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REGULACIJA EKSPRESIJE EKTOPEPTIDAZA I OPIODNIH RECEPTORA



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REGULACIJA EKSPRESIJE EKTOPEPTIDAZA I OPIODNIH RECEPTORA

REGULATION OF ECTOPEPTIDASES AND OPIOID RECEPTORS EXPRESSION
Voditeljica projekta: dr. sc. Jelka Gabrilovac

Tel. ++385 1 4561 082   e-mail: gabril@irb.hr



Suradnici
Davorka Breljak, doktorica biol. znanosti, znanstvena novakinja u suradničkom zvanju više asistentice (do 31.5.2005.)
Barbara Čupić, dipl. ing. molekularne biologije, znanstvena novakinja u suradničkom zvanju asistentice
Jelka Gabrilovac, doktorica biol. znanosti, znanstvena savjetnica
Irena Martin-Kleiner, doktorica kem. znanosti, znanstvena suradnica
Tamara Stipčević, doktorica biol. znanosti, znanstvena novakinja u suradničkom zvanju više asistentice

Tehnički suradnici
Margareta Cvetkovski

Vanjski suradnici
Milivoj Boranić, doktor med. znanosti, znanstveni savjetnik u mirovini

Program rada i rezultati na projektu:

U nastavku istraživanja membranskih peptidaza ispitivana je uloga glukokortikoida na ekspresiju neutralne endopeptidaze (NEP; CD10) na nezrelim B limfocitma. Ispitivan je učinak niskih, fiziološki relevantnih koncentracija glukokortiokoida, na ekspresiju NEP/CD10 na nezrelim B limfocitima. Kao model korištene su stanice nezrele B-linije, NALM-6. Rezultati su pokazali smanjenje NEP/CD10 ekspresije na transkripcijskoj, proteinskoj i funkcionalnoj razini. Učinak je bio posredovan glukokortikoidnim receptorima, i nije uključivao selekciju CD10low stanica. Budući da je sazrijevanje normalnih B limfocita povezano sa smanjenom ekspresijom NEP/CD10, rezultati upućuju na zaključak da glukokortiokoidi u fiziološki relevantnim koncentracijama, kao što su one u stanju akutnog stresa, pozitivno reguliraju sazrijevanja B limfocita

Ispitivana je uloga još jedne ektopeptidaze – aminopeptidase N (APN/CD13) na stanicama mijelo-monocitnog porijekla. Koristeći svoju enzimatsku aktivnost APN podešava lokalnu koncentraciju peptidnih medijatora (kemokina, citokina, faktora rasta), te tako utječe na rast, aktivaciju i smrt stanica. Ispitivali smo stoga ulogu T-staničnog citokina interferona-gama, (IFN-gama), na ekspresiju APN na stanicama mijelo-monocitne linije HL-60. Rezultati su pokazali da IFN-gama modulira APN ekspresiju, ovisno o koncentraciji i vremenu tretmana i to na razini mRNA, membranskog proteina, te njegove enzimske aktivnosti. Promjene u APN ekspresiji inducirane pomoću IFN-gama, ne uključuju de novo sintezu TGF-beta. Dobiveni rezultati ukazuju na moguću ulogu APN u modulaciji upalnih reakcija.

Research programme and results:

Neutral endopeptidase is a membrane bound enzyme with various functions depending on cell type or tissue origin. Normal development and differentiation of immature B cells depends on expression of CD10/NEP on B cell progenitors and bone marrow stromal cells. We investigated the effect of low, physiologically relevant concentrations of glucocorticoids, on expression of differentiation marker CD10/NEP on immature B cells. Concentration- and time-dependent down-regulation of CD10/NEP was observed at transcriptional, membrane protein and functional level. The observed CD10/NEP down-regulation was mediated via glucocorticoid receptors (GR), as it was fully abrogated by a GR antagonist, RU 38486 at all three levels. The mechanism of dex-induced CD10/NEP down-regulation is not likely to include selection of cells that are CD10low, since the effect was partly reversible after the removal of glucocorticoids. However, glucocorticoid-induced CD10/NEP down-regulation did include decreased transcription of the CD10 mRNA. Transcriptional inhibitor actinomycin D completely reversed glucocorticoid-induced CD10/NEP down-regulation. Since differentiation of normal B lymphocytes is associated with down-regulation of CD10/NEP, the data suggest that low, physiologically relevant concentrations of glucocorticoid (such as observed in acutetress) may play regulatory role in normal development and maturation of B lymphocytes.

Membrane-bound peptidases play important roles in the regulation of local concentrations of various signaling peptides such as the growth factors, hormones, chemokines and cytokines. We investigated the effects of a T-cell derived cytokine, interferon-gamma (IFN-gamma) on the activity of aminopeptidase N (APN), an ectoenzyme processing several signal peptides. Cells of a myelo-monocytic cell line HL-60 were used as a model system, and APN was assayed at the levels of mRNA, its membrane marker CD13, and the enzyme activity. Regulation of CD13/APN by IFN-gamma was found at all three levels. The direction of regulation was time-dependent: an initial down-regulation seen 24 and 48 hrs after the onset of treatment with IFN-gamma was replaced by an up-regulation after 72 and/or 96 hrs. The delayed up-regulation of CD13/APN (observed after 72 and/or 96 hrs), required de novo protein synthesis as it could be abrogated by cycloheximide, an inhibitor of protein synthesis. Possible role of endogenous (IFN-gamma-induced) TGF-beta in mediating CD13/APN up-regulation could be excluded, since no TGF-beta was found in supernatants of IFN-gamma-treated HL-60 cells. Thus, we have shown regulation of CD13/APN on cells of myelo-monocytic origin by a T-cell derived cytokine, IFN-gamma. A similar mechanism might play a role in inflammation.

Three RT-PCR based methods: semi-quantitative, competitive and real-time RT-PCR for relative quantification of mRNA were compared. Aminopeptidase N expressed on human promyeloid HL-60 cell line, at basal and IFN-gamma-activated state, served as a model for comparison. Total cellular RNA was isolated, reverse transcribed to cDNA and semi-quantitative, competitive and real-time RT-PCR were performed to obtain the relative levels of mRNA for aminopeptidase N. The data obtained showed that all three RT-PCR based methods gave reliable and comparable results. Thus, in spite of rapid advances made in the area of real-time RT-PCR, end-point RT-PCR such as competitive and semi-quantitative RT-PCR, although laborious and time consuming, may still remain useful techniques for relative mRNA quantification when small number of samples are to be analyzed.




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