• Title, Summary, Keyword: Far-Western blotting

Search Result 9, Processing Time 0.036 seconds

Elevated Aurora Kinase A Protein Expression in Diabetic Skin Tissue

  • Cho, Moon Kyun;An, Je Min;Kim, Chul Han;Kang, Sang Gue
    • Archives of Plastic Surgery
    • /
    • v.41 no.1
    • /
    • pp.35-39
    • /
    • 2014
  • Background Aurora kinase A (Aurora-A) plays an important role in the regulation of mitosis and cytokinesis. Dysregulated Aurora-A leads to mitotic faults and results in pathological conditions. No studies on Aurora-A expression in human diabetic skin tissue have been reported. In light of this, we explored the expression of Aurora-A in human diabetic skin tissue. Methods Aurora-A protein was evaluated by western blotting in 6 human diabetic skin tissue and 6 normal skin specimens. Results Increased expression of Aurora-A protein was detected in all diabetic skin tissue samples in both western blot analysis and immunohistochemical staining. However, in the case of the normal skin tissue, no bands of Aurora-A protein were detected in either the western blotting analysis or the immunohistochemical staining. Conclusions Thus far, there have been no studies on the expression of Aurora-A in diabetic skin tissue. However, we believe that oxidative DNA damage related to the expression of Aurora-A protein and Aurora-A could be involved inhuman diabetic skin tissue.

A NELL-1 Binding Protein: Vimentin

  • Chae, Hwa-Sung;Kim, Young-Ho
    • Journal of Korean Dental Science
    • /
    • v.4 no.1
    • /
    • pp.6-13
    • /
    • 2011
  • Purpose: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. NELL-1 is a novel molecule overexpressed during premature cranial suture closure in human CS. From a functional perspective, NELL-1 has been reported to accelerate chondrocyte maturation and modulate calvarial osteoblast differentiation and apoptosis pathways. The mechanism through which NELL-1 induces these phenomena, however, remains unclear. The purpose of this study is to identify the NELL-1 binding protein(s) through which the biologic mechanism of NELL-1 can be further investigated. Materials and Methods: Far-Western and Immunoprecipitation (IP) assays were performed, independently and in sequence, followed by mass spectrometry to identify the NELL-1 binding proteins. Reverse IP was used to verify and confirm candidate binding protein. Results: The only confirmative protein from current experimentation was vimentin. Vimentin is the major structural component of the intermediate filaments. Conclusion: The present study identified and confirmed vimentin as a NELL-1 binding protein, which opened up a new window to mechanistically facilitate studies on this CS-associated molecule.

Analysis of Pigments and Thylakoid Membrane Proteins in Photosystem I - Mutants from Synechocystis sp. PCC6803 (Synechocystis sp. PCC6803을 이용한 Photosystem I- mutants의 색소 및 틸라코이드막 단백질 분석)

  • 전은경;장남기
    • Asian Journal of Turfgrass Science
    • /
    • v.11 no.1
    • /
    • pp.45-58
    • /
    • 1997
  • Pigments and thylakoid membrane proteins were investigated in wild type and PS I- mutants from Synechocystis sp. PCC6803 Comparing morphological features, B2 was less fluorescent than the other strains. The contents of chlorophyll a were propotional to the FNR activity in thylakoid membrane. The FNR activity of mutants was lower than that of wild type. In the result of pigments analysis, mutants had smaller cholophyll a than that of wild type. The major carotenoid was found to he $\beta$-caroene, but aeaxanthin was barely detected in thylakoid membrane of mutants. The polypeptide, 14.8kD was detected by electrophoresis in mutants. It was considered to be the modification of 15.4kD in wild type. Membrane polypeptides of 17.6 and 19.7kD were not detected in mutants. In the result of western blotting, subunit I was detected in all strains, but subunit II was barely detected in mutants. Subunit II was not detected in B2 at all. In view of the results so far achieved, the changes of contents of chlorophyll and zeaxanthin were affected by the defficiency or modification of functional domain in subunit I. Also the modification in subunit I affected the subunit II- binding site in PS I. As the result, efficiency of photosynthesis was decreased. Key words: Synechoystis sp. PCC6803, PS I - mutant, Photosynthetic efficiency, Pigment,Thylakoid membrane proteins, Subunit I, II.

  • PDF

Anti-inflammatory effect of Geranium thunbergii on lipopolysaccharide-stimulated RAW 264.7 cells

  • Kwon, Tae-Hyung;Lee, Su-Jin;Kim, You-Jeong;Park, Jung-Ja;Kim, Taewan;Park, Nyun-Ho
    • Korean Journal of Food Science and Technology
    • /
    • v.48 no.6
    • /
    • pp.618-621
    • /
    • 2016
  • Geranium thunbergii is a perennial plant commonly used as an oriental medicine for prevention of diarrhea, constipation, and gastrointestinal disorders. However, its anti-inflammatory effect has not been evaluated thus far. Therefore, the present study aimed to investigate the anti-inflammatory effect of G. thunbergii. In this study, G. thunbergii extracted with methanol; this methanol extract was further partitioned using various solvents, and G. thunbergii ethyl acetate fraction (GTEF) was obtained. To determine the anti-inflammatory activity of G. thunbergii, the effects of GTEF on nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 cells were evaluated. GTEF suppressed NO production in a dose-dependent manner without any toxic effects. In addition, western blotting was performed to examine the effect of GTEF on expression of inducible nitric oxide synthase and cyclooxygenase-2. These results suggest that GTEF as a phytoextract may be useful for the prevention or treatment of inflammation.

Cross-reactivity of Human Polyclonal Anti-GLUT1 Antisera with the Endogenous Insect Cell Glucose Transporters and the Baculovirus-expressed GLUT1

  • Lee, Chong-Kee
    • Biomedical Science Letters
    • /
    • v.7 no.4
    • /
    • pp.161-166
    • /
    • 2001
  • Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

  • PDF

Anti-inflammatory effect and contents from the aerial part and root of the various Taraxacum spp. distributed in Korea (국산 5종 포공영(蒲公英)의 항염 효과 및 성분 함량 비교 연구)

  • Lee, Mi-Hwa;Song, Sun-Ho;Ham, In-Hye;Bu, Young-Min;Kim, Ho-Cheol;Choi, Ho-Young
    • The Korea Journal of Herbology
    • /
    • v.25 no.4
    • /
    • pp.77-84
    • /
    • 2010
  • Objectives : Taraxaci Herba et Radix (THR) is widely used as a food and medicinal herb in Korea. It has been used for treatment of virus inflammatory disease, liver diseases and gastritis. So far, anti-inflammatory effects and constituents of various species in THR has not been studied for comparison. The aim of this study is to compare the anti-inflammatory effects of the aerial part and root from various THR. Also, we have compared the contents of its known constituents with each. Methods : In this study, we estimated anti-inflammatory effect and compared their constituent by HPLC. For the determination of anti-inflammatory effects, we investigated NO and $PGE_2$ production by ELISA. The expressions of iNOS was determined by western blotting in LPS-induced RAW 264.7 macrophage cells. And, standard compounds which are methyl gallate, gallic acid, syringic acid and esculetin of THR were analyzed by HPLC using a $C_{18}$ column. Results : Methanol extracts of THR decreased NO and $PGE_2$ production. The expressions of iNOS protein were also decreased in methanol extracts of THR. As a result, HPLC analysis showed that they showed similar patterns. Methyl gallate and esculetin showed the highest content. Methyl gallate was included over 10% content in each aerial part and root of THR. Conclusions : These results indicate that most of THR distributed in Korea might represent therapeutic agent for treatment of inflammatory diseases.

Limb-girdle Muscular Dystrophy (지대형 근이양증)

