Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg220 to Ala and Thr232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg220 and Thr232, in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements

Short-term Efficacy of Topical Immunosuppressive Agents on the Survival of Cultivated Allo-Conjunctival Equivalents

PURPOSE: To investigate the short-term efficacy of topical immunosuppressive agents on the survival of cultivated allo-conjunctival equivalents. METHODS: Twenty-five eyes of New Zealand white rabbits were included. Temporal conjunctivae were trephined to a diameter of 7.5 mm, and then cultured allo-conjunctival epithelial cells on amniotic membrane were transplanted onto them. Various immunosuppressants including steroid, cyclosporine, and rapamycin were applied topically four times a day for a week. Epithelial defects and graft edema were graded daily. Numbers of inflammatory cells were measured in H&E. PKH26 and cytokeratin 4 and 7 were immunostained. RESULTS: Earlier epithelialization was observed in 1% steroid-treated eyes and defects persisted significantly in 0.5% CsA applied eyes. In histology, PKH26 positive cells considered as donor cells were only found in 1% steroid or 0.01% rapamycin applied eyes. 1% steroid- or 0.01% rapamycin-applied eyes both showed positive staining for keratin-4 and -7. Inflammatory cells were less found in 1% steroid or 0.01% rapamycin treated eyes. CONCLUSIONS: Topical steroid or rapamycin can help to suppress acute inflammation and enhance the acute survival of transplanted conjunctival cells.Y

DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease

A logit analysis of hospital choice behavior in Chollabukdo province of Korea

This paper analyzes hospital choice behavior of inpatients in Chollabukdo Province of the Republic of Korea. By applying a binary logit model on the disaggregate basis, we examine how individual inpatients select tertiary hospitals with high-level services as opposed to secondary low-level hospitals. From the analysis, we have found significant differences in the hospital choice behavior among the different socioeconomic groups of population. Especially meaningful differences were observed between urban and rural residents. The present policy focusing on the construction of low-level hospitals is thought to be reasonable to cope with the needs of rural residents, whereas increasing demand for high-level services is expected in the urban area.hospital choice logit model Korea

The Effect of Electrolytes on the Preparation of an Extraction Replica in 3 wt. % Si Steel

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Identification of alpha-Gal and non-Gal Epitopes in Pig Corneal Endothelial Cells and Keratocytes by Using Mass Spectrometry

Purpose: To investigate the expression of alpha-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry. Methods: The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immuno chemical staining was added to confirm the non-Gal antigen expression in pig corneal cells. Results: Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of alpha-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed. Conclusions: As well as alpha-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the alpha-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.Gil GC, 2008, ANAL BIOCHEM, V379, P45, DOI 10.1016/j.ab.2008.04.039Kim YG, 2008, PROTEOMICS, V8, P2596, DOI 10.1002/pmic.200700972Lee HI, 2007, XENOTRANSPLANTATION, V14, P612, DOI 10.1111/j.1399-3089.2007.00433.xAlvarez-Manilla G, 2007, GLYCOBIOLOGY, V17, P677, DOI 10.1093/glycob/cwm033ZHIQIANG P, 2007, XENOTRANSPLANTATION, V14, P603Hering BJ, 2006, NAT MED, V12, P301, DOI 10.1038/nm1369Kim YG, 2006, PROTEOMICS, V6, P1133Chen G, 2005, NAT MED, V11, P1295, DOI 10.1038/nm1330Lee CS, 2005, BIOTECHNOL BIOPROC E, V10, P212Kuwaki K, 2005, NAT MED, V11, P29, DOI 10.1038/nm1171Yamada K, 2005, NAT MED, V11, P32, DOI 10.1038/nm1172Kang P, 2005, RAPID COMMUN MASS SP, V19, P3421, DOI 10.1002/rcm.2210Kolber-Simonds D, 2004, P NATL ACAD SCI USA, V101, P7335Komoda H, 2004, XENOTRANSPLANTATION, V11, P237, DOI 10.1111/j.1399-3089.2004.00121.xMiwa Y, 2004, XENOTRANSPLANTATION, V11, P247, DOI 10.1111/j.1399-3089.2004.00126.xMorelle W, 2004, RAPID COMMUN MASS SP, V18, P2451, DOI 10.1002/rcm.1640Ciucanu I, 2003, J AM CHEM SOC, V125, P16213, DOI 10.1021/ja035660tAmano S, 2003, CURR EYE RES, V26, P313Phelps CJ, 2003, SCIENCE, V299, P411, DOI 10.1126/science.1078942Banerjee S, 2003, EXPERT OPIN INV DRUG, V12, P29Zhu A, 2002, XENOTRANSPLANTATION, V9, P376Gouw JW, 2002, RAPID COMMUN MASS SP, V16, P905, DOI 10.1002/rcm.654Zeng YJ, 2001, J BIOMECH, V34, P533QIAN Y, 2001, EXPERT REV MOL MED, P1Morozumi K, 1999, TRANSPLANT P, V31, P942Rocha G, 1998, CRIT REV IMMUNOL, V18, P305Viseux N, 1997, ANAL CHEM, V69, P3193Inohara H, 1996, CANCER RES, V56, P4530JAGER MJ, 1995, TRANSPL IMMUNOL, V3, P135COOPER DKC, 1994, IMMUNOL REV, V141, P31ORIOL R, 1993, TRANSPLANTATION, V56, P1433GALILI U, 1993, IMMUNOL TODAY, V14, P480COOPER DKC, 1993, TRANSPL IMMUNOL, V1, P198GOOD AH, 1992, TRANSPLANT P, V24, P559CORNIL I, 1990, J CELL BIOL, V111, P773HAZLETT LD, 1989, J HISTOCHEM CYTOCHEM, V37, P1215STREILEIN JW, 1979, NATURE, V282, P326

하대정맥 막성폐쇄의 초음파소견 - 하대정맥조영술과의 비교를 중심으로

Membranous obstruction of upper cava is one of the frequent causes of inferior vena caval obstruction in this country. Since it can be cured by transcardiac membranotomy or other interventions, accurate diagnosis of the disease is important. Sonographic findings in 6 cases of membranous obstruction of inferior vena cava were analysed and correlated with the findings of the inferior venacavography. At the obstructed site, the membrane was detected as a high echogenic focal or segmental obliteration of the lumen sonographically. The lumen of vena cava below the obstruction was easily delineated without normal respiratory changes. Venacavography was superior in demonstrating all collateral channels except transhepatic collaterals which were depicted well in sonography