• 생명과학회지
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• 제22권1호
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• pp.98-109
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• 2012

큰입우럭(Micropterus salmoides) 조직의 젖산탈수소효소(EC 1.1.1.27, LDH)의 특성 및 골격근과 심장조직의 monocarboxylate 수송체 1, 2, 4의 발현을 연구하였다. Native-PAGE 결과골격근에서 LDH $A_4$, 심장, 간, 눈 및 뇌조직에서 $A_4$, $A_2B_2$, $B_4$, 눈조직에서 eye-specific $C_4$ 동위효소가 발현되었다. 9월에 심장조직에서 LDH $B_4$ 동위효소의 활성이 강하고 다른 조직에서는 $A_4$의 활성이 강했으나, 11월에 심장조직에서 $A_4$ 동위효소의 활성이 강하게 확인되었다. 골격근과 심장조직에서 LDH/CS로 조직의 혐기적 대사 비율이 높게 확인되었으며, 면역 침강 후 native-PAGE에 의해 LDH eye-specific $C_4$ 동위효소가 $B_4$보다 $A_4$ 동위효소에 더 유사한 것으로 확인되었다. LDH $A_4$ 동위효소가 affinity chromatography에 의해 정제되었고, 하부단위체 A의 분자량은 37.200이었다. 피루브산 10 mM에서 조직 LDH의 활성이 11.05-28.32% 남아 저해 정도가 컸고, 눈조직 LDH의 $K_m^{PYR}$이 낮았으며, 조직의 최적 pH는 7.5~8.0이였다. 골격근 미토콘드리아에서 LDH $A_4$ 동위효소, 심장조직의 미토콘드리아에서 $B_4$와 $A_2B_2$ 동위효소가 확인되었고, 골격근과 심장조직의 원형질막과 미토콘드리아에서 MCT 1, 2, 4가 확인되었다. 골격근 MCT 1, 2, 4 골격근 MCT 1 60 kDa, MCT 2 54~38 kDa, MCT 4 63 kDa, 심장조직 MCT 1 57 kDa, MCT 2 54~38 kDa 및 MCT 4 55.5 kDa이었다. 실험 결과, 큰입우럭이 저산소 조건에 적응되어져 혐기적 대사가 우세하고, 활성이 큰 골격근과 심장조직에서 원형질막과 미토콘드리아 MCT 1, 2, 4를 통해 젖산과 피루브산이 유입되고 유출되며 LDH에 의해 에너지 생성을 효율적으로 조정하는 것으로 사료된다.

• BMB Reports
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• 제40권5호
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• pp.731-739
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• 2007

The purpose of this study was to develop paclitaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles coated with cationic SM5-1 single-chain antibody (scFv) containing a polylysine (SMFv-polylys). SM5-1 scFv (SMFv) is derived from SM5-1 monoclonal antibody, which binds to a 230 kDa membrane protein specifically expressed on melanoma, hepatocellular carcinoma and breast cancer cells. SMFv-polylys was expressed in Escherichia coli and purified by cation-exchange chromatography. Purified SMFv-polylys was fixed to paclitaxel-loaded PLGA nanoparticles to form paclitaxel-loaded PLGA nanoparticles coated with SMFv-polylys (Ptx-NP-S). Ptx-NP-S was shown to retain the specific antigen-binding affinity of SMFv-polylys to SM5-1 binding protein-positive Ch-hep-3 cells. Finally, the cytotoxicity of Ptx-NP-S was evaluated by a non-radioactive cell proliferation assay. It was demonstrated that Ptx-NP-S had significantly enhanced in vitro cytotoxicity against Ch-hep-3 cells as compared with non-targeted paclitaxel-loaded PLGA nanoparticles. In conclusion, our results suggest that cationic SMFv-polylys has been successfully generated and may be used as targeted ligand for preparing cancer-targeted nanoparticles.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권3호
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• pp.200-208
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• 2006

Amino acids are required for protein synthesis and energy sources in all living cells. The amino acid transport system L is a major nutrient transport system that is responsible for $Na^+$-independent transport of neutral amino acids including several essential amino acids. In malignant tumors, the L-type amino acid transporter 1 (LAT1), the first isoform of system L, is highly expressed to support tumor cell growth. In the present study, the expression and functional characterization of amino acid transport system L were, therefore, investigated in Saos2 human osteogenic sarcoma cells. RT-PCR and western blot analyses have revealed that the Saos2 cells expressed the LAT1 and the L-type amino acid transporter 2 (LAT2), the second isoform of system L, together with their associating protein heavy chain of 4F2 antigen (4F2hc) in the plasma membrane, but the expression of LAT2 was very weak. The uptakes of [${14}^C$]L-leucine by Saos2 cells were $Na^+$-independent and were completely inhibited by the system L selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [${14}^C$]L-leucine uptake and the inhibition profiles of [${14}^C$]L-leucine uptake by various amino acids in the Saos2 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [${14}^C$]L-leucine uptake is, therefore, mediated by LAT1 in the Saos2 cells. These results suggest that the transports of neutral amino acids including several essential amino acids into Saos2 human osteogenic sarcoma cells are for the most part mediated by LAT1. Therefore, the Saos2 human osteogenic sarcoma cells are excellent tools for examine the properties of LAT1. Moreover, the specific inhibition of LAT1 in tumor cells might be a new rationale for anti-tumor therapy.

