4 research outputs found
Az autofágia mechanizmusának, szabályozásának, sejt és fejlődésbiológiai szerepének komplex jellemzése sejtbiológiai és genetikai módszerekkel Caenorhabditis elegansban = Complex characterisation of the mechanism, regulation, cellular and development role of autophagy in Caenorhabditis elegans by methods of cell biology and genetics
Az autofágiát Ă©s autofág gĂ©nek hatását jellemeztĂĽk C. elegansban vad, mutáns, RNAi-vel csendesĂtett, gfp-vel jelzett autĂłfág fehĂ©rjĂ©ket expresszálĂł törzsekben. Legfontosabb eredmĂ©nyeink a következĹ‘k. (1) A bec-1/Atg6 eszenciális gĂ©n; a BEC-1 egyĂĽttműködik az autofágiában szereplĹ‘ III tĂpusĂş PI3-kinázzal (LET-512/Vps34); komplexet kĂ©pez az antiapoptotikus CED-9/Bcl-2-vel Ă©s csökkent szintje CED-3/Caspase-fĂĽggĹ‘ programozott sejthalált indukál; a bec-1 az autofágia Ă©s az apoptĂłzis közötti kapocskĂ©nt szerepelhet. (2) MutáciĂłs Ă©s RNAi analĂzis szerint az autofágia szabályozásában szereplĹ‘ inzulin/IGF szignalizáciĂł sextĹ‘l fĂĽggĹ‘ Ă©s korrelál a C. elegans tanulási kĂ©pessĂ©gĂ©vel. (3) Az unc-51/Atg1, bec-1 Ă©s a lgg-1/Atg8 autofág gĂ©nek inaktiválása gátolja a toxikus ioncsatorna-fĂĽggĹ‘ neurondegeneráciĂłt; a CeTOR által közvetĂtett autofágiát gátlĂł jelátviteli Ăşt vĂ©di a sejteket a nekrotikus pusztulástĂłl; a fokozott autofágiával járĂł Ă©hezĂ©s segĂti a nekrĂłzist. (4) Az unc-51 and bec-1 gĂ©n inaktiválása kis testmĂ©retet eredmĂ©nyez; az unc-51 Ă©s a bec-1 funkciĂłvesztĂ©ses mutáciĂłja szupresszálja nagy testmĂ©retű insulin/IGF-1 vagy TGF-? mutánsok fenotĂpusát; az autofág gĂ©neknek szerepĂĽk van a testmĂ©ret szabályozásában. (5) Az autofágiában szereplĹ‘ több gĂ©n (unc-51, bec-1, let-512) mutáciĂłiban fellĂ©pĹ‘ membránátalakulások (pl. A multilammelláris Ă©s -vezikuláris testek expanziĂłja, az ER tubularizáciĂłja) ezen gĂ©neknek az endomembrán szerkezetre gyakorolt eddig ismeretlen hatását tĂĽkrözik. | We have studied autophagy and related genes in wild type C. elegans, strains treated with RNAi and expres- sing gfp-tagged proteins. Our most important results are the following. (1) The Atg6/bec-1 is essential for development; BEC-1 interacts with the class III PI3 kinase LET-512 with important role in autophagy; BEC-1 forms a complex with the antiapoptotic protein CED-9/Bcl-2; its depletion triggers CED-3/Caspase-dependent programmed cell death; bec-1 may represent a link between autophagy and apoptosis. (2) RNAi and mutation analysis shows that the insulin/IGF pathway implicated in the regulation of autophagy, is sex-dependent and correlates with the learning ability of the worm. (3) The inactivation of the autophagy genes unc-51/Atg1, bec-1 and lgg-1/Atg8 suppresses neurodegeneration in toxic ion channel mutants; the CeTOR signaling that downregulates autophagy protects neurons from necrotic cell death; nutrient deprivation promotes nec- rosis. (4) Mutational inactivation of unc-51 and bec-1 results in small body size; loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutants with aberrant insulin/IGF-1 or TGF-? signaling; autophagy genes have role in regulation of body size. (5) Mutations of several autophagy genes (unc-51, bec-1, let-512) lead to special endomembrane changes (e.g. expansion of multilamellar and multivesicular bodies, tubularization of ER) pointing to hitherto unknown effect of these genes on the endo- membranes
In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.
Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24Â h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties