Východiska: N-myc downstream-regulovaný gen 1 (NDRG1) má významnou funkci při progresi nádorů. U karcinomu prostaty (prostate cancer – PCa) však regulační mechanizmus NDRG1 zůstává nejasný. Materiál a metody: Hladiny exprese miR-96-5p a NDRG1 byly hodnoceny v buněčných liniích PCa a v tkáních prostaty a validovány ve veřejných databázích pomocí polymerázové řetězové reakce v reálném čase, analýzy western blot a imunohistochemie. Funkce miR-96-5p a NDRG1 byla zkoumána pomocí testů hojení ran a transwell testů in vitro a testu myšího xenoimplantátu in vivo. Dráha regulovaná pomocí NDRG1 byla testována technikou sekvenování nové generace. K detekci vztahu mezi miR-96-5p, NDRG1 a NF-kB dráhou byl použit imunofluorescenční test a test s luciferázou. Výsledky: Nadměrná exprese NDRG1 potlačuje migraci, invazi a epiteliálně-mezenchymální přechod (EMT) in vitro a inhibuje metastázy in vivo. Navíc miR-96-5p přispívá k deficitu NDRG1 a podporuje migraci a invazi buněk PCa. Kromě toho ztráta NDRG1 aktivuje dráhu NF-kB, která stimuluje fosforylaci p65 a IKBa a indukuje EMT v PCa. Závěr: MiR-96-5p podporuje migraci a invazi PCa tím, že cílí na NDRG1 a reguluje dráhu NF-kB.

Background: The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). Methods: The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. Results: Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. Conclusions: MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

• Klíčová slova
• NDRG1, miR-96-5p,
• MeSH
• epitelo-mezenchymální tranzice MeSH
• genetické techniky MeSH
• imunohistochemie metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty * genetika   patofyziologie   MeSH
• NF-kappa B * MeSH
• pohyb buněk MeSH
• polymerázová řetězová reakce metody   MeSH
• sekvenční analýza MeSH
• signální transdukce MeSH
• transfekce metody   MeSH
• western blotting metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• klinická studie MeSH

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

• MeSH
• buněčné linie MeSH
• DNA chemie   ultrastruktura   MeSH
• exprese genu MeSH
• fluorescenční protilátková technika MeSH
• genetické vektory * chemie   ultrastruktura   MeSH
• kationty chemie   MeSH
• kovové nanočástice chemie   ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil metody   MeSH
• molekulární struktura MeSH
• průtoková cytometrie MeSH
• reportérové geny MeSH
• technika přenosu genů * MeSH
• transfekce MeSH
• viabilita buněk MeSH
• železnaté sloučeniny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

• MeSH
• aktiny metabolismus   MeSH
• Caco-2 buňky MeSH
• centrozom metabolismus   MeSH
• forminy metabolismus   MeSH
• genový knockout MeSH
• lidé MeSH
• melanom experimentální metabolismus   patologie   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• nádory kůže metabolismus   patologie   MeSH
• polymerizace MeSH
• profiliny genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transfekce MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action.

• MeSH
• buněčné linie MeSH
• chuť * MeSH
• chuťová percepce * MeSH
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• myši inbrední C57BL fyziologie   MeSH
• myši MeSH
• oxid deuteria analýza   MeSH
• receptory spřažené s G-proteiny metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

• MeSH
• buněčné linie MeSH
• buňky Hep G2 MeSH
• exprese genu MeSH
• genový knockout MeSH
• hepatocyty imunologie   metabolismus   MeSH
• interferonové regulační faktory genetika   metabolismus   MeSH
• interferony nedostatek   genetika   metabolismus   MeSH
• interleukiny nedostatek   genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• receptor interleukinu-10 - beta-podjednotka nedostatek   genetika   metabolismus   MeSH
• receptory interferonů nedostatek   genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The ABCG2 gene is a well-established hyperuricemia/gout risk locus encoding a urate transporter that plays a crucial role in renal and intestinal urate excretion. Hitherto, p.Q141K-a common variant of ABCG2 exhibiting approximately one half the cellular function compared to the wild-type-has been reportedly associated with early-onset gout in some populations. However, compared with adult-onset gout, little clinical information is available regarding the association of other uricemia-associated genetic variations with early-onset gout; the latent involvement of ABCG2 in the development of this disease requires further evidence. We describe a representative case of familial pediatric-onset hyperuricemia and early-onset gout associated with a dysfunctional ABCG2, i.e., a clinical history of three generations of one Czech family with biochemical and molecular genetic findings. Hyperuricemia was defined as serum uric acid (SUA) concentrations 420 μmol/L for men or 360 μmol/L for women and children under 15 years on two measurements, performed at least four weeks apart. The proband was a 12-year-old girl of Roma ethnicity, whose SUA concentrations were 397-405 µmol/L. Sequencing analyses focusing on the coding region of ABCG2 identified two rare mutations-c.393G>T (p.M131I) and c.706C>T (p.R236X). Segregation analysis revealed a plausible link between these mutations and hyperuricemia and the gout phenotype in family relatives. Functional studies revealed that p.M131I and p.R236X were functionally deficient and null, respectively. Our findings illustrate why genetic factors affecting ABCG2 function should be routinely considered in clinical practice as part of a hyperuricemia/gout diagnosis, especially in pediatric-onset patients with a strong family history.

• MeSH
• ABC transportér z rodiny G, člen 2 genetika   metabolismus   MeSH
• dítě MeSH
• dna (nemoc) komplikace   genetika   MeSH
• dospělí MeSH
• fenotyp MeSH
• genetická predispozice k nemoci MeSH
• HEK293 buňky MeSH
• hyperurikemie krev   komplikace   genetika   MeSH
• jednonukleotidový polymorfismus * MeSH
• kyselina močová krev   MeSH
• lidé MeSH
• mutace MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• přenašeče organických aniontů genetika   metabolismus   MeSH
• rodokmen MeSH
• transfekce MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.

• MeSH
• aktivace enzymů genetika   MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• cyklin A2 genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 2 nedostatek   genetika   MeSH
• cytoplazma metabolismus   MeSH
• fosforylace genetika   MeSH
• G2 fáze genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitóza genetika   MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinkinasa CDC2 nedostatek   genetika   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• S fáze genetika   MeSH
• signální transdukce genetika   MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.

• MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• myši MeSH
• rekombinasa Rad51 biosyntéza   genetika   metabolismus   MeSH
• replikace DNA * MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 μM) and IAD (IC50 10 μM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.

• MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• indoly metabolismus   MeSH
• kultivované buňky MeSH
• kyseliny indoloctové metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• nádorové buněčné linie MeSH
• P-glykoproteiny genetika   metabolismus   MeSH
• pregnanový X receptor agonisté   genetika   MeSH
• reportérové geny MeSH
• střevní mikroflóra * MeSH
• střevní sliznice * metabolismus   mikrobiologie   MeSH
• transfekce MeSH
• tryptofan metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

• MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• genetická transkripce MeSH
• imidazoly farmakologie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• melanom genetika   metabolismus   patologie   MeSH
• MFC-7 buňky MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• piperaziny farmakologie   MeSH
• pluripotentní kmenové buňky metabolismus   MeSH
• proteiny buněčného cyklu biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny biosyntéza   genetika   metabolismus   MeSH
• roskovitin farmakologie   MeSH
• sulfonamidy farmakologie   MeSH
• synergismus léků MeSH
• transfekce MeSH
• triaziny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH