RESUMO
Cereal grains are an important source of food and feed. To provide comprehensive spatiotemporal information about biological processes in developing seeds of cultivated barley (Hordeum vulgare L. subsp. vulgare), we performed a transcriptomic study of the embryo, endosperm, and seed maternal tissues collected from grains 4-32 days after pollination. Weighted gene co-expression network and motif enrichment analyses identified specific groups of genes and transcription factors (TFs) potentially regulating barley seed tissue development. We defined a set of tissue-specific marker genes and families of TFs for functional studies of the pathways controlling barley grain development. Assessing selected groups of chromatin regulators revealed that epigenetic processes are highly dynamic and likely play a major role during barley endosperm development. The repressive H3K27me3 modification is globally reduced in endosperm tissues and at specific genes related to development and storage compounds. Altogether, this atlas uncovers the complexity of developmentally regulated gene expression in developing barley grains.
Assuntos
Endosperma , Regulação da Expressão Gênica de Plantas , Hordeum , Sementes , Transcriptoma , Hordeum/genética , Hordeum/crescimento & desenvolvimento , Hordeum/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transcriptoma/genética , Endosperma/genética , Endosperma/metabolismo , Endosperma/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Regulação da Expressão Gênica no Desenvolvimento , Epigênese Genética , Histonas/metabolismo , Histonas/genéticaRESUMO
Prioritization of self-related information (e.g. self-face) may be driven by its extreme familiarity. Nevertheless, the findings of numerous behavioral studies reported a self-preference for initially unfamiliar information, arbitrarily associated with the self. In the current study, we investigated the neural underpinnings of extremely familiar stimuli (self-face, close-other's face) and stimuli newly assigned to one's own person and to a close-other (abstract shapes). Control conditions consisted of unknown faces and unknown abstract shapes. Reaction times (RTs) to the self-face were shorter than to close-other's and unknown faces, whereas no RTs differences were observed for shapes. P3 amplitude to the self-face was larger than to close-other's and unknown faces. Nonparametric cluster-based permutation tests showed significant clusters for the self-face vs. other (close-other's, unknown) faces. However, in the case of shapes P3 amplitudes to the self-assigned shape and to the shape assigned to a close-other were similar, and both were larger than P3 to unknown shapes. No cluster was detected for the self-assigned shape when compared with the shape assigned to the close-other. Thus, our findings revealed preferential attentional processing of the self-face and the similar allocation of attentional resources to shapes assigned to the self and a close-other.
Assuntos
Face , Reconhecimento Visual de Modelos , Humanos , Atenção , Tempo de Reação , Reconhecimento PsicológicoRESUMO
Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.
Assuntos
Técnicas Biossensoriais , Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais/métodos , Técnicas de Microbalança de Cristal de Quartzo , Imunoensaio , Tetraspaninas , Vesículas Extracelulares/química , Biomarcadores , Tetraspanina 28 , Tetraspanina 30/análise , Tetraspanina 29/análiseRESUMO
Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.
Assuntos
Hordeum , Hordeum/genética , Endosperma/genética , Núcleo Celular/genética , Histonas/genética , Centrômero/genéticaRESUMO
This paper reports the synthesis and characterization of novel monoferrocenylsumanenes obtained by means of the Sonogashira cross-coupling or click chemistry reaction as well as their application in cesium cation electrochemical sensors. A new synthetic protocol based on Sonogashira cross-coupling was developed for the synthesis of monoferrocenylsumanene or ethynylsumanene. The click chemistry reaction was introduced to the sumanene chemistry through the synthesis of 1,2,3-triazole containing monoferrocenylsumanene. The designed synthetic methods for the modification of sumanene at the aromatic position proved to be efficient and proceeded under mild conditions. The synthesized sumanene derivatives were characterized by detailed spectroscopic analyses of the synthesized sumanene derivatives. The supramolecular interactions between cesium cations and the synthesized monoferrocenylsumanenes were spectroscopically and electrochemically investigated. Furthermore, the design of the highly selective and sensitive cesium cation fluorescence and electrochemical sensors comprising the synthesized monoferrocenylsumanenes as receptor compounds was analyzed. The tested cesium cation electrochemical sensors showed excellent limit of detection values in the range of 6.0-9.0 nM. In addition, the interactions between the synthesized monoferrocenylsumanenes and cesium cations were highly selective, which was confirmed by emission spectroscopy, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and cyclic voltammetry.
RESUMO
The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Subunidades Proteicas/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Óvulo Vegetal/genética , Pólen/genética , Ligação Proteica , Sementes/genética , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
In every pandemic, it is critical to test as many people as possible and keep track of the number of new cases of infection. Therefore, there is a need for novel, fast and unambiguous testing methods. In this study, we designed a sandwich-type voltammetric immunosensor based on unlabeled- and labeled with a redox probe antibodies against virus spike protein for fast and ultrasensitive detection of SARS-CoV-2. The process of the preparation of the sensor layer included chemisorption of cysteamine layer and covalent anchoring of antibody specific for the S1 subunit of the S protein. The source of the voltametric signal was the antibody labeled with the redox probe, which was introduced onto biosensor surface only after the recognition of the virus. This easy-to-handle immunosensor was characterized by a wide analytical range (2.0·10-7 to 0.20 mg·L-1) and low detection limit (8.0·10-8 mg·L-1 ≡ 0.08 pg·mL-1 ≡ 4 virions·µL-1). The utility of the designed device was also evidenced by the detection of SARS-CoV-2 in the clinical samples. Moreover, the main advantage and a huge novelty of the developed device, compared to those already existing, is the moment of generating the analytical signal of the redox probe that appears only after the virus recognition. Thus, our diagnostic innovation may considerably contribute to controlling the COVID-19 pandemic. The as-developed immunosensor may well offer a novel alternative approach for viral detection that could complement or even replace the existing methods.
RESUMO
Targeted drug delivery by nanocarriers molecules can increase the efficiency of cancer treatment. One of the targeting ligands is folic acid (FA), which has a high affinity for the folic acid receptors, which are overexpressed in many cancers. Herein, we describe the preparation of the nanoconjugates containing quantum dots (QDs) and ß-cyclodextrin (ß-CD) with foliate-targeting properties for the delivery of anticancer compound C-2028. C-2028 was bound to the nanoconjugate via an inclusion complex with ß-CD. The effect of using FA in QDs-ß-CD(C-2028)-FA nanoconjugates on cytotoxicity, cellular uptake, and the mechanism of internalization in cancer (H460, Du-145, and LNCaP) and normal (MRC-5 and PNT1A) cells was investigated. The QDs-ß-CD(C-2028)-FA were characterized using DLS (dynamic light scattering), ZP (zeta potential), quartz crystal microbalance with dissipation (QCM-D), and UV-vis spectroscopy. The conjugation of C-2028 with non-toxic QDs or QDs-ß-CD-FA did not change the cytotoxicity of this compound. Confocal microscopy studies proved that the use of FA in nanoconjugates significantly increased the amount of delivered compound, especially to cancer cells. QDgreen-ß-CD(C-2028)-FA enters the cells through multiple endocytosis pathways in different levels, depending on the cell line. To conclude, the use of FA is a good self-navigating molecule in the QDs platform for drug delivery to cancer cells.
Assuntos
Acridinas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Ácido Fólico/farmacologia , Acridinas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Nanoconjugados/química , Nanoestruturas , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Pontos Quânticos/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologiaRESUMO
Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.
Assuntos
Dano ao DNA , Epigênese Genética , Inativação Gênica , Adenosina/análogos & derivados , Adenosina/farmacologia , Arabidopsis/genética , Azacitidina/farmacologia , Aberrações Cromossômicas/efeitos dos fármacos , Citidina/análogos & derivados , Citidina/farmacologia , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Decitabina/farmacologia , Epigênese Genética/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Heterocromatina/efeitos dos fármacos , Interferência de RNA/efeitos dos fármacos , Sequências de Repetição em Tandem/efeitos dos fármacosRESUMO
Seeds are complex biological systems comprising three genetically distinct tissues: embryo, endosperm, and maternal tissues (including seed coats and pericarp) nested inside one another. Cereal grains represent a special type of seeds, with the largest part formed by the endosperm, a specialized triploid tissue ensuring embryo protection and nourishment. We investigated dynamic changes in DNA content in three of the major seed tissues from the time of pollination up to the dry seed. We show that the cell cycle is under strict developmental control in different seed compartments. After an initial wave of active cell division, cells switch to endocycle and most endoreduplication events are observed in the endosperm and seed maternal tissues. Using different barley cultivars, we show that there is natural variation in the kinetics of this process. During the terminal stages of seed development, specific and selective loss of endoreduplicated nuclei occurs in the endosperm. This is accompanied by reduced stability of the nuclear genome, progressive loss of cell viability, and finally programmed cell death. In summary, our study shows that endopolyploidization and cell death are linked phenomena that frame barley grain development.
