beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.

• MeSH
• adenokarcinom metabolismus   MeSH
• alkalická fosfatasa metabolismus   MeSH
• androstadieny metabolismus   MeSH
• beta-katenin genetika   metabolismus   MeSH
• butyráty metabolismus   MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace MeSH
• imunohistochemie MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• kinasy ribozomálního proteinu S6, 70-kDa metabolismus   MeSH
• kinasy ribozomálního proteinu S6 metabolismus   MeSH
• kolorektální nádory metabolismus   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• nádorové buněčné linie MeSH
• serin metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• tyrosin metabolismus   MeSH
• Check Tag
• lidé MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace MeSH
• interleukin-2 metabolismus   MeSH
• lidé MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• proteinkinasy metabolismus   MeSH
• ribozomální proteiny chemie   metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfatidylseriny metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• proteinové domény MeSH
• steroidní receptory chemie   metabolismus   MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

BACKGROUND: The PI*S variant is one of the most prevalent mutations within alpha-1 antitrypsin deficiency (AATD). The risk of developing AATD-related lung disease in individuals with the PI*SS genotype is poorly defined despite its substantial prevalence. Our study aimed to characterize this genotype and its risk for lung disease and compare it with the PI*ZZ and PI*SZ genotypes using data from the European Alpha-1 antitrypsin Deficiency Research Collaboration international registry. METHOD: Demographic, clinical, functional, and quality of life (QoL) parameters were assessed to compare the PI*SS characteristics with the PI*SZ and PI*ZZ controls. A propensity score with 1:3 nearest-neighbour matching was performed for the most important confounding variables. RESULTS: The study included 1007 individuals, with PI*SS (n = 56; 5.6%), PI*ZZ (n = 578; 57.4%) and PI*SZ (n = 373; 37.0%). The PI*SS population consisted of 58.9% men, with a mean age of 59.2 years and a mean FEV1(% predicted) of 83.4%. Compared to PI*ZZ individuals they had less frequent lung disease (71.4% vs. 82.2%, p = 0.037), COPD (41.4% vs. 60%, p = 0.002), and emphysema (23.2% vs. 51.9%, p < 0.001) and better preserved lung function, fewer exacerbations, lower level of dyspnoea, and better QoL. In contrast, no significant differences were found in the prevalence of lung diseases between PI*SS and PI*SZ, or lung function parameters, exacerbations, dyspnoea, or QoL. CONCLUSIONS: We found that, as expected, the risk of lung disease associated with the PI*SS genotype is significantly lower compared with PI*ZZ, but does not differ from that observed in PI*SZ individuals, despite having higher serum AAT levels. TRIAL REGISTRATION: www. CLINICALTRIALS: gov (ID: NCT04180319).

• MeSH
• alfa-1-antitrypsin * genetika   MeSH
• deficit alfa1-antitrypsinu * genetika   epidemiologie   diagnóza   MeSH
• genotyp * MeSH
• kvalita života MeSH
• lidé středního věku MeSH
• lidé MeSH
• plicní nemoci genetika   epidemiologie   diagnóza   MeSH
• registrace MeSH
• rizikové faktory MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates.

• Publikační typ
• časopisecké články MeSH

Activated phosphoinositide 3-kinase delta (PI3Kδ) syndrome (APDS) is an inborn error of immunity with clinical manifestations including infections, lymphoproliferation, autoimmunity, enteropathy, bronchiectasis, increased risk of lymphoma, and early mortality. Hyperactive PI3Kδ signaling causes APDS and is selectively targeted with leniolisib, an oral, small molecule inhibitor of PI3Kδ. Here, 31 patients with APDS aged ≥12 years were enrolled in a global, phase 3, triple-blinded trial and randomized 2:1 to receive 70 mg leniolisib or placebo twice daily for 12 weeks. Coprimary outcomes were differences from baseline in the index lymph node size and the percentage of naïve B cells in peripheral blood, assessed as proxies for immune dysregulation and deficiency. Both primary outcomes were met: the difference in the adjusted mean change (95% confidence interval [CI]) between leniolisib and placebo for lymph node size was -0.25 (-0.38, -0.12; P = .0006; N = 26) and for percentage of naïve B cells, was 37.30 (24.06, 50.54; P = .0002; N = 13). Leniolisib reduced spleen volume compared with placebo (adjusted mean difference in 3-dimensional volume [cm3], -186; 95% CI, -297 to -76.2; P = .0020) and improved key immune cell subsets. Fewer patients receiving leniolisib reported study treatment-related adverse events (AEs; mostly grades 1-2) than those receiving placebo (23.8% vs 30.0%). Overall, leniolisib was well tolerated and significant improvement over placebo was notable in the coprimary endpoints, reducing lymphadenopathy and increasing the percentage of naïve B cells, reflecting a favorable impact on the immune dysregulation and deficiency seen in patients with APDS. This trial was registered at www.clinicaltrials.gov as #NCT02435173.

• MeSH
• dvojitá slepá metoda MeSH
• fosfatidylinositol-3-kinasy třídy I MeSH
• fosfatidylinositol-3-kinasy * MeSH
• lidé MeSH
• pyridiny MeSH
• pyrimidiny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

• MeSH
• barva MeSH
• barvicí látky chemie   MeSH
• chemická frakcionace MeSH
• isoelektrická fokusace metody   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• peptidy analýza   MeSH
• proteom analýza   MeSH
• sérový albumin analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The most sensitive cell structure - a DNA molecule, is the common target of cancer therapy. DNA damage response (controlled by enzymes from the phosphatidylinositol 3-kinase-related kinases family - PIKK) presents many encouraging targets for improving both conventional cytotoxic anticancer therapy and individualized monotherapy. DNA-dependent protein kinase (DNA-PK) is a member of the PIKK superfamily and plays an important role in the detection and repair of DNA double-strand breaks via the non-homologous end-joining pathway. The ability of cancer cells to repair DNA damage is an important element determining their sensitivity to radio- or chemo-therapy. The overactivation of DNA-PK in cancers can result in resistance to anticancer therapy. The inhibition of DNA-PK is a very promising target in anticancer research. However, the specific DNA-PK inhibitors currently known are limited by poor solubility and high metabolic lability in vivo, leading to a short serum half-life. Construction of new compounds based on existing drugs is the most important strategy to improve drug efficacy, pharmacokinetic parameters and to reduce toxicity. This review will describe small molecule inhibitors and summarize their efficacy in synergizing radio- and chemotherapy in vitro.

• MeSH
• fosfatidylinositol-3-kinasy MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• poškození DNA MeSH
• proteinkinasa aktivovaná DNA antagonisté a inhibitory   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Cardiovascular complications are a side effect of cancer therapy, potentially through reduced blood vessel function. ONC201 (TIC10) is currently used in phase 2 clinical trials to treat high-grade gliomas. TIC10 is a phosphatidylinositol 3-kinase (PI3K)/AKT/extracellular signal-regulated kinase (ERK) inhibitor that induces apoptosis via upregulation of TNF-related apoptosis-inducing ligand, which via stimulation of FOXO and death receptor could increase eNOS upregulation. This has the potential to improve vascular function through increased NO bioavailability. Our aim was to investigate the role of TIC10 on vascular function to determine if it would affect the risk of CVD. Excised abdominal aorta from White New Zealand male rabbits were cut into rings. Vessels were incubated with TIC10 and AS1842856 (FOXO1 inhibitor) followed by cumulative doses of acetylcholine (Ach) to assess vessel function. Vessels were then processed for immunohistochemistry. Incubation of blood vessels with TIC10 resulted in enhanced vasodilatory capacity. Combination treatment with the FOXO1 inhibitor and TIC10 resulted in reduced vascular function compared to control. Immunohistochemical analysis indicated a 3-fold increase in death receptor 5 (DR5) expression in the TIC10-treated blood vessels but the addition of the FOXO1 inhibitor downregulated DR5 expression. The expression of DR4 receptor was not significantly increased in the presence of TIC10; however, addition of the FOXO1 inhibitor downregulated expression. TIC10 has the capacity to improve the function of healthy vessels when stimulated with the vasodilator Ach. This highlights its therapeutic potential not only in cancer treatment without cardiovascular side effects, but also as a possible drug to treat established CVD.

• MeSH
• fosfatidylinositol-3-kinasy MeSH
• imidazoly * MeSH
• králíci MeSH
• lidé MeSH
• protein FOXO3 * MeSH
• protein TRAIL MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We evaluated the impact of ataxia-telangiectasia mutated kinase inhibitor KU55933, DNA-dependent protein kinase inhibitor NU7441 and ataxia telangiectasia and rad3-related kinase inhibitor VE821 in human peripheral lymphocytes in vitro. The lymphocytes were divided into 5 groups: non-irradiated control, irradiated group (2 Gy) and 3 groups pretreated with inhibitors 30 min before irradiation. We used flow cytometry to evaluate phosphorylated H2AX (γ-H2AX) and cytotoxicity (Apoptest). Micronucleus assay was used to assess genotoxicity. After irradiation, γ-H2AX, incidence of micronuclei (MN), nucleoplasmatic bridges (NPBs) and nuclear buds in binuclear cells, MN in mononuclear cells and apoptosis were increased. KU55933 decreased γ-H2AX and inhibited ionizing radiation-induced cytotoxicity. NU7441 showed no effect on γ-H2AX but it significantly increased MN and NPBs in binuclear cells and apoptosis. VE821 decreased γ-H2AX, whereas genotoxicity and cytotoxicity were not affected. In conclusion, KU55933 protected lymphocytes, which might be employed to preserve the immune system during anticancer therapy. NU7441 radiosensitized lymphocytes, thus, undesirable side effects toward immune system could be expected. VE821 showed decrease of γ-H2AX with no radiosensitizing effects in our model likely due to p53 positive status, which underlies the concept of its application in p53 negative environment.

• Klíčová slova
• Účinky ionizujícího záření způsobené inhibitory ATM (KU55933), DNA-PK (NU7441) a ATR (VE821) na lymfocyty periferní krve,
• MeSH
• ATM protein účinky záření   MeSH
• biomedicínský výzkum MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy účinky záření   MeSH
• fosforylace imunologie   účinky záření   MeSH
• histony chemie   účinky záření   MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• ionizující záření * MeSH
• krevní a imunitní systémy cytologie   imunologie   účinky záření   MeSH
• lidé MeSH
• lymfocyty * cytologie   imunologie   účinky záření   MeSH
• statistika jako téma MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH