found in the endometrium (cervix, corpus and cornu uteri) in both estrus and diestrus. MHC II+ cells were found in the surface epithelium of the cervix uteri in diestrus, but in the corpus uteri in both estrus and diestrus and in the cornu uteri in estrus. MHC II+ cells were also found freely in the lumen of the glands and between the gland epithelia of the corpus and cornu uteri in both estrus and diestrus. There were also MHC II+ cells in the connective tissue of the myometrium and perimetrium (outside the endometrium) and around the blood vessels. Endothelial cells were frequently positive for MHC II staining. More MHC II+ cells were found in the endometrium than outside the endometrium in both estrus and diestrus (p<0.001). However, there was no difference in the numbers of positive cells between estrus and diestrus either in the endometrium or outside it. These results are the first evidence for HLA-DP-like MHC II+ cells in the bovine uterus. They indicate that antigen presentation by HLA-DP-like MHC II+ cells of the uterus is not influenced by hormonal status.

• Proceedings of the Korean Vacuum Society Conference
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• 2011.08a
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• pp.389-389
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• 2011

최근 생체분자 구조 연구가 의료진단, 생명 현상 규명 및 의약품 개발 등 다양한 분야에 응용되고 있으나 대부분의 분석방법이 제한적이어서 새로운 기술 개발의 필요성이 증대하고 있다. 종래의 DNA 등의 생체분자의 분석은 형광염료를 이용한 방법이 주로 이용되었다. 형광염료는 단백질을 포함한 여러 물질들에 대해 반응하지 않기 때문에 분석에 제한이 있으며, 이와 같은 단점을 보완하는 방법으로 SPR (surface plasmon resonance) 분석법이 연구되었다. SPR은 형광염료 분석에 필수적인 레이블링(labeling) 등의 전처리 과정 없이 높은 민감도로 분석이 가능한 장점이 있다. 한편, 그래핀은 뛰어난 전자기적 성질과 기계적 성질 을 가지는 반금속(semimetal)으로, 실험실 규모에서 안정적인 합성이 실현되면서 그 응용 분야에 대한 연구가 활발히 이루어 지고 있다. 그래핀은 큰 표면적 대 부피비를 가지며, 이는 검출물질과의 반응성이 좋아야 하는 센서기술에 있어서 장점으로 작용한다. 특히, 비금속성을 띠는 단층 그래핀을 여러 장 겹치면 금속성을 갖게 되기 때문에 SPR 센서의 금속 필름으로 응용이 가능하다. 본 연구에서는 SPR 현상을 이용하는 광섬유 센서의 감도와 정확도를 증진시키기 위해 광섬유 표면에 그래핀을 적용하였다. 광섬유는 상부 피복과 클래딩을 제거하여 코어를 노출시킨 후, 다층 그래핀 필름을 코팅함으로써 검출부를 구성한다. 그 후, DNA-biotin 용액, DNA-biotin 용액, 그리고 Streptavidin 단백질 복합 용액에 대한 검출기 신호를 분석하였다. 구성된 센서에 각 용액을 1 ${\mu}{\ell}$ 씩 반응시켜 분광계로 파장에 따른 광강도를 측정하는 실험을 수행했으며, 450 nm에서 460 nm 범위의 푸른빛의 광원을 사용하였다. 그래핀 필름의 유무에 따라 확연히 구분되는 경향을 보이는 결과를 얻었고 그래핀 필름이 기존 SPR 센서의 금속박막을 대체 할 수 있음을 확인하였다.