Coffee is one of the most familiar beverages to modern human adults, but its bio-physiological effect has not been clearly elucidated. It was known that more than one thousand chemicals were included in the ordinary coffee extract. Among them, the caffein and chlorogenic acid (caffeoylquinic acids) are most abundant and have been investigated by many authors so far. In order to know the real cellular effect of whole coffee extract elements, the dialyzed coffee extract (DCE)1) was made to get coffee elements less than 1000 Da molecular weight, which are freely absorable through gastrointestinal tract. It was directly treated in the culture of RAW 264.7 cells, a murine macrophage lineage. RAW 264.7 cells were treated with DCE equivalent to 2.5 cups of coffee (DCE-2.5), DCE-5, and DCE-10 for 12 hours, and their protein extracts were examined by histological observation and immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the proliferation-related proteins depending on the dose of DCE. DCE-2.5 and DCE-5 enhanced the cellular growth of RAW 264.7 cells by increasing the expression of β-actin, PCNA, Ki-67, MPM2, MAX, cMyc, E2F-1, and Rb-1, and by decreasing the expression of MAD and p21. These proliferation-related proteins were rarely affected by DCE-10. DCE-2.5 and DCE-5 induced the cellular proliferation of RAW 264.7 cells by the signaling of E2F-1 and cMyc, respectively, but these cellular effects almost disappeared in DCE-10. Therefore, it was presumed that the low dose of coffee, DCE-2.5 and DCE-5 might be effective for the proliferation of murine macrophages, RAW264.7 cells, contrast to the high dose of coffee, DCE-10. It was also suggested that the low dose of DCE-2.5 and DCE-5 be helpful to increase the innate immunity in vivo by increasing the cell number of macrophages in contrast to the high dose of DCE-10.

Recent studies suggested that gut symbionts modulate insect development and reproduction. However, how gut symbionts modulate host physiologies and what types of molecules are involved in these changes are still unclear. When we analyzed hemolymph proteins and transcriptional levels of host insects, hexamerin-α (Hex-α), hexamerin-β (Hex-β) and vitellogenin-1 (Vg-1) were highly expressed in symbiotic insects (Sym) compared to aposymbiotic insects (Apo). Depletion of Hex-β by RNA interference in 2nd Sym-nymphs delayed adult emergence, whereas Hex-α and Vg-1 RNA interference in 5th nymphs decreased reproduction of female insects and caused loss of color of laid eggs. Also, the levels of JHSBIII of Riptortus host were 3-fold higher in the Sym-female insects compared to the Apo-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production through regulating the expression of three host storage proteins by controlling of brain hormone.

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation.To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and 1 μg/ml) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except 1 μg/ml (p<0.001).Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or 0.01 μg/ml AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and 0.1 μg/ml AFP (p<0.05), but increased with 1 μg/ml AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

본 연구에서는 돈지육 및 돈육 조직 내에 열안정성 수용성 단백질의 존재 여부를 확인하고 항체 생산에 있어항원으로의 사용 가능 여부를 확인하고자 하였다. 이를 위해 돈지육 및 돈육을 생(raw) 시료와 조리된(cooked) 시료로 구분하여 비열처리 및 열처리법으로 단백질을 추출한 후 단백질 존재여부를 단백질 정량과 SDS-PAGE로 확인하였다. 그 결과 돈지육과 돈육 모두 생 시료를 비열처리법으로 추출한 시료의 경우 25~100 kDa 사이의 다양한 단백질이 확인된 반면 시료를 가열하거나 추출 시 열처리를 한경우 돈지육에는 100 kDa 이상의 단백질과 30 kDa 및 15 kDa 이하의 일부 단백질이, 돈육에는 100 kDa 이상과 30 kDa 이하의 단백질이 확인되어 돈지육과 돈육에 열안정성 수용성 단백질이 존재하는 것으로 확인되었다. 이들 열안정성 수용성 단백질을 마우스에 면역 후 항혈청 역가를 측정한 결과 면역한 모든 마우스에서 높은 역가를 나타내었고, 생산된 혈청은 돈지육과 돈육에 각각 특이적인 반응성을 보인 반면 다른 축육과 지방육에 대해서는 반응성이 상대적으로 낮았다. 이러한 연구결과를 볼 때 돈지육 및 돈육에 존재하는 열안정성 수용성 단백질이 돈지육과 돈육에 특이적으로 반응하는 항체를 개발하는데 유용한 마커로서 활용이 가능하며, 열안정성 수용성 단백질에 대한 항체개발은 열처리된 축육 가공품 중 돈지육 및 돈육의 분석에도 매우 유용하게 활용할 수 있을 것으로 판단된다.

현재의 단백질 분리 공정들은 비용과 시간이 많이 들고 오래 걸리는 단점을 가지고 있다. 이러한 단점을 해결하기 위해서 분리막을 이용한 방법들이 계속적으로 연구되고 있다. 먼저, 단백질 분리공정에는 단백질 크기에 따른 분리공정 과 pH에 따른 단백질 표면의 전하 차이를 이용한 분리 공정이 있다. 그 중에서도 본 연구는 유사한 크기의 단백질을 분리하기 위해 폴리술폰을 개질하여 전하를 부여하였으며, 상전환법을 이용하여 분리막을 제조하였고 제조된 분리막 표면의 Zeta potential을 측정하여 pH에 따른 표면의 전위차를 확인하였다. FT-IR 과 1HNMR을 이용하여 화학구조를 분석하였으며, UV Spectrometer를 이용하여 단백질의 농도를 측정하였다.

Early pregnancy results in th production of various signal molecules such as steroids, prostaglandins, and many protein factors. The proteins especially produced by the placenta have been used to detect pregnancy for many years in other species. More recently, pregnancy-specific protein B, which is a placental glycoprotein can be measured by RIA or proteomic methods in serum of pregnant cow. And 2D Fluorescence difference gel electrophoresis (DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. For this reason, we are analyzed serum of bovine. The purpose of this study was to apply DIGE technique for identification of bovine pregnancy-specific proteins using bovine pregnant and non-pregnant serum samples. Serums of 2 pregnant Holstein dairy cattle at day 21 after AI and those of 2 non-pregnant were used in this study. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labeling of non-pregnancy and pregnant serum proteins which are then mixed and labeled with Cy2 were used as an internal standard. Two pools of proteins are labeled with Cy3 and Cy5 fluorescent dyes, respectively. Labeled proteins with Cy2, Cy3 and Cy5 mixed together and separated in same gel and then were detected by fluorescence image analyzer. The 2D DIGE analysis using fluorescence CyDye flour showed higher sensitivity and better reproducible results than conventional 2D gel electrophoresis. Approximately 1,500 protein spots were detected by 2D DIGE. The differentially expressed proteins were identified by MALDI-TOF Mass spectrometer. Total 16 protein spots differentially expressed in the pregnant serum were detected, among which 7 spots were up-regulated proteins identified as conglutinin precursor, modified bovine fibrinogen, IgG1 etc, and 6 spots were down-regulated proteins identified as hemoglobin, complement component 3, bovine fibrinogen, IgG2a etc. These results indicated that DIGE system could be advantageous for the analysis of serum proteomics diversified by physiological conditions.

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. We found that actin nucleation promoting factor called WASP homolog-associated protein with actin, membranes and microtubules (WHAMM) play crucial roles in mouse oocyte maturation by generation of ER-associated actin filaments during meiotic spindle migrations. We also investigate regulatory mechanism of actin nucleator spire and discovered the novel roles of Zinc in regulating spire localization and cytoplasmic actin mesh formation. Another class of actin-binding proteins including cofilin, tropomyosin, capping proteins and tropomodulin, are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. The heterodimeric actin-capping protein (CP) binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. When CP is knockdowned or inhibitory component was overexpressed, asymmetric divisions of oocytes have been compromised. It turns out that knockdown or inhibition of CP deplete cytoplasmic actin mesh level, which have been known to be essential for maintain cytoplasmic actin mesh. Another actin binding proteins, tropomodulin 3 (Tmod3), binds to the slow-growing end of actin filaments and knockdown or expression deletion mutant of Tmod3 also decrease actin mesh level in maturing oocyte and it severely ablated asymmetric division of oocyte. Finally, tropomyosin 3, actin filament binding proteins protect actin filament from depolymerization, is also important to maintain cortex integrity in maturing oocyte, therefore showed the importance maintenance of actin filaments during oocyte maturation. Taken together, our study on various actin nucleator and actin binding study showed the importance of actin dynamics in mammalian oocyte maturation and early embryonic developments.

The Y chromosome is a type of sex chromosome existing primarily in male mammalian species. The Y chromosome passes through the male gamete and determines male sex in humans, non-human primates, and other mammals. The mammalian Y chromosome varies from the X chromosome and the rest of the chromosomes primarily by size and its male sex-determining/spermatogenesis function. In the Y chromosome, male sex-determining function is exclusively located on the short arm, while the spermatogenesis function is distributed widely on the short and long arm. Deletions or mutations particularly in the male-specific region of Y chromosome (MSY) may cause male infertility. During the last few decades, researchers put forth an enormous effort to discover Y chromosome specific genes, and their encoded RNAs and proteins in humans, primates, and rodents. As a result, most of the genes and encoded proteins responsible for male-sex determination, testis development, and spermatogenesis have been discovered in humans, however not well established in non-human primates and rodents. Also, there might be a percent of proteins missing in human Y chromosome. The aim of this study is to annotate the proteins that encoded on the Y chromosome of humans, chimpanzee, and mouse using extensive bioinformatics tools. The human (annotation release 107), chimpanzee (annotation release 103), and mouse (annotation release 105) proteins were first retrieved from the National Center for Biotechnology Information (NCBI) eukaryotic genome annotation resource database. Then, the annotated human proteins (66 proteins) were compared with the core databases of human proteome project such as neXtProt, PeptideAtlas, and the Human Protein Atlas. The X-homologous of human Y chromosome-encoded proteins were searched using the NCBI Protein BLAST program. The cellular pathways and protein-protein interactions involving human Y chromosome-encoded proteins were searched using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping database, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the Pathway Studio software. Finally, the human Y chromosome-encoded protein homologs/orthologs in chimpanzee and mouse were analyzed using the NCBI bl2seq program. This analysis resulted a significant number of homologous/orthologous proteins between human, chimpanzee and mouse. Our findings provide the scientific community with updated information on the Y chromosome-encoded proteins in humans, chimpanzee, and mouse.

Dental pulp is a highly vascularized tissue with high regenerative potential. Revascularization of severed vasculature in the tooth is required for pulp healing during avulsed tooth treatment. In this study, the relative expression of angiogenesis-related proteins was determined in human dental pulp cells using a human angiogenesis proteome profiler array. The proteome profiler array detected differentially expressed angiogenesis-related factors under conditions of hypoxia, which enhances the angiogenic potential of dental pulp cells. We confirmed that hypoxia regulates the mRNA expression of angiogenesis-related factors, including CXCL16 in dental pulp cells. Furthermore, conditioned media of hypoxic pulp cells induced tube-like structures of vascular endothelial cells, which were reduced by the neutralization of CXCL16 function. In conclusion, our data show that angiogenesisrelated factors are differentially expressed by hypoxia in dental pulp cells and suggest that CXCL16 may involve in the revascularization of hypoxic dental pulp.

현재 수준의 단백질 분리는 공정비용이 많이 들고 시간이 오래 걸린다는 단점이 있다. 이러한 단점들을 해결하기 위해서 공정이 비교적 간단하고 친환경적인 분리막을 이용한 방법들이 연구되고 있다. 먼저, 단백질 분리공정을 두 가지 정도로 나눌 수 있고, 이는 단백질 크기에 따른 분리공정과 pH에 따른 단백질 표면의 전하 차이를 이용한 분리 공정을 들 수 있다. 본 연구는 폴리술폰의 표 면전하를 개질하여서 유사한 크기의 단백질을 분리하고자 하였으며, 용매를 이 용한 고분자용액을 만들고 비용매에 침전하여 분리막을 제조하는 상변환법을 이용하여 제조하였고, 제조된 분리막 표면의 Zeta potential을 측정하여 pH에 따른 표면의 전위차를 확인하였다. 폴리술폰의 표면개질을 확인하고자 FT-IR 과 1HNMR을 이용하여 화학구조를 분석하였으며, UV Spectrometer를 이용하여 단 백질의 농도를 측정하였다.

연산오계는 오래전부터 건강기능 증진 및 치료 효능이 높은 것으로 알려져 왔다. 최근 건강 기능식품 소재로 기능성 펩타이드 효능이 알려짐에 따라, 연산오계 다리육으로부 올리고 펩타이드 최적 생산 공정 및 생성물 특성에 대하여 연구를 수행하였다. 최적 효소가수 분해 공정 표면반응 분석을 이 용하여 수행하였다. 최적 공정 조건을 확립하기 위해서 온도 (40, 50, 60℃), pH (pH 6.0, 7.0, 8.0), 효 소 (1, 2, 3%) 범위에서 수행을 하였다. 생성물에 대한 가수분해도, 유리아미노산, 분자량 분포를 분석 하였다. 효소 가수분해 최적 온도는 58℃, pH 7.5, 효소의 농도는 3% 이었다. 최적 조건에서 2 시간 효소 가수분해를 한 결과 75-80% 이었다. 유리 아미노산 총량은 168.131 mg/100 g 이었다. 분자량를 MALDI-TOF 으로 분석을 한 결과 90% 이상이 300-1,000 Da 분포를 보여주었다.

The bacterial lipopolysaccharide (LPS) mainly contributes to the structural integrity, survival and protection barrier against harsh environments. Therefore, the early stages in LPS or lipid A biosynthesis are attractive targets in the identification and development of inhibitors which would be effective against infections caused by Gram-negative bacteria. The bacterial outer membrane proteins (OMPs) meanwhile function as maintenance for structure, adhesion to other cells and substances, as well as development of resistance to antimicrobials. The LPS and LPS-related molecules, and OMPs are important immunogenic components of several important pathogens including Brucella, which have been extensively used in immunological studies and in the diagnosis of diseases. Here we review the importance, structure, functions and immunogenic aspects of LPS and OMPs particularly of Brucella which can be targeted for the prevention and diagnosis of brucellosis.

현재 사용되는 단백질 분리공정은 비용이 많이 들고 시간이 오래 걸리는 단점을 가지고 있다. 이러한 문제점을 보완하기 위해서 분리막을 이용한 분리 공정들이 계속적으로 연구되고 있으며, 단백질의 크기에 따른 분리공정과 pH에 따른 단백질 표면의 전하 차이를 이용한 분리공정이 있다. 본 연구에서는 유사한 크기의 단백질을 분리하기 위해 폴리술폰을 개질하여 전하를 부여하였으며, 상전환법을 이용하여 분리막을 제조하였다. 분리막 표면의 Zeta potential을 측정하여 pH에 따른 표면의 전위차를 확인하였다. FT-IR과 1HNMR을 이용하여 화학구조를 분석하였으며, UV Spectrometer를 이용하여 단백질의 농도를 측정하였다.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs with high mortality. PEDV is belong to Coronavirus, enveloped, single-stranded, positive-sense RNA virus. PEDV particles were composed of four structure proteins such as a glycosylated peplomer (spike, S) protein, envelope (E), glycosylated membrane (M) protein, and unglycosylated RNA-binding nucleocapsid (N) protein. Many of previous studies talk about this four structure proteins have a great potential to diagnosis and prevent PEDV. In this study we investigated expression of these structure proteins using the bacterial and baculovirus expression system. In bacterial expression system, our results showed that structure proteins fused polyhedrin and intein gene were expressed higher than non-fusion structure proteins. The expressed fusion proteins were used to immune mice for generating a polyclonal antibodies. In baculovirus expression system, co-infection of insect cells with these four recombinant baculoviruses led to self-assembly of virus-like particles as demonstrated by Transmission electron microscopy. They were confirmed by western blot analysis using pre-made polyclonal antibodies. Finding in this study may provide important information for vaccine and diagnostic development.

Lonicera japonica 에탄올 추출물의 처리에 의하여 LPS에 의해서 활성화된 RAW 264.7 세포에서 NO의 생성량과 TNF-α, IL-1β 및 IL-6와 같은 염증성 사이토카인의 분비를 억제하였고, MAPK family인 ERK, p38 및 JNK의 인산화와 Iκ-Bα의 분해를 억제하였다. LPS로 유도한 endotoxin shock 동물실험에서 Lonicera japonica 에탄올 추출물 20 mg/kg에서 LPS로 유도한 endotoxin shock에 대한 생존율을 3배 이상 증가시켰으며, 생존시간도 1.3~1.4배 증가시켰다.

현재 인간이 일반적으로 이용하는 의료용 단백질은 대개 동물세포 배양기술에 의하여 생산되고 있다. 그러나 제한된 특정 시설에서 생산되기 때문에 생산 단가가 높아서 전세계의 수요가 증가함에도 불구하고 일반화하는 데는 한계가 있는 실정이다. 이러한 문제를 극복하기 위하여 오래전부터 식물시스템을 이용하여 의료용 단백질을 생산하는 연구가 관심을 끌고 있다. 즉 쉽게 규모화할 수 있고 생산비용 효과도 경제적이고 동물세포보다 더 안전하게 의료용 단백질 생산할 수 있는 수단이 될 수 있기 때문이다. 이를 위하여 식물에 안정적으로 유전자를 핵 게놈과 엽록체 게놈에 형질전환 시키는 기술과 바이러스 운반체를 이용하여 일시적 발현을 유도함으로 의료용 단백질을 생산하는 기술이 개발되고 있다. 최근 해체 바이러스 기반의 운반체 개발은 재조합 단백질을 빠르고 일시적 발현으로 대량생산을 가능하게 하고 있다. 따라서 이 바이러스 발현시스템이 적절한 식물기반의 대량생산을 위한 플렛폼을 제공하고 있다. 따라서 본 총설은 식물로부터 의료용 단백질을 생산하는데 있어서 식물 바이러스발현 시스템 개발 및 이용에 대하여 기술하였다.

단백질을 분리하는데 있어 현재 사용되는 공정들은 비용이 많이 들고 시간이 오래 걸리는 단점을 가지고 있다. 이러한 문제점을 보완하기 위해서 분리막을 이용한 분리 공정들이 계속적으로 연구되고 있으며, 단백질의 크기에 따른 분리공정과 pH에 따른 단백질 표면의 전하 차이를 이용한 분리 공정이 있다. 본 연구에서는 유사한 크기의 단백질을 분리하기 위해 폴리술폰을 개질하여 전하를 부여하였으며, 상전환법을 이용하여 분리막을 제조하였다. 분리막 표면의 Zeta potential을 측정하여 pH에 따른 표면의 전위차를 확인하였다. FT-IR 과 1HNMR을 이용하여 화학구조를 분석하였으며, UV Spectrometer를 이용하여 단백질의 농도를 측정하였다.

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 μM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 μM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

Cows may suffer impaired ovarian function, often accompanied by reduced conception rates and increased embryonic loss. Cystic ovarian disease (COD) is one of the most frequently diagnosed gynecological findings in dairy cattle. It causes temporary infertility and is likely to affect reproduction as well as production parameters in cattle. Therefore, the purpose of this study was to determine the expression patterns of apoptosis (Bcl-2, Bax), implantation (E-cadherin) and immune related proteins (TNF-α, IL-10) in uterine endometrium of Hanwoo (Korean native cattle) with ovarian cyst and normal ovarian follicles. In the Western blot analysis, the expression of anti-apoptotic Bcl-2 protein was significantly higher in endometrium with normal ovarian follicles, whereas expression of pro-apoptotic Bax protein was significantly lower. Also, the expressions of E-cadherin and TNF-α proteins were significantly higher in uterine endometrium with normal ovarian follicles. On the other hand, the expression of IL-10 protein was significantly lower in uterine endometrium with normal ovarian follicles. Taken together, our results provided that the expressions of apoptosis, adhesion and immune related proteins in uterine endometrium with ovarian cyst were showed the aberrant patterns, and we suggest that different expression changes of these proteins may be affect to pregnancy ability of cattle.