The glycoprotein clusterin (CLU) is involved in cell proliferation and DNA damage repair and is highly expressed in tumor cells. Here, we aimed to investigate the effects of CLU dysregulation on two human astrocytic cell lines: CCF-STTG1 astrocytoma cells and SV-40 immortalized normal human astrocytes. We observed that suppression of CLU expression by RNA interference inhibited cell proliferation, triggered the DNA damage response, and resulted in cellular senescence in both cell types tested. To further investigate the underlying mechanism behind these changes, we measured reactive oxygen species, assessed mitochondrial function, and determined selected markers of the senescence-associated secretory phenotype. Our results suggest that CLU deficiency triggers oxidative stress-mediated cellular senescence associated with pronounced alterations in mitochondrial membrane potential, mitochondrial mass, and expression levels of OXPHOS complex I, II, III and IV, indicating mitochondrial dysfunction. This report shows the important role of CLU in cell cycle maintenance in astrocytes. Based on these data, targeting CLU may serve as a potential therapeutic approach valuable for treating gliomas.
- MeSH
- Astrocytes * metabolism pathology MeSH
- Clusterin * metabolism genetics MeSH
- Humans MeSH
- Membrane Potential, Mitochondrial * physiology MeSH
- Mitochondria * metabolism MeSH
- Cell Line, Tumor MeSH
- Oxidative Stress physiology MeSH
- Oxidative Phosphorylation MeSH
- DNA Damage MeSH
- Cell Proliferation * MeSH
- Reactive Oxygen Species metabolism MeSH
- Cellular Senescence * physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
Background: In recent years, significant resistance of microorganisms to antibiotics has been observed. A biofilm is a structure that significantly aids the survival of the microbial population and also significantly affects its resistance. Methods: Thyme and clove essential oils (EOs) were subjected to chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with a flame ionization detector (GC-FID). Furthermore, the antimicrobial effect of these EOs was tested in both the liquid and vapor phases using the volatilization method. The effect of the EOs on growth parameters was monitored using an RTS-8 bioreactor. However, the effect of the EOs on the biofilm formation of commonly occurring bacteria with pathogenic potential was also monitored, but for less described and yet clinically important strains of Arcobacter spp. Results: In total, 37 and 28 compounds were identified in the thyme and clove EO samples, respectively. The most common were terpenes and also derivatives of phenolic substances. Both EOs exhibited antimicrobial activity in the liquid and/or vapor phase against at least some strains. The determined antimicrobial activity of thyme and clove oil was in the range of 32-1024 μg/mL in the liquid phase and 512-1024 μg/mL in the vapor phase, respectively. The results of the antimicrobial effect are also supported by similar conclusions from monitoring growth curves using the RTS bioreactor. The effect of EOs on biofilm formation differed between strains. Biofilm formation of Pseudomonas aeruginosa was completely suppressed in an environment with a thyme EO concentration of 1024 μg/mL. On the other hand, increased biofilm formation was found, e.g., in an environment of low concentration (1-32 μg/mL). Conclusions: The potential of using natural matrices as antimicrobials or preservatives is evident. The effect of these EOs on biofilm formation, especially Arcobacter strains, is described for the first time.
- Publication type
- Journal Article MeSH
Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.
- MeSH
- Leukemia, Myeloid, Acute * genetics MeSH
- Mutation MeSH
- Mice MeSH
- Tumor Suppressor Protein p53 * genetics MeSH
- DNA Damage MeSH
- Cell Proliferation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.
A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics.
Apolipoprotein J (clusterin) is a component of high-density lipoproteins, the high level of which is reversely correlated with the risk of coronary heart disease. In addition, it exerts anti-inflammatory and anti-apoptotic effects on endothelial cells and inhibits smooth muscle cell migration and proliferation, indicating that it may play a protective role in cardiovascular disease. However, the exact mechanisms by which this occurs remain unclear. This study aimed to clarify these underlying protective mechanisms by researching the inhibitory effects of apolipoprotein J via the NOD-like receptor protein 3 pathway on the inflammation induced by cholesterol crystals in THP‐1 macrophages. In culture, THP-1 macrophages were infected with adenoviral vectors containing apolipoprotein J genes and subsequently treated with cholesterol crystals. The inflammatory cytokines interleukin‐1β, interleukin 18 and tumour necrosis factor α were quantitatively measured with ELISA kits. NOD-like receptor protein 3, cysteinyl aspartate specific proteinase 1 and interleukin 1β were evaluated by Western blot and PCR analysis. As a result, apolipoprotein J expression was found to remarkably decrease the levels of inflammatory cytokines, including tumour necrosis factor α, interleukin 18 and interleukin 1β, secreted by THP‐1 macrophages. It was also found capable of inhibiting the levels of NOD-like receptor protein 3, cysteinyl aspartate-specific proteinase 1 and interleukin 1β both at the protein and mRNA levels. In the current study, we revealed that over-expression of apolipoprotein J attenuated the inflammation induced by cholesterol crystals through inhibition of the NOD-like receptor protein 3 inflammasome pathway.
- MeSH
- Cholesterol metabolism MeSH
- Cytokines metabolism MeSH
- Endothelial Cells metabolism pathology MeSH
- Inflammasomes * metabolism pharmacology MeSH
- Interleukin-18 metabolism MeSH
- Interleukin-1beta metabolism MeSH
- Clusterin metabolism pharmacology MeSH
- Aspartic Acid metabolism pharmacology MeSH
- Humans MeSH
- Macrophages metabolism MeSH
- Peptide Hydrolases metabolism pharmacology MeSH
- NLR Family, Pyrin Domain-Containing 3 Protein * metabolism MeSH
- Tumor Necrosis Factor-alpha metabolism MeSH
- Inflammation pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
In current study is done antioxidant, anticholinesterase, and carbonic anhydrase isoenzymes I and II inhibition assays, screening of biological active compounds and electronic microscopy analysis of secretory canals of fruits, flowers, roots, and aerial parts extracts and essential oils of Angelica purpurascens. Phenolic constituents, antioxidant, and anti-lipid peroxidation potentials of variants were estimated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and thiobarbituric acid (TBA) processes. Cholinesterase inhibition effect was detected through Ellman's method. The GC/ Mass Spectrometry (MS) and gas chromatography (GC)-flame Ionization Detector (FID) was used for essential oils analysis. NMR techniques was used for identification of the isolated compounds. The fruit hexane and dichloromethane fractions exhibited a greater antioxidant capacity and total phenolic content. The dichloromethane fraction of fruit demonstrated the most higher acetylcholinesterase inhibition (39.86 ± 2.63%), while the fruit hexane fraction displayed the best inhibition towards butyrylcholinesterase (84.02 ± 1.28%). Cytosolic isoenzymes of human carbonic anhydrase (hCA) I, and II isoenzymes were influentially suppressed by flower and fruit dichloromethane fractions with 1.650 and 2.020 µM IC50 values, respectively. The electronic microscopy analysis of secretory canals found that the small number of secretory canals were at leaf while the largest shape of secretory canals was at the fruit. The secretory canals of roots, aerial parts, and fruits include more monoterpene hydrocarbons, while the canals, existing in the flowers are qualified by a higher presence of sesquiterpenes β-caryophyllene (12.1%), germacrene D (4.5%) and ether octyl acetate (11.9%). The highest level of monoterpene β-phellandrene (47.6%) and limonene (8.2%) were found in the fruit essential oil. The next isolated compounds from fruits of A. purpurascens like stigmasterol, β-sitosterol, bergapten, and oxypeucedanin have shown high anticholinesterase and antioxidant activities.
- Publication type
- Journal Article MeSH
Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.
- MeSH
- Apoptosis * MeSH
- Cell Cycle MeSH
- Radiation, Ionizing MeSH
- Lymphoma metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Neoplasms, Radiation-Induced metabolism MeSH
- DNA Repair MeSH
- DNA Damage MeSH
- Cell Proliferation MeSH
- Protein Phosphatase 2C physiology MeSH
- Thymocytes cytology metabolism MeSH
- Thymus Gland * cytology metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.
- MeSH
- Acetonitriles chemistry MeSH
- Chemical Fractionation methods MeSH
- Chromatography, Liquid methods MeSH
- Phosphopeptides chemistry MeSH
- Phosphoproteins metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Proteome MeSH
- Proteomics MeSH
- Tandem Mass Spectrometry methods MeSH
- Titanium chemistry MeSH
- Pressure MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