Assuntos
Hordeum , Ciclo Celular , Endorreduplicação , Endosperma/genética , Hordeum/genética , Sementes/genéticaRESUMO
Visual objects are typically perceived as parts of an entire visual scene, and the scene's context provides information crucial in the object recognition process. Fundamental insights into the mechanisms of context-object integration have come from research on semantically incongruent objects, which are defined as objects with a very low probability of occurring in a given context. However, the role of attention in processing of the context-object mismatch remains unclear, with some studies providing evidence in favor, but other against an automatic capture of attention by incongruent objects. Therefore, in the present study, 25 subjects completed a dot-probe task, in which pairs of scenes-congruent and incongruent or neutral and threatening-were presented as task-irrelevant distractors. Importantly, threatening scenes are known to robustly capture attention and thus were included in the present study to provide a context for interpretation of results regarding incongruent scenes. Using N2 posterior-contralateral ERP component as a primary measure, we revealed that threatening images indeed capture attention automatically and rapidly, but semantically incongruent scenes do not benefit from an automatic attentional selection. Thus, our results suggest that identification of the context-object mismatch is not preattentive.
Assuntos
Atenção/fisiologia , Encéfalo/fisiologia , Potenciais Evocados/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Adulto , Eletroencefalografia , Feminino , Humanos , Masculino , Percepção Visual , Adulto JovemRESUMO
The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4-naphtoquinone derivative, plumbagin (up to 106.63 mg × g-1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli and up to 7% against Pseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. KEY POINTS: ⢠Rhizobium-mediated transformation created Dionaea muscipula teratomas. ⢠Transformed plants had highly increased synthesis of phenolic compounds. ⢠The MBC value was connected with plumbagin and phenolic acid concentrations.
Assuntos
Droseraceae , Agrobacterium/genética , Antibacterianos/farmacologia , Humanos , FenóisRESUMO
Nearly half of patients with advanced and metastatic melanomas harbor a BRAF mutation. Vemurafenib (VEM), a BRAF inhibitor, is used to treat such patients, however, responses to VEM are very short-lived due to intrinsic, adaptive and/or acquired resistance. In this context, we present the action of the B-Raf serine-threonine protein kinase inhibitor (vemurafenib) on the glycans structure and metallomics profiles in melanoma cells without (MeWo) and with (G-361) BRAF mutations. The studies were performed using α1-acid glycoprotein (AGP), a well-known acute-phase protein, and concanavalin A (Con A), which served as the model receptor. The detection of changes in the structure of glycans can be successfully carried out based on the frequency shifts and the charge transfer resistance after interaction of AGP with Con A in different VEM treatments using QCM-D and EIS measurements. These changes were also proved based on the cell ultrastructure examined by TEM and SEM. The LA-ICP-MS studies provided details on the metallomics profile in melanoma cells treated with and without VEM. The studies evidence that vemurafenib modifies the glycans structures and metallomics profile in melanoma cells harboring BRAF mutation that can be further implied in the resistance phenomenon. Therefore, our data opens a new avenue for further studies in the short-term addressing novel targets that hopefully can be used to improve the therapeutic regiment in advanced melanoma patients. The innovating potential of this study is fully credible and has a real impact on the global patient society suffering from advanced and metastatic melanomas.
Assuntos
Melanoma/metabolismo , Metais/metabolismo , Mutação , Polissacarídeos/química , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/farmacologia , Concanavalina A/química , Concanavalina A/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Metais/análise , Orosomucoide/química , Orosomucoide/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
This work presents a new look at the application of cyclodextrins (CD) as a drug nanocarrier. Two different cyclodextrins (αCD, ßCD) were covalently conjugated to branched polyethylenimine (PEI), which was additionally functionalized with folic acid (PEI-ßCD-αCD-FA). Here, we demonstrated that the combination of αCD and ßCD enabled to load and control release of two anticancer drugs: doxorubicin (DOX) and beta-lapachone (beta-LP) (DOX in ß-CD and beta-LP into α-CD) via host-guest inclusion. The PEI-ßCD(DOX)-αCD-FA nanoconjugate was used to transport anticancer drugs into A549 lung cancer cells for estimation the cytotoxic and antitumor effect of this nanoconjugate. The presence of FA molecules should facilitate the penetration of studied nanoconjugate into the cell. Whereas, the non-cellular experiments proved that the drugs are released from the carrier mainly in the pH 4.0. The release mechanism is found to be anomalous in all studied cases.
Assuntos
Ciclodextrinas/química , Doxorrