Selective inhibition of histone deacetylase 6 (HDAC6) is being recognized as a therapeutic approach for cancers. In this study, we designed a new HDAC6 inhibitor, named Suprastat, using in silico simulations. X-ray crystallography and molecular dynamics simulations provide strong evidence to support the notion that the aminomethyl and hydroxyl groups in the capping group of Suprastat establish significant hydrogen bond interactions, either direct or water-mediated, with residues D460, N530, and S531, which play a vital role in regulating the deacetylase function of the enzyme and which are absent in other isoforms. In vitro characterization of Suprastat demonstrates subnanomolar HDAC6 inhibitory potency and a hundred- to a thousand-fold HDAC6 selectivity over the other HDAC isoforms. In vivo studies reveal that a combination of Suprastat and anti-PD1 immunotherapy enhances antitumor immune response, mediated by a decrease of protumoral M2 macrophages and increased infiltration of antitumor CD8+ effector and memory T-cells.

• MeSH
• fenylmočovinové sloučeniny chemická syntéza   metabolismus   terapeutické užití   MeSH
• histondeacetylasa 6 antagonisté a inhibitory   metabolismus   MeSH
• imunologické faktory chemická syntéza   metabolismus   terapeutické užití   MeSH
• imunoterapie MeSH
• inhibitory histondeacetylas chemická syntéza   metabolismus   terapeutické užití   MeSH
• jaterní mikrozomy metabolismus   MeSH
• krysa rodu rattus MeSH
• krystalografie rentgenová MeSH
• kyseliny hydroxamové chemická syntéza   metabolismus   terapeutické užití   MeSH
• lidé MeSH
• melanom farmakoterapie   terapie   MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• racionální návrh léčiv MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

• MeSH
• Aspergillus fumigatus metabolismus   MeSH
• biologické markery analýza   MeSH
• chromatografie kapalinová metody   MeSH
• data mining metody   MeSH
• datové soubory jako téma MeSH
• ferrichrom analogy a deriváty   izolace a purifikace   metabolismus   MeSH
• fixace tkání metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• invazivní plicní aspergilóza diagnóza   mikrobiologie   MeSH
• kryoultramikrotomie metody   MeSH
• krysa rodu rattus MeSH
• kyseliny hydroxamové izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• siderofory izolace a purifikace   metabolismus   MeSH
• software MeSH
• železité sloučeniny izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the current study, pyroglutamic acid (pGlu), a natural amino acid derivative, has efficiently inhibited the catalytic activities of three important enzymes, namely: Human recombinant phosphodiesterase-5A1 (PDE5A1), human angiotensin-converting enzyme (ACE), and urease. These enzymes were reported to be associated with several important clinical conditions in humans. Radioactivity-based assay, spectrophotometric-based assay, and an Electrospray Ionization-Mass Spectrometry-based method were employed to ascertain the inhibitory actions of pGlu against PDE5A1, ACE, and urease, respectively. The results unveiled that pGlu potently suppressed the activity of PDE5A1 (half-maximal inhibitory concentration; IC50 = 5.23 µM) compared with that of standard drug sildenafil citrate (IC50 = 7.14 µM). Moreover, pGlu at a concentration of 20 µg/mL was found to efficiently inhibit human ACE with 98.2% inhibition compared with that of standard captopril (99.6%; 20 µg/mL). The urease-catalyzed reaction was also remarkably inactivated by pGlu and standard acetohydroxamic acid with IC50 values of 1.8 and 3.9 µM, respectively. Remarkably, the outcome of in vitro cytotoxicity assay did not reveal any significant cytotoxic properties of pGlu against human cervical carcinoma cells and normal human fetal lung fibroblast cells. In addition to in vitro assays, molecular docking analyses were performed to corroborate the outcomes of in vitro results with predicted structure-activity relationships. In conclusion, pGlu could be presented as a natural and multifunctional agent with promising applications in the treatment of some ailments connected with the above-mentioned anti-enzymatic properties.

• MeSH
• angiotensin konvertující enzym chemie   genetika   metabolismus   MeSH
• buněčné linie MeSH
• cyklické nukleotidfosfodiesterasy, typ 5 chemie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• inhibiční koncentrace 50 MeSH
• kaptopril chemie   metabolismus   MeSH
• kyselina pyrrolidonkarboxylová chemie   metabolismus   toxicita   MeSH
• kyseliny hydroxamové antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• rekombinantní proteiny biosyntéza   chemie   izolace a purifikace   MeSH
• sildenafil citrát chemie   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• spektrofotometrie MeSH
• terciární struktura proteinů MeSH
• ureasa antagonisté a inhibitory   metabolismus   MeSH
• vazebná místa MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• biologický transport MeSH
• endocytóza fyziologie   MeSH
• houby patogenita   fyziologie   MeSH
• interakce hostitele a patogenu MeSH
• kyseliny hydroxamové metabolismus   MeSH
• lidé MeSH
• molekulární zobrazování metody   trendy   MeSH
• mykózy diagnóza   metabolismus   MeSH
• radioizotopy galia MeSH
• siderofory metabolismus   MeSH
• železité sloučeniny metabolismus   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1β to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPβ to UV-irradiated chromatin.

• MeSH
• acetylace MeSH
• buněčné linie MeSH
• buňky 3T3 MeSH
• časosběrné zobrazování MeSH
• chromatin metabolismus   účinky záření   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• FRAP MeSH
• histony metabolismus   MeSH
• inhibitory histondeacetylas metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kyseliny hydroxamové metabolismus   MeSH
• lidé MeSH
• myši MeSH
• poškození DNA MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity. The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. Furthermore, we estimated the reduced presence of HDAC2 and HDAC3 proteins by NaB and TSA treatment in AR-negative DU145 cell line. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor protein.

• MeSH
• androgenní receptory genetika   metabolismus   MeSH
• butyráty metabolismus   farmakologie   terapeutické užití   MeSH
• časové faktory MeSH
• histondeacetylasa 2 metabolismus   MeSH
• histondeacetylasy metabolismus   MeSH
• imunoprecipitace MeSH
• inhibitory histondeacetylas terapeutické užití   MeSH
• korepresor 2 jaderného receptoru genetika   metabolismus   MeSH
• kyseliny hydroxamové metabolismus   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• vazba proteinů genetika   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• MeSH
• Escherichia genetika   metabolismus   MeSH
• finanční podpora výzkumu jako téma MeSH
• kosmidy analýza   genetika   MeSH
• kyseliny hydroxamové metabolismus   MeSH
• techniky in vitro MeSH

Thirty seven strains of Escherichia coli isolated from the urine of patients with acute symptomatic urinary tract infection were examined for siderophore production: hydroxamate (aerobactin) and phenolate (enterochelin). All the strains were found to produce varying amounts of enterochelin. With the chemical assay, 24.3% strains were aerobactin producers, while 43.2% were positive in the bio-assay. All the aerobactin producers carried the aerobactin receptor on their surface. Attempts to correlate siderophore production with growth in minimal and iron-depleted medium showed that there was a positive quantitative correlation between enterochelin production and growth of organisms under iron depletion. Aerobactin production failed to give an additional advantage of growth to strains producing enterochelin.

• MeSH
• chelátory železa metabolismus   MeSH
• enterochelin metabolismus   MeSH
• Escherichia coli účinky léků   růst a vývoj   izolace a purifikace   MeSH
• infekce močového ústrojí mikrobiologie   MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• kyseliny hydroxamové metabolismus   MeSH
• lidé MeSH
• proteiny vnější bakteriální membrány * MeSH
• receptory imunologické analýza   MeSH
• serin analogy a deriváty   MeSH
• siderofory MeSH
• železo metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH