Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.
- Klíčová slova
- Brassica napus, Brassinosteroids, Enzyme immunoassay, Immunoaffinity chromatography, Liquid chromatography-tandem mass spectrometry, Monoclonal antibodies,
- MeSH
- Brassica napus chemie MeSH
- brassinosteroidy analýza izolace a purifikace MeSH
- chromatografie afinitní metody MeSH
- imobilizační protilátky chemie MeSH
- imunosorbenty chemie MeSH
- regulátory růstu rostlin analýza izolace a purifikace MeSH
- rostlinné extrakty chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- brassinosteroidy MeSH
- imobilizační protilátky MeSH
- imunosorbenty MeSH
- regulátory růstu rostlin MeSH
- rostlinné extrakty MeSH
A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3β-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).
- Klíčová slova
- 28-homocastasterone, brassinosteroids, enantiospecific reaction, tritium dehalogenation, tritium labeling,
- MeSH
- cholestanony chemie MeSH
- izotopové značení MeSH
- katalýza MeSH
- stereoizomerie MeSH
- tritium chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cholestanony MeSH
- homocastasterone MeSH Prohlížeč
- tritium MeSH
In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 μg/mL.
- Klíčová slova
- enzyme-linked immunosorbent assay, ghrelin, lipopeptides, liquid chromatography mass spectrometry, monolithic columns,
- MeSH
- chromatografie kapalinová * MeSH
- ghrelin analogy a deriváty MeSH
- kalibrace MeSH
- lipopeptidy krev MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ghrelin MeSH
- lipopeptidy MeSH
Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.
- MeSH
- antagonisté antidiuretického hormonu izolace a purifikace metabolismus MeSH
- Formicidae MeSH
- lidé MeSH
- mutační analýza DNA MeSH
- neuropeptidy genetika izolace a purifikace metabolismus MeSH
- receptory vasopresinů agonisté MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- substituce aminokyselin MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antagonisté antidiuretického hormonu MeSH
- neuropeptidy MeSH
- receptory vasopresinů MeSH
- rekombinantní proteiny MeSH
The radioactively labelled 6-amino-5-nitroso-uracil (1) and 5-acetyl-6-amino-1,3-dimethyl-uracil (2) were required for metabolic studies to assess their suitability as drug candidates. A common precursor for both compounds was [cyano-14 C]cyanoacetic acid (6), readily prepared from potassium [14 C]cyanide. ACS reagents, namely, diethyl ether, acetic acid and acetic anhydride, had to be rigorously repurified to achieve a successful synthesis of 14 C-labelled compounds on a tenth-of-a-milligramme scale. 6-Amino-5-nitroso-[6-14 C]uracil (1-14 C) (0.55 mCi) was prepared with radiochemical purity > 98% and specific activity (SA) = 55.6 mCi/mmol. 5-Acetyl-6-amino-1,3-dimethyl-[6-14 C]uracil (2-14 C) (8 mCi) was prepared with radiochemical purity > 97% and SA = 55.6 mCi/mmol. It has been shown that a SA assay can be made from standard 13 C NMR spectra, thus avoiding the need to perform lengthier inverse-gated 13 C NMR experiments.
- Klíčová slova
- 6-amino-uracil derivatives, [cyano-14C]cyanoacetic acid, carbon 14, specific activity assay,
- MeSH
- acetáty chemie MeSH
- izotopové značení MeSH
- radioizotopy uhlíku chemie MeSH
- techniky syntetické chemie MeSH
- uracil analogy a deriváty chemická syntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6-aminouracil MeSH Prohlížeč
- acetáty MeSH
- cyanoacetic acid MeSH Prohlížeč
- radioizotopy uhlíku MeSH
- uracil MeSH
3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.
- Klíčová slova
- C─H activation, borotritides, hydrogen/deuterium exchange, iridium catalyst, tritium-labeled γ-hydroxybutyric acid,
- MeSH
- alkeny chemie MeSH
- bor chemie MeSH
- deuterium chemie MeSH
- hydroxybutyráty chemie MeSH
- iridium chemie MeSH
- izotopové značení MeSH
- katalýza MeSH
- ligandy MeSH
- oxidace-redukce MeSH
- tritium chemie MeSH
- vodík-deuteriová výměna * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 4-hydroxybutyric acid MeSH Prohlížeč
- alkeny MeSH
- bor MeSH
- deuterium MeSH
- hydroxybutyráty MeSH
- iridium MeSH
- ligandy MeSH
- tritium MeSH
7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.
- MeSH
- analýza přežití MeSH
- DNA genetika metabolismus MeSH
- fibroblasty MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie genetika metabolismus patologie MeSH
- permeabilita buněčné membrány účinky léků MeSH
- poškození DNA účinky léků MeSH
- proliferace buněk účinky léků MeSH
- proteosyntéza účinky léků MeSH
- protinádorové látky chemie metabolismus farmakologie MeSH
- regulace genové exprese u nádorů MeSH
- sbalování proteinů účinky léků MeSH
- tubercidin analogy a deriváty chemie metabolismus farmakologie MeSH
- výsledek terapie MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- messenger RNA MeSH
- protinádorové látky MeSH
- tubercidin MeSH
6-Chloropurines substituted at the position 9 with variously modified bicyclic skeletons represent promising antiviral and anticancer agents. This work aimed to investigate the transport mechanisms of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP) and their relationship to the metabolism and biological activity of the compound. Transport experiments were conducted in CCRF-CEM cells using radiolabeled compound ([(3)H]NCP). The pattern of the intracellular uptake of [(3)H]NCP in CCRF-CEM cells pointed to a combination of passive and facilitated diffusion as prevailing transport mechanisms. NCP intracellular metabolism was found to enhance its uptake by modifying NCP concentration gradient. The transport kinetics reached steady state under the conditions of MRP and MDR proteins blockade, indicating that NCP is a substrate for these efflux pumps. Their inhibition also increased the cytotoxicity of NCP. Our findings suggest that the novel nucleoside analog NCP has potential to become a new orally available antileukemic agent due to its rapid membrane permeation.
- Klíčová slova
- CCRF-CEM cells, Carbocyclic nucleoside analogs, MRP proteins, P-glycoprotein, facilitated diffusion,
- MeSH
- ABC transportéry antagonisté a inhibitory genetika metabolismus MeSH
- biologický transport MeSH
- buthionin sulfoximin farmakologie MeSH
- chinoliny farmakologie MeSH
- dibenzocyklohepteny farmakologie MeSH
- exprese genu MeSH
- kinetika MeSH
- kyselina ethakrynová farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- permeabilita buněčné membrány účinky léků MeSH
- propionáty farmakologie MeSH
- protinádorové látky chemická syntéza metabolismus farmakologie MeSH
- puriny chemická syntéza metabolismus farmakologie MeSH
- T-lymfocyty účinky léků metabolismus patologie MeSH
- tritium MeSH
- usnadněná difuze MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 9-norbornyl-6-chloropurine MeSH Prohlížeč
- ABC transportéry MeSH
- buthionin sulfoximin MeSH
- chinoliny MeSH
- dibenzocyklohepteny MeSH
- kyselina ethakrynová MeSH
- propionáty MeSH
- protinádorové látky MeSH
- puriny MeSH
- tritium MeSH
- verlukast MeSH Prohlížeč
- zosuquidar trihydrochloride MeSH Prohlížeč
AIM: 6-Chloropurines substituted at position 9 with bicyclic skeletons represent promising chemotherapeutic agents. We explored the metabolism and membrane transport of 9-norbornyl-6-chloropurine (NCP) aiming to understand its mechanism of action. MATERIALS AND METHODS: The metabolism of NCP was studied in vitro in whole cells (CCRF-CEM), cellular extracts, subcellular fractions and purified enzymes. Transport experiments were conducted in Caco-2 cell monolayers. RESULTS: Three metabolites were identified, a glutathione conjugate (NCP-GS), NCP-cysteinylglycine and NCP-cysteine. Both glutathione-S-transferase inhibition and glutathione (GSH) depletion prevented metabolite formation and increased the cytotoxicity of NCP. Transepithelial transport (Caco-2) indicated good permeability, with Papp (12.6±0.3) ×10(-5) cm/s. Importantly, the drug induced glutathione depletion in treated cells and affected the activity of several GSH-dependent enzymes. CONCLUSION: The novel nucleoside analog NCP represents a promising orally available antileukemic agent, acting through lowering of GSH levels in tumor cells.
