- Publikační typ
- abstrakt z konference MeSH
We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.
- MeSH
- adenosintrifosfát farmakologie MeSH
- centrifugace - gradient hustoty MeSH
- frakcionace buněk metody MeSH
- glukosylceramidasa metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- intracelulární membrány účinky léků metabolismus MeSH
- kyseliny metabolismus MeSH
- lidé MeSH
- lyzozomy účinky léků metabolismus MeSH
- subcelulární frakce účinky léků metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.
- MeSH
- buněčné linie MeSH
- cystein metabolismus MeSH
- embryonální vývoj MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- lipoylace MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- myši MeSH
- protein Wnt1 genetika metabolismus MeSH
- protein Wnt3 MeSH
- protein Wnt3A MeSH
- proteiny Wnt genetika metabolismus MeSH
- proteiny Xenopus MeSH
- sekvence aminokyselin MeSH
- serin metabolismus MeSH
- substituce aminokyselin MeSH
- Xenopus embryologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.
- MeSH
- beta-katenin metabolismus MeSH
- buněčné linie MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- genetická transkripce MeSH
- genový knockdown MeSH
- lidé MeSH
- myši MeSH
- promotorové oblasti (genetika) MeSH
- proteiny vázající RNA antagonisté a inhibitory genetika metabolismus MeSH
- proteiny Wnt metabolismus MeSH
- regulace genové exprese MeSH
- transkripční faktory BHLH-Zip MeSH
- transkripční faktory chemie metabolismus MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Endolymphatic sac tumor (Heffner tumor) (ELST) is a very rare nonmetastasizing, locally aggressive low-grade adenocarcinoma of endolymphatic sac origin, which is linked to von-Hippel-Lindau disease (VHLD). VHLD is an autosomal dominant disorder characterized by an inherited genetic abnormality of the VHL gene located on the short arm of chromosome 3 (3p26-p25). VHL gene mutations have been shown both in ELSTs associated with VHLD and in sporadic cases. Because of the rarity of ELST, only a small number of cases have been subjected to molecular genetic analysis. We have encountered two patients with ELST, one of whom presented with a medical and family history of VHLD. The second was a sporadic case, the patient having no symptoms of VHLD. The tissues obtained from Heffner tumor and cerebellar hemangioblastoma from the patient with inherited VHLD possess a point mutation in exon 1 of VHL gene. This mutation is a C to T exchange at position 194, resulting in amino acid exchange S65L. No mutation was found in any of the three exons analyzed and in the exon-intron junctions of the VHL gene in the sporadic case.
- MeSH
- adenokarcinom genetika chirurgie patologie MeSH
- bodová mutace MeSH
- dospělí MeSH
- exony MeSH
- genetická predispozice k nemoci MeSH
- introny MeSH
- invazivní růst nádoru MeSH
- lidé MeSH
- mutační analýza DNA MeSH
- nádorový supresorový protein VHL genetika MeSH
- nádory lebky genetika chirurgie patologie MeSH
- nádory ucha genetika chirurgie patologie MeSH
- regulace genové exprese u nádorů MeSH
- rodokmen MeSH
- saccus endolymphaticus chirurgie patologie MeSH
- senioři MeSH
- spánková kost chirurgie patologie MeSH
- střední ucho chirurgie patologie MeSH
- von Hippelova-Lindauova nemoc genetika patologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- Publikační typ
- abstrakt z konference MeSH
The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller-Dieker syndrome. HIC1 encodes a zinc-finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N-terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/beta-catenin-mediated transcription in Wnt-stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF-4 and to recruit TCF-4 and beta-catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF-binding elements of the Wnt-responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/beta-catenin signaling.
- MeSH
- beta-katenin genetika metabolismus MeSH
- buněčné jádro metabolismus MeSH
- cytoskeletální proteiny genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- financování organizované MeSH
- genetická transkripce MeSH
- kultivační média speciální MeSH
- lidé MeSH
- myši MeSH
- promotorové oblasti (genetika) MeSH
- proteiny Wnt genetika metabolismus MeSH
- regulace genové exprese MeSH
- RNA interference MeSH
- signální transdukce fyziologie MeSH
- transkripční faktory Krüppel-like MeSH
- transkripční faktory TCF genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
Blood platelet and monocyte adhesion was studied in vitro with respect to the influence of hydrophilic polymer chemical functional groups and their charge. The results showed that the strongest adhesion of human monocytes was to coverslips covered with cationic polymer. Platelet adhesion to all tested polymers proved to be negligible; no differences related to the charge of the polymers used were observed. These results show the obvious difference between the biocompatibility and haemocompatibility in vitro which must be taken into consideration during polymer biological properties testing before clinical trials.
- MeSH
- adhezivita trombocytů * MeSH
- hydrogely * MeSH
- lidé MeSH
- monocyty * MeSH
- polymery * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
- techniky in vitro MeSH
