• 대한의생명과학회지
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• 제2권2호
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• pp.175-185
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• 1996

모든 진핵세포에 존재하며 세포의 성장 및 분화에 주로 관계되는 신호전달물질의 하나인 Mitogen-activated protein(MAP)kinase의 mitogen에 의한 핵내 활성화와 기질 인산화에 대해 알아보기 위해 본 실험을 수행하였다. P388세포를 10% fetal bovine serum이 첨가된 DMEM배지에 배양한 후, 혈청이 들어있지 않은 배지에서 24시간 더 배양하고 serum 및 PMA를 농도별로 처리하여 세포성 장을 위한 최적 농도를 확인한 결과 serum은 5-20% 농도에서 세포성장을 촉진시켰고 PMA는 실험한 모든 농도에서 세포성장을 거의 촉진시키지 못하는 경향을 확인하였다. 이어 P388 세포를 serum 및 PMA로 10 분간 활성화하여 파쇄한후 세포질분획과 핵분획으로 분리하여 각 분획을 10% gel 상에서 전기영동 하여 nitrocellulose paper에 옳긴 후 anti-ERKI antibody를 이용해 확인해본 결과 serum, PMA로 처리된 세포 모두에서 MAP kinase의 핵내 이동이 관찰되었으며 특히 세포질 내에 주로 존재하는 42, 44 Kd의 MAP kinase isoform중 42 Kd의 isoform이 주로 핵내로 이동되는 것이 관찰되었다. MAP kinase의 기질인산화 실험을 위해 serum으로 활성화시킨 세포를 파쇄하여 SP-sephadex C-50, Phenyl superose, Mono Q column의 순서로 chromatography를 시 행하여 MAP kinase를 부분분리 하였다. 이와 같이 얻은 MAP kinase를 가지고 면역 T세포에 존재하는 tyrosine kinase인 $p56^{lck}$ 의 N-terminal peptide로 구성된 GST-fusion protein에 대한 인산화를 확인하였다. 또한 세포에서 분리한 MAP kinase를 가지고 transcription factor의 하나인 c-Jun protein에 대한 인산화실험을 실시한 결과 MAP kinase에 의해 인산화 됨이 확인되었다. 이상의 결과를 통해 P388세포는 (1)세포 성장시 외부 신호를 G-protein-coupled receptor/protein kinase C/MAP kinase의 경로보다는 주로 tyrosine kinase receptor protein/Ras/MAP kinase의 경로를 이용하여 핵으로 전달하는 것으로 추측되 며 (2) mitogen의 처리로 활성화된 MAP kinase중 주로 42 Kd isoform이 핵내로 이동하고, 분리한 MAP kinase가 GST-fusion protein과 transcription factor인 c-Jun을 모두 인산화 시키는 결과로 보아 MAP kinase의 isoform에 따라 표적 compartment가 다르고 결과적으로 표적 기질에 차이가 있을지 모른다고 간접적으로 추론할 수 있다.

• Molecules and Cells
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• 제40권12호
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• pp.954-965
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• 2017

Mammalian genomes are well established, and highly conserved regions within odorant receptors that are unique from other G-protein coupled receptors have been identified. Numerous functional studies have focused on specific conserved amino acids motifs; however, not all conserved motifs have been sufficiently characterized. Here, we identified a highly conserved 18 amino acid sequence motif within transmembrane domain seven (CAS-TM7) which was identified by aligning odorant receptor sequences. Next, we investigated the expression pattern and distribution of this conserved amino acid motif among a broad range of odorant receptors. To examine the localization of odorant receptor proteins, we used a sequence-specific peptide antibody against CAS-TM7 which is specific to odorant receptors across species. The specificity of this peptide antibody in recognizing odorant receptors has been confirmed in a heterologous in vitro system and a rat-based in vivo system. The CAS-TM7 odorant receptors localized with distinct patterns at each region of the olfactory epithelium; septum, endoturbinate and ectoturbinate. To our great interests, we found that the CAS-TM7 odorant receptors are primarily localized to the dorsal region of the olfactory bulb, coinciding with olfactory epithelium-based patterns. Also, these odorant receptors were ectopically expressed in the various non-olfactory tissues in an evolutionary constrained manner between human and rats. This study has characterized the expression patterns of odorant receptors containing particular amino acid motif in transmembrane domain 7, and which led to an intriguing possibility that the conserved motif of odorant receptors can play critical roles in other physiological functions as well as olfaction.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.98-98
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• 2003

${\beta}$-(1,3)-D-Glucans have been known to exhibit antitumor and antimicrobial activities. The presence of dectin-1,${\alpha}$, ${\beta}$-glucan receptor of dendritic cell, on macrophage has been controvertial. RT-PCR analysis led to the detection of dectin-1${\alpha}$ and ${\beta}$ in murine macrophage Raw264.7 cell line. Among the various organs of mouse, dectin-1${\alpha}$ and ${\beta}$ were detected in the thymus, lung, spleen, stomach and intestine. To analyze gene expression modulated by ${\beta}$-glucan treated murine Raw264.7 macrophage, total mRNA was applied to cDNA microarray to interrogate the expression of 7,000 known genes. cDNA chip analysis showed that ${\beta}$-glucan of P. osteatus increased gene expressions of immunomodulating genes, membrane antigenic proteins, chemokine ligands, complements, cytokines, various kinases, lectin associated genes and oncogenes in Raw 264.7 cell line. When treated with ${\beta}$-glucan of P. osteatus and LPS, induction of gene expression of TNF-${\alpha}$ and IFN-R1 was confirmed by RT-PCR analysis. Induction of TNF-R type II expression was confirmed by FACS analysis. IL-6 expression was abolished by EDTA in ${\beta}$-glucan and LPS treated Raw264.7 cell line, indicating that ${\beta}$-glucan binds to dectin-l in a Ca$\^$＋＋/ -dependent manner. To increase antitumor efficacy of ${\beta}$-glucan, ginsenoside Rh2 (GRh2) was co-treated with ${\beta}$-glucan in vivo and in vitro tests. IC$\sub$50/ values of GRh2 were 20 and 25 $\mu\textrm{g}$/$m\ell$ in SNU-1 and B16 melanoma F10 cell line, respectively. Co-treatment with ${\beta}$-glucan and GRh2 showed synergistic antitumor activity with cisplatin and mitomycin C both in vitro and in vivo. Single or co-treatment with ${\beta}$-glucan and GRh2 increased tumor bearing mouse life span. Co-treatment with ${\beta}$-glucan and GRh2 showed more increased life span with mitomycin C than that with cisplatin. Antitumor activities were 67% and 72 % by co-injection with ${\beta}$-glucan and GRh2 in the absence or presence of mitomycin C, respectively.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.135-141
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• 1997

The early studies demonstrated that the relative amount of FSH was important for stimulating normal ovarian activity and demonstrated the existence of a threshold level for FSH, above which follicular growth was activated. It was found that only a modest increase in circulating FSH level above the threshold (between 10 and 30%) was required to stimulate folliculogenesis. In addition, FSH is primary responsible for initiating estradiol production through the activation of the aromatase enzyme system in granulosa cells, follicular secretion and growth. LH on the other hand, plays a supportive role in ovarian steroidogenesis, stimulating the ovarian thecal cells to produce androgen, the precursor for estradiol synthesis. But there is now an increasing number of reports in the literature demonstrating an adverse effect of LH on fertility and miscarriage in infertile and fertile women. So HP-FSH is the drug of a highly purified FSH preparation which has a higher specific activity and far fewer impurities than FSH. This study was performed to evaluate the efficacy and safety of HP-FSH administered (SC; subcutaneous) versus FSH(IM; intramuscular) for ovulation induction. 20 candidates patients for ovulation induction were participated. All patients underwent pituitary desensitizing with a long gonadotropin-releasing hormone (GnRH) agonist protocol and ovulation induction was started with HP-FSH SC (10 patients; group I) or FSH IM (10 patients; group II). After ovulation, outcome of ovulation induction and local reaction of injection site were compared. There were no difference of outcome of ovulation in two groups except pregnancy rate/embryo transfer. Group I had a higher pregnancy rate/ embryo transfer than Group II (44.4% Vs 28.6%). Pain, redness, tenderness, bruising and itching when the injection received on the first 5 days of treated (50 SC and 50 IM injections) were assessed. There were no significant difference (P>0.05) in the incidence of tenderness, bruising and itching between the IM and SC injection. But IM injection (FSH) had a tendency of higher above incidence. The number of reports of pain, redness were significantly increased in IM injection group (P<0.05). These results indicate that SC administration of HP-FSH has been shown to be as effect for superovulation as traditional gonadotropins, with an improved safety profile due to the removal of extaneous proteins.