• Journal of Oral Medicine and Pain
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• 제38권4호
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• pp.313-318
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• 2013

수면은 외부의 자극에 대해 반응하지 않는 상태로 만성통증을 지닌 환자는 수면 장애를 겪고 있으며 수면장애는 통각과 민한 상태를 유발하고 통증인지에 영향을 미친다. 구강작열감 증후군은 구강점막에 화끈거리는 감각을 포함하는 만성 안면통증으로 감각과 통증 역치가 변성되고 신경병증 징후를 보인다. 구강작열감 증후군은 첫 번째로 국소적, 전신적 요인 즉, 흡연, 투약 등 작열감 증상의 원인으로 가능한 요인을 제거하고, 두 번째로는 환자를 안심시키는데 있다. 수면박탈 내인성, 외인성 아편유사제의 진통효과는 수면 계속성 즉, 선택적인 렘수면 박탈은 아편유사제 진통효과를 방해 하고 opioid protein synthesis 억제를 야기하고 ${\mu}$ 와 ${\delta}$ opioid 수용체 친화력을 감소시킨다. enkephalinase-inhibitor와 MAO-B inhibitor 적용으로 나타나는 아편유사제의 진통상승효과는 렘수면 박탈시에 무효화 된다. 렘수면 박탈후에는 5-HT 와 5-hydroxy indole acetic acid 레벨이 유의하게 감소되고, 아편유사체에 활성에 의해 유발되는 통증억제를 지지하는 세로토닌 체계를 가능하지 않게 한다. 구강작열감 증후군은 설말단 신경섬유에서 추삭변성 변화가 있고 특히, 열자극, 냉자극 및 미각 유해수용성 자극의 인지를 포함한 감각변화가 폐경기후 생식스테로이드의 급격한 감소가 신경활성 스테로이드 생성에 변성을 일으켜며, 이것은 말초성 변성와 중추성 변성이 존재함을 제시한다. 구강작열감 증후군은 기본적으로 가능한 요인을 제거하는 것이 중요하므로 구강작열감의 진행을 방지하고, 만정적인 작열감의 경감을 위하여 증상 경과에 영향을 미칠 수 있는 수면에 대하여 고찰하고자 한다.

• 생명과학회지
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• 제17권2호통권82호
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• pp.185-191
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• 2007

Akt/PKB는 세포의 증식, 분화, 사멸, 혈관신생 등 매우 많은 생리활성 조절에 있어 매우 중요한 역할을 수행한다. 우리는 Akt/PKB의 tyrosine잔기의 인산화가 $Thr^{\308}$ 인산화에 필수적임을 밝혔다. COS-7 세포주에 EGF를 처 리하면 Akt/PKB의 tyrosine 잔기에 인산화가 촉진되었으며 이러한 인산화 촉진은 Akt/PKB에 myristoylation site를 이용해 세포막으로 이동시키면 더욱 더 증가하였다. 특히, 분리된 Akt/PKB와 EGF 수용체를 이용해 인산화 반응을 실시하면 tyrosine잔기의 인산화뿐만 아니라 $Ser^{\473}$에 대한 인산화도 증가하였다. 더욱이 tyrosine잔기에 인산화 된 Akt/PKB는 활성화된 EGF 수용체와 직접적인 결합을 이루고 있음을 확인하였다. 마지막으로 예측되는 tyrosine 잔기인 $(Tyr^{\326})$을 Alanine으로 치환하면 정상 Akt/PKB뿐만 아니라 활성화된 Akt/PKB의 EGF에 의한 $Thr^{\308}$ 인산화가 사라짐을 확인하였다. 이러한 결과들을 바탕으로 EGF 수용체에 의한 직접적인 Akt/PKB의 tyrosine 인산화는 EGF에 의한 많은 생리활성 조절기전의 또 다른 기전이라 볼 수 있다.

• 한국미생물·생명공학회지
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• 제40권3호
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• pp.274-277
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• 2012

본 연구에서는 다제내성을 보이는 인체 병원균의 하나인 S. aureus에서 유래된 FtsZ 단백질의 유전자를 클로닝하고 대장균에 형질전환하여 재조합 단백질을 만들고, in vitro 상에서 폴리머 형성 활성을 측정하였다. Bradford 방법을 이용하여 SA FtsZ단백질의 농도를 측정한 후, SA FtsZ단백질의 폴리머 형성 활성을 확인하기 위해 형광계를 이용하여 excitation 방향과 $90^{\circ}$의 방향에서 산란되는 빛의 양을 측정하는 방법을 사용하였을 때에 대조군에서는 빛이 산란되지 않았고, SA FtsZ 단백질에 GTP와 $Mg^{2+}$를 처리한 실험군에서만 빛이 산란되는 현상을 관찰하였다. 1분여의 시간이 지난 이후에는 다시 산란되는 빛이 줄어드는 것을 볼 수 있는데, 이것은 SA FtsZ 단백질의 아미노말단 도메인의 GTPase 활성에 의해서 GTP가 분해되어서 SA FtsZ 단백질의 폴리머가 단량체로 분해되었기 때문이라고 예측된다. 본 연구를 통하여 확립된 SA FtsZ 활성 측정 방법은 향후 SA FtsZ 단백질의 폴리머 형성을 저해하는 방식으로 S. aureus를 표적으로 하는 항생제 후보물질 도출을 위한 스크리닝 방법으로 사용될 수 있을 것이다.

• 미생물학회지
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• 제32권2호
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• pp.147-154
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• 1994

Serratia marcescens가 세포외로 분비하는 nuclease의 유전자가 발현된 Escherichia coli JM107을 배양하여 다량의 효소를 정제하였다. Matrex green gel과 heparin agarose gel column chromatography법으로 약 50배 정제한 효소는 분자량이 29KDa였으며, 전기영동 상에서 단일 띠를 보였다. 이 단백질을 이용하여 polyclonal antibody를 만들고, 면역조직화학법으로 세포내의 분포를 조사하였다. Nuclease는 주로 세포막에 존재하였고, 이를 토대로 효소가 세포질에서 합성된 후 세포막으로 빠르게 이동함을 알 수 있었다. 이 결과는 세포의 막분획에서 효소의 활성의 대부분이 회수되며, 면역블럿 방법으로 효소의 대부분이 세포막에서 검출된다는 결과와 일치하였다.

• Bulletin of the Korean Chemical Society
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• 제14권6호
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• pp.687-691
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• 1993

Complexation of $Cd^{2+},\;Pb^{2+}\;and\;Hg^{2+}$ ions with four cryptands were studied by potentiometry and solution calorimetry in various weight percent methanol-aqueous solvent at 25${\circ}$C under $CO_2$free nitrogen atmosphere. The stabilities of the complexes were dependent on the cavity size of macrocycles. The $Hg^{2+}$ ion stability constants are higher than those of $Cd^{2+}\;and\;Pb^{2+}$ ion. All the cryptands formed complexes having 1 : 1 (metal to ligand) mole-ratio except for $Hg^{2+}-L_1$ (cryptand 1,2b: 3,5-benzo-9,14,17-trioxa-1,7-diazabicyclo-(8,5,5) heptadecane) and $Cd^{2+}-L_2$ (cryptand 2,2b: 3,5-benzo-10,13,18,21-tetraoxa-1,7-diazabicyclo (8,5,5) eicosane) complexes. $Hg^{2+}-L_1$ complex was a sandwitch type, and the $Cd^{2+}-L_2$ complex showed two stepwise reactions. Thermodynamic parameters of the $Cd^{2+}-L_2$ complex were $6.08(log\;K_1)$, -7.28 Kcal/mol $({\Delta}H_1)$, and $4.78\;(log\;K_2)$, -4.62 Kcal/mol $({\Delta}H_2)$, respectively, for 1 : 1 and 2: 1 mole-ratio. The sequences of the selectivity were increased in the order of $Hg^{2+}\;>Pb^{2+}\;>Cd^{2+}$ ion for $L_3\;and\;L_4$ macrocycles, and the $L_2$-macrocycle has a selectivity for $Cd^{2+}$ ion relative to $Zn^{2+},\;Ni^{2+},\;Pb^{2+}\;and\;Hg^{2+}$ ions. Thus, it is expected that the $L_2$ can be used as carrier for seperation of the post transition metals by macrocycles-mediated liquid membrane because $L_2$ is not soluble in water, and the difference of stability constants of the metal complexes with $L_2$ are large as compared with the other transition metal complexes. The $^1H\;and\;^{13}C-NMR studies indicated that the nitrogen atoms of cryptands have greater affinity to the post transition metal ions than the oxygen atoms, and that the planarities of the macrocycles were lost by complexation with the metal ions because of the perturbation of ring current of benzene molecule attached to macrocycles and counter-anions.

• Journal of Animal Science and Technology
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• 제66권5호
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• pp.1021-1033
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• 2024

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.10-12
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.