  • Kim, Dae-Seong
    • Annals of Clinical Neurophysiology
    • /
    • v.6 no.2
    • /
    • pp.65-74
    • /
    • 2004
  • Limb-girdle muscular dystrophy (LGMD) is a heterogeneous group of inherited muscle disorders caused by the mutations of different genes encoding muscle proteins. In the past, when the molecular diagnostic techniques were not available, the subtypes of muscular dystrophies were classified by the pattern of muscle weakness and the mode of inheritance, and LGMD had been considered as a 'waste basket' of muscular dystrophy because many unrelated heterogeneous cases with 'limb-girdle' weakness were put into the category of LGMD. With the advent of molecular genetics at the end of the last century, it has been known that there are many subtypes of LGMD caused by the mutation of different genes, and now, LGMD is classified according to the results of the linkage analysis and the genes or proteins affected. Only small proportion (probably less than 10%) of LGMD is dominantly inherited, and autosomal dominant LGMD (AD-LGMD) consists of six subtypes (LGMD1A to 1F) so far. In autosomal recessive LGMD (AR-LGMD), more than 10 subtypes (LGMD2A to 2J) have been linked and most of the causative genes have been identified. Among AR-LGMDs, LGMD2A (calpain 3 deficiency), 2B (dysferlin deficiency), and sarcoglycanopathy (LGMD2C-2F) are major subtypes. The defective proteins in LGMDs are components of nuclear envelope, cytosol, sarcomere, or sarcolemma, and seem to play a different role in the pathogenesis of muscular dystrophy. It is notable that many causative genes of LGMDs are also responsible for other categories of muscular dystrophy or diseases affecting other tissue. However, by which mechanism they produce such a broad phenotypic variability is still unknown. The identification of mutation in the relevant gene is confirmative for the diagnosis, and is essential for genetic counseling and antenatal diagnosis of LGMD. Because many different genes are responsible for LGMD, differentiation of subtypes using immunohistochemistry and western blotting is the essential step toward the detection of mutation. For the effective research and medical care of the patients with muscular dystrophy in Korea, a research center with a medical facility supported by the government seems to be needed.

  • PDF

FNC, a Novel Nucleoside Analogue, Blocks Invasion of Aggressive Non-Hodgkin Lymphoma Cell Lines Via Inhibition of the Wnt/β-Catenin Signaling Pathway

  • Zhang, Yan;Wang, Chen-Ping;Ding, Xi-Xi;Wang, Ning;Ma, Fang;Jiang, Jin-Hua;Wang, Qing-Duan;Chang, Jun-Biao
    • Asian Pacific Journal of Cancer Prevention
    • /
    • v.15 no.16
    • /
    • pp.6829-6835
    • /
    • 2014
  • Chemotherapy is the primary therapy for malignant lymphoma (ML). However, the clinical outcome is still far from satisfactory. Consequently, an understanding of the mechanism of modulating cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. FNC, 2'-deoxy-2'-${\beta}$-fluoro-4'-azidocytidine, a novel cytidine analogue, has demonstrated significantly inhibitory effects on proliferation of several non-Hodgkin lymphoma (NHL) cell lines. A previous study indicated that FNC effectively inhibited the growth of Raji and JeKo-1 cells in dose-time dependent effects with $IC_{50}$ values of $0.2{\mu}M$ and $0.097{\mu}M$, respectively. This study was focused on investigating the anti-invasive properties of FNC on NHL cells and its potential mechanisms of action. Cell adhesion and transwell chamber assays were utilized to investigate the anti-invasive effects of FNC on Raji and JeKo-1 cells. Real-time PCR and Western blotting were employed to qualify the expression of ${\beta}$-catenin, the glycogen synthase kinase-3 beta (GSK-$3{\beta}$), E-cadherin vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The results revealed that FNC remarkably inhibited the adhesion, migration and invasion of two human aggressive non-Hodgkin lymphoma cell lines in a dose dependent manner. Furthermore, ${\beta}$-catenin, MMP-2, MMP-9, VEGF mRNA and protein levels were decreased after FNC treatment, while GSK-$3{\beta}$ and E-cadherin increased. Our studies thus provide evidence and a rationale that FNC may offer an effective chemotherapeutic agent by regulating the invasion and metastasis of aggressive non-Hodgkin lymphoma via inhibition of the Wnt/${\beta}$-catenin signaling pathway.

Study on the expression and detection of the p53 mutation in Korean colon cancer cell lines (한국인의 대장암 세포주에서 p53 돌연변이의 발견과 발현에 관한 연구)

  • Jung, Ji-Yeon;Oh, Sang-Jin
    • IMMUNE NETWORK
    • /
    • v.1 no.2
    • /
    • pp.151-161
    • /
    • 2001
  • Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.

  • PDF