• Asian-Australasian Journal of Animal Sciences
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• 제8권5호
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• pp.463-470
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• 1995

An experiment was conducted to evaluate the effects of chromium picolinate on growth performance, nutrient utilizability, carcass composition, serum traits and in vitro protein synthesis of 3 day old Arbor Acres broiler chickens when dietary crude protein levels were varying in diets. Six replicates of eight chicks each (average initial weitht = 59.4 g) were randomly assigned to three levels (low, medium, high) of dietary crude protein at two levels of chromium (0, 200 ppb Cr/kg diet) as chromium picolinate. Six chicks/treatment were randomly chosen for analyses of carcass composition, six additional chicks/treatment were randomly chosen for analyses of serum components, and a chick/treatment was chosen for in vitro culture of liver tissue. Chromium picolinate did not affect feed intake, protein and fat utilizability, regradless of dietary crude protein level. But feed/gain ratio were more improved in groups fed the low protein diets added with chromium picolinate compared with groups fed the medium and high protein diets with chromium picolinate. Carcass fat tended to decrease whereas carcass protein tended to increase when added with chromium picolinate. Broilers fed diets with chromium picolinate exhibited lower serum triglyceride and nonesterified fatty acid concentrations than those fed without chromium picolinate (p < 0.05). Both secreted and retained proteins in cultured acinar cell were higher in groups fed diets with chromium picolinate than those fed diets without chromium picolinate (p < 0.05). It could be suggested that chromium picolinate was effective in improving weight gain and nutrient utilizability when dietary crude protein was low (p < 0.05), and also effective in manipulating carcass fat when dietary crude protein level was high (p < 0.05).

• Archives of Pharmacal Research
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• 제29권3호
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• pp.224-234
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• 2006

We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.

• 한국동물위생학회지
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• 제42권4호
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• pp.257-264
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• 2019

Mastoparan V1 was used as adjuvant of formalin-inactivated Salmonella Gallinarum whole cells vaccine against fowl typhoid in a chicken model. The 75 brown nick chickens were equally divided into 5 groups, and all chickens of each group were immunized at 6 weeks of age (0 WPPI; weeks prime post immunization), and at 9 weeks of age (3 WPPI) (except group B). Group A chickens were intramuscularly (IM) inoculated with 500 uL of sterile phosphate-buffered saline (PBS), and group B chickens were subcutaneously immunized with 0.2 ml containing 5×10⁷ viable vaccine strain/bird. The chickens in groups C~E were IM inoculated with approximately 3×10⁹ cells/0.5 mL of formalin-inactivated the S. Gallinarum whole cells, approximately 3×10⁹ cells/0.5 mL of formalin-inactivated the S. Gallinarum whole cells with mastoparan V1 as adjuvant, and 0.5 mL of PBS, respectively. S. Gallinarum outer membrane proteins-specific serum IgG titers were considerably higher in groups B~D than in groups A and E. However, the levels of IFN-γ in groups B and D only than in groups A and E were significantly higher. Following oral challenge with virulent wild-type S. Gallinarum, no chicken in groups A (no challenge group) and B was dead, and only 30% of chickens in group D was dead. However, 70% of chickens in group C and all chickens in group E were dead after oral challenge. The results of this study demonstrated that IM immunization with approximately 3×10⁹ of the formalin-inactivated S. Gallinarum whole cells containing mastoparan V1 induced robust antibody and cell-mediated immune responses in chickens. The whole cells also conferred protection against infection with wild-type S. Gallinarum.

