To prepare calcium-binding peptides as calcium supplement, barley proteins were hydrolyzed using Flavourzyme for 18 h and the hydrolysates were ultra-filtered under 3 kDa as a molecular weight. The resultant filtered peptides were fractionated using ion exchange and normal-phase high performance liquid chromatography. Then each fraction that was obtained was determined for its calcium-binding activity to isolate the calcium-binding peptides. As a result, the highest calcium-binding peptide fraction was obtained, and the results suggest that barley protein hydrolysates can be used as a calcium supplement.

Nesfatin-1/NUCB2, which is secreted from the brain, is known to control appetite and energy metabolism. Recent studies have been shown that nesfatin-1/NUCB2 was expressed not only in the brain, but it was also expressed in the gastric organs and adipose tissue. However, little is known about the expression of nesfatin-1/NUCB2 in the male reproductive system. Therefore, we examined whether the nesfatin-1/NUCB2 and its binding site exists in the male reproductive organs. Nesfatin-1/NUCB2 mRNA and protein were detected in the mouse testis and epididymis by PCR and Western blot analysis. As a result of the immunohistochemistry staining, the nesfatin-1 protein was localized at the interstitial cells and Leydig cells in the testis. Nesfatin-1 binding sites were also displayed at boundary cells in the tunica albuginea. Furthermore, in order to examine if the expression of nesfatin-1/NUCB2 mRNA in the testis and epididymis were affected by gonadotropin, its mRNA expression was analyzed after PMSG administration into mice. NUCB2 mRNA expression levels were increased in both of the testis and epididymis after PMSG administration. These results demonstrated for the first time that nesfatin-1 and its binding site were expressed in the mouse testis and epididymis. In addition, nesfatin-1/NUCB2 mRNA expression was controlled by gonadotropin, suggesting a possible role of nesfatin-1 in the male reproductive organs as a local regulator. Due to this, further study is needed to elucidate the functions of nesfatin-1 on the male reproductive system.

Lhx8 is a member of the LIM-homeobox transcription factor family expressed in the mouse ovary. We discovered that Lhx8 knockout females lose oocytes within 7 days after birth. Lhx8–/–ovaries fail to maintain the primordial follicles and growing follicles. Lhx8–/–ovaries misexpress numerous oocyte-specific genes such as H1foo and Nlrp14. The molecular mechanism of there gulation of Lhx8 in the oocyte has not been described. We examined to characterize Lhx8 DNA binding elements and to identify its direct target genes in the oocyte. CAST was performed using glutathione transferase Lhx8 homeodomain fusion protein (GST-LHX8HD). A 15-bp random sequence flanked by 20-bp of fixed sequences were incubated with purified GST-LHX8HD protein. Unbound DNA was washed with binding reaction buffer. Bound DNA was eluted and re-amplified by PCR for the next round of CAST. Final PCR products were cloned and sequenced to derive consensus binding sequence. EMSA was performed using 32P-labeled oligomers. Binding reactions were conducted by incubating 32P-labeled probes with purified protein. Dual luciferase assays were carried out with extracts of total HEK293 cell which was transfected by the pGL4-promoter vector containing three artificial repeats of LBE(3xLBE-Luc) and overexpression vector carrying the Lhx8 homeodomain as recommended by Promega. We identified several cis-acting sites, TGATTG as Lhx8 DNA binding elements (LBE) using a library of randomly generated oligonucleotides by CAST. EMSA reslut shows that Lhx8 preferentially binds to the oligomer including Lhx8 binding element (TGATTG) with high affinity. In addition, we found that the relative luciferase activity of reporter construct containing three copies of TGATTG was increased by 2.3-fold with Lhx8 overexpression. These results suggest that Lhx8 preferentially binds Lhx8 DNA binding element, TGATTG, and can transactivate reporter genes through the LBE. The transcription of Lhx8 target gene in oocytes directly might be regulated by its during early folliculogenesis.