- Klíčová slova
- Substituted 6-chloropurines, carbocyclic nucleoside analogs, glutathione depletion, glutathione-S-transferase,
- MeSH
- biologický transport účinky léků MeSH
- Caco-2 buňky MeSH
- epitelové buňky účinky léků metabolismus MeSH
- glutathion metabolismus MeSH
- glutathiontransferasa antagonisté a inhibitory metabolismus MeSH
- inhibitory cytochromu P450 MeSH
- leukemie farmakoterapie patologie MeSH
- lidé MeSH
- protinádorové látky chemie farmakologie terapeutické užití MeSH
- puriny chemie farmakologie terapeutické užití MeSH
- screeningové testy protinádorových léčiv MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- xanthinoxidasa antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6-chloropurine MeSH Prohlížeč
- 9-norbornyl-6-chloropurine MeSH Prohlížeč
- glutathion MeSH
- glutathiontransferasa MeSH
- inhibitory cytochromu P450 MeSH
- protinádorové látky MeSH
- puriny MeSH
- systém (enzymů) cytochromů P-450 MeSH
- xanthinoxidasa MeSH
Neuropeptide FF (NPFF) belongs to the RF-amide family of peptides bearing the identical C-terminal amino acid sequence (R-F-NH2). In addition to NPFF, prolactin-releasing peptide (PrRP), another RF-amide, binds to NPFF receptors with high affinity. A selective antagonist of PrRP has not yet been identified, but a selective antagonist of NPFF, 1-adamantanecarbonyl-RF-NH2 (RF9), was recently reported to antagonize the hyperalgesic effect of NPFF after central administration to mice. In the present study, RF9 competed with NPFF analog D-Y-L-(N-Me)-F-Q-P-Q-R-F-NH2 (1DMe) in binding to CHO-K1 cell membranes transfected with the human NPFF2 receptor. In rat pituitary RC-4B/C cells, where the expression of the NPFF2 receptor was proved by immunodetection, RF9 did not reverse the phosphorylation of MAPK/ERK1/2 induced by [Tyr(1)]NPFF. In vivo experiments with fasted mice confirmed that centrally injected [Tyr(1)]NPFF significantly lowered food intake. However, RF9, a putative NPFF2 antagonist, did not reverse the anorectic effect of [Tyr(1)]NPFF. Paradoxically, RF9 itself exhibited an anorectic effect in fasted mice not only after intracerebroventricular but also after subcutaneous administration. This finding casts doubt on claims that RF9 is an NPFF antagonist.
- MeSH
- adamantan analogy a deriváty farmakologie MeSH
- anorektika farmakologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- dipeptidy farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- kompetitivní vazba MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- nádorové buněčné linie MeSH
- oligopeptidy metabolismus MeSH
- přijímání potravy účinky léků MeSH
- receptory neuropeptidů metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adamantan MeSH
- adamantylcarbonyl-arginyl-phenylalaninamide MeSH Prohlížeč
- anorektika MeSH
- dipeptidy MeSH
- extracelulárním signálem regulované MAP kinasy MeSH
- neuropeptide FF receptor MeSH Prohlížeč
- neuropeptide FF, Tyr(1)-N-methyl-Phe(3)- MeSH Prohlížeč
- oligopeptidy MeSH
- receptory neuropeptidů MeSH