• 한국식품영양과학회지
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• 제39권2호
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• pp.203-209
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• 2010

본 연구를 통하여 여주 추출물은 세포 증식을 제외하고는 조골세포에 긍정적인 영향을 미치지 못하였지만 머위 추출물은 세포의 증식, ALP 활성, bone nodule의 형성이 대조군과 비슷한 결과를 나타내거나 높은 경향을 나타냄으로써 조골세포의 골 형성 과정인 증식, 기질의 성숙, 기질의 석회화의 세 단계에서 유효성을 증명하였다. 또한 OPG mRNA의 2배 이상의 증가는 조골세포의 골 형성에 주요 매개 물질로서 가능성이 있음을 밝혔다. 따라서 머위 추출물은 골수의 미세 환경에서 세포의 조절작용을 하는 물질로 여겨지며, 골다공증을 포함한 각종 골 결손 질환의 예방과 치료약 개발에 긍정적인 가능성을 제시할 것이라 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제26권5호
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• pp.654-660
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• 2013

Three Korean native steers ($779{\pm}24$ kg) fitted with duodenal cannulas were used in a $3{\times}3$ Latin square design to investigate the influence of oral administration of soluble proteins, intact casein (IC) and acid hydrolyzed casein (AHC), on gastro-intestinal hormone (GIH) secretion in the blood and pancreatic ${\alpha}$-amylase activity in the duodenum. Oral treatment consisted of a basic diet (control), IC (C+100% protein), or AHC (C+80% amino acid, 20% peptide) for 21 d. Blood and duodenum samples were collected for measurement of serum GI hormones, and pancreatic ${\alpha}$-amylase activity was determined at 900, 1030, 1330, 1630, and 1930 h after feeding on d 21 of treatment. The levels of serum cholecystokinin (CCK) and secretin in the IC treatment group were higher compared to the other treatment groups (p<0.05). In addition to the changes in CCK and secretin levels upon IC treatment, the pancreatic ${\alpha}$-amylase activity in the duodenum was higher in the IC group compared to the control diet group (p<0.05). The response of serum ghrelin to IC and AHC treatment was in accordance with the response of serum secretin. The level of peptide fragments flowing in the duodenum was higher in the IC treatment group than the other treatment groups (p<0.05). In conclusion, this study demonstrated that an increase in duodenal CCK and secretin upon IC oral administration increased pancreatic ${\alpha}$-amylase secretion. In addition, ghrelin may be associated with GI hormone secretion in Korean native steers.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.174-183
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• 2001

During the rapid phase of gonadal development of the freshwater teleost, the catfish (Silurus asotus), the influence of hCG upon the inducement of final oocyte maturation and spawning was investigated electrophoretically and ultrastructurally. The electrophoretic patterns obtained were different in the presence and absence of some of the major or minor zones, because of the hormone level in catfish. The vitellogenin of hormone-treated fish was stained more intensively than that of sham-treated fish. These proteins showed some minor or main bands of egg extracts which migrated at positions corresponding to molecular weights of approximately 90,000. However, the thickness of electrophoretic band in molecular weight for hCG-treated fish was slightly lower than that for saline control. It seemed the plasma protein with molecular weight of approximately 45,000 in hCG-treated fish disappeared. In contrast to the control fish, the ovaries in the catfish treated with hCG shows a marked ultrastructural change under the electron microscope. No dilated profiles were seen in the granulosa cells of the mature oocyte before ovulation. After germinal vesicle breakdown (GVBD), the zona radiata interna (ZRI) becomes more compact, and there is a loss of all the processes from the pore canals. There is a wide space between the vitelline membrane and zona radiata. Also, during final maturation, the microvillar processes from the oocyte are seen no longer to penetrate deeply into the extracellular spaces of the overlying granulosa cells, and the reticulate patterns of the zona radiata interna becomes occluded, giving the zona radiata a more solid appearance. It has been possible to initiate 100% oocyte maturation in yolk granules and follicles in vivo by treatment with hCG and a high water temperature ($27^{\circ}C$). In hCG-treated fish, the percentages of successful artificial fertilization and hatching were maximal at 15 h after a single injection. It seems clear that a long acting preparation containing hCG can be successfully used in prespawning fish to advance the final events of gonadal maturation and initiate spawning. Further studies are necessary to evaluate the potential of hCG to either stimulate or inhibit the reproductive development of fish at other stages of the seasonal reproductive cycle.