최근 시상하부에서 생성되는 nesfatin-1/NUCB2가 섭식과 에너지 대사를 조절한다는 사실이 새롭게 밝혀졌다. 본 연구에서는 이러한 단백질이 생쥐의 생식기관에서도 발현을 하는지, 그리고 그 수용체가 생식기관 내에 존재하는 지를 확인함으로써 nesfatin-1이 생식기능에 미칠 수 있는 가능성을 알아보고자 하였다. 암컷 생쥐에서 난소와 자궁을 획득하여 conventional PCR 방법으로 NUCB2 mRNA 발현을 조사하였고, real-time PCR 방법으로 상대적인 NUCB2 mRNA 발현량을 비교 분석하였다. 난소 내 nesfatin-1 단백질의 발현 위치를 조사하기 위하여 nesfatin-1 항체를 이용한 면역조직화학염색법을 수행하였으며, biotin conjugated nesfatin-1을 이용하여 nesfatin-1 결합 부위를 확인하였다. 또한 생식소 내 NUCB2 mRNA 발현이 성선자극호르몬에 의해 영향을 받는지 알아보기 위해 PMSG 투여 후 NUCB2 mRNA 발현량을 조사하였다. 실험 결과, 생쥐의 난소와 자궁에서 확인된 NUCB2 유전자가 시상하부에서 만큼이나 많은 양이 발현되고 있었다. 면역조직화학적 염색 결과, nesfatin-1 단백질은 협막세포와 대부분의 기질세포에서 발현되었고, 일부 황체세포에서도 발현이 확인되었다. 반면, 난포 내 과립세포에서는 발현되지 않았으나, 특정 난포 내 난자에서는 발현됨을 확인하였다. 한편, nesfatin-1 단백질의 결합 부위는 난소 백막 주위의 기질세포와 협막세포에서 관찰되었다. 또한 PMSG 투여 후 난소와 자궁에서 NUCB2 mRNA의 발현이 유의하게 증가함을 확인하였다. 이상의 결과에서 난소 내 nesfatin-1 단백질의 발현과 그 결합 부위의 존재는 nesfatin-1이 뇌에서 뿐만 아니라 생식기관에서도 국부조절인자로써 중요한 역할을 할 것으로 사료되며, 앞으로 생식기관에 미치는 nesfatin-1의 역할을규명하기위한더많은연구가필요하다고판단된다.

청미래덩굴 뿌리(Smilacis rhizoma)로부터 칼슘과 결합하는 물질을 분리하고자 열수로 추출한 추출물을 ion exchange, normal-phase HPLC 및 gel filtration chromatogarphy를 이용하여 칼슘 결합 물질을 순차적으로 분리하였다. 그 결과 ion exchange chromatography에서 7개의 major peaks를 얻었으며, 이 중 F6 fraction이 0.083 mM로 칼슘과 가장 높은 결

Mammalian spermatogenesis takes place in the seminiferousepithelium, which is composed of Sertoli cells and germ cells. The interaction between spermatogenic and Sertoli cells as well as elongated spermatids and Sertoli cells is tightly regulated by junctional adhesion molecules (JAMs). JAMs, which are cell adhesion molecules, are known to play roles in various biological processes such as fertilization, neurogenesis, cancer progression, and spermatogenesis. Members of the JAM family have a unique structure: they contain an N-terminal signal peptide domain, immunoglobulin (Ig)-like domains, transmembrane and cytoplasmic tail domains, each of which has distinct functions. The extracellular Ig-like domains interact in a homophilic or heterophilic manner, whereas cytoplasmic tail domain mediates the tight junction assembly. Although members of the JAM family are exclusively present in or restricted to the testis, their precise roles in spermatogenesis and fertilization have not yet been completely explored. The functional roles of Nectin-2, Nectin-3, JAM-C, cell adhesion molecule1 (CADM1), coxsackie and adenovirus receptor (CAR) have been evaluated by analysis of null mutant mice. Unfortunately, CAR-deficient mice had an embryonic lethal phenotype; this demonstrates the importance of CAR in development, but its physiological role in spermatogenesis is not known. The loss of CADM1, Nectin-3 and JAM-C resulted in male infertility caused by loss of adhesion between germ and Sertoli cells. A variety of JAMs participate in the interaction between germ and Sertoli cells. Recently, human VSIG1 has been characterized, which was originally known as A34, as a new member of the JAM family; VSIG1 is composed of two extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain. However, this molecule has not been functionally characterized, so this was one of the aims of our present study. RT-PCR and immunoblot analyses were used to study VSIG1 expression, VSIG1 was specifically expressed in testicular germ cells but not in sperm. Pull-down assay with glutathione S-transferase (GST) or His-fused first Ig and second Ig domains of VSIG1 and SDS-PAGE under mild non-reducing conditions demonstrated that VSIG1 functions as an in vitro homophilic adhesion molecule. Furthermore, cells expressing a deletion of the C-terminus of VSIG1 failed to interact with ZO-1, the central structural protein of the tight junction. These findings suggest mouse VSIG1 interacts with an unknown molecule in Sertoli cells via its extracellular domain, while its cytoplasmic domain is needed for binding to ZO-1. Thus, we suggest mouse VSIG1 may play an important role in spermatogenesis rather than fertilization by forming heterophilic complex with a molecule similar to JAM family.

The perilla [Perilla frutescens (L.)], which belongs to the family Lamiaceae, have been used as a not only important traditional source of oil but also used traditional herbal medicine for treating various disease including depression, anxiety, tumor, cough, bacterial and fungal infections, allergy, detoxication and some intestinal disorders in east asian countries. In this context, luteolin isolated from the P. frutescens inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC50 of 5.0 μM. To investigate the inhibitory effect of luteolin on dioxygenase enzyme, we assayed soybean lipoxygenase-1 activity with the inhibitor. Soybean lipoxygenase-1 showed time-dependent inhibition in the presence of luteolin. Increasing luteolin concentrations led to the decrease in both the initial velocity (vi) and the steady-state rate (vs) in the progress curve. Thus luteolin showed a simple reversible noncompetitive slow-binding inhibition against soybean lipoxygenase-1 with kinetic parameter (k3 = 0.056 μM-1min-1, k4,= 0.006 μM-1min-1, Ki app = 0.106 μM).

A cDNA clone encoding a MDR-like ABC transporter protein was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (named as Brmdr 1; GenBank accession no.: DQ296184 ) had a total length of 4222 bp with an open reading frame of 3900 bp, and encoded a predicted polypeptide of 1300 amino acids with a molecular weight of 143.1 kDa. The BrMDR1 protein shared 71.0, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAN28720), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Brmdr1 was a low-copy gene. Expression pattern analysis revealed that Brmdr1 constitutively expressed in the root, stem petals and stamens, but with lower expression in leaves and open flowers. The domains analysis showed that BrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that BrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

BPES(Blepharophimosis/Ptosis/Epicanthus inversus Syndrome)는 FOXL2 유전자의 돌연변이에 의해 유발되는 상염색체 우성질환이다. 눈꺼풀이 갈라지거나 쳐지고 넓은 미간이 나타나는 특징이 있으며, 여성의 조기 난소 부전증(prema-ture ovarian failure, POF)을 일으켜 불임을 유발한다. FOXL2는 forkhead family에 속하는 전사인자로서 FOXL2가 결여된 난소에서는 granulosa cell의 분화가 진행되지 않아 난포 성숙과정의 멈춤과 난자의 폐쇄증을 유발한다. FOXL2를 bait로 하여 rat의 난소 cDNA 라이브러리의 yeast two-hybrid screening을 시행하여 FOXL2 단백질과 상호작용을 하는 small ubiquitin-related modifier(SUMO)-conjugating E2 효소인 UBE2I 단백질을 찾았다. UBC9이라고도 알려진 UBE2I 단백질은 SUMO 변형 과정을 위한 필수적인 단백질이다. Sumoylation은 수 많은 전사인자의 전사능력의 조절을 포함하여 다양한 신호전달체계에 관여하는 번역 후 변형과정이다. 본 연구에서 인간세포인 293T 내에서 면역침전반응 실험을 통해 FOXL2와 UBE2I의 단백질-단백질간의 상호작용을 확인하고, FOXL2의 돌연변이형을 제작하여 yeast two-hybrid system을 이용해 UBE2I와 결합에 필요한 FOXL2의 부분을 규명하였다. 따라서, FOX2에 상호작용하는 UBE2I의 규명은 sumoylation에 의한 FOXL2의 새로운 조절 메커니즘을 시사한다.

Sequential passes through columns were used to select phages that displays ligands for dextran (-1,6 linked linear chains) from a phage antibody library. Those phages that bound to the in each iteration were replicated in E. coli. A phage preparation isolated on the third round selection produced 5.4 nephelos turbidity units (NTU) in a dextran specific immunonephelometric assay, a 2.2 fold higher value than the phage preparation from the first round selection. This phage gave normalized intensity (N.I.) in a dip-stick assay against high molecular size dextran (T2000, 30\;{\pm}\;610^4\beta-galactosidase linked assay and polymerase chain reaction.

본 연구에서는 보통포틀랜드시멘트(OPC: ordinary Portland cement), 플라이애쉬(PFA: pulverised fly ash), 고로슬래그미분말(GGBFS: ground granulated blast furnace slag), 실리카퓸(SF: Silica fume)등의 각종 결합재를 적용한 시멘트 페이스트의 염소이온 고정화능력에 관하여 연구하였다. 각각의 사용 시멘트 페이스트는 40%의 물/결합재로 PFA, GGBFS 및 SF 혼화제의 각기 다른 치환률을 갖도록 하였으며 미리 혼합수내에 결합재 중량당 0.1～0.3%의 염소이온을 배합수내에 혼입 포함시켜 배합되어 제조되었다. 염소이온의 측정은 7일간 양생 후 수분 추출 방법을 이용하여 측정하였다. 실험을 통해 염소이온 고정화 능력이 결합재 종류 및 치환률에 의존하고 있음을 확인하였고, 총 염소이온량의 증가는 염소이온 고정화능력을 제한하여 결론적으로 염소이온 고정화를 감소시키고 있음을 보였다. 본 연구에서 최대 30%의 치환율을 가진 PFA와 60%의 치환률을 가진 GGBFS의 경우는 OPC보다 염소이온고정화 능력이 작았으며, SF의 치환률의 증가는 고정화를 감소시키고 있음을 확인하였으며, 이는 포졸란계 재료의 잠재 수화반응 혹은 공극수의 pH 저하등의 이유로 판단된다. 재령 7일에서의 염소이온의 고정화능력은 염해부식에 대한 저항성으로 나타내어지며, 염분을 혼입한 경우의 고정화능력의 순서는 30%PFA > 10%SF > 60%GGBFS > OPC로 나타났다. 더욱이 염소이온의 고정화 거동은 Langmuir isotherm 및 Freundlich isotherm으로 잘 표현될 수 있음을 보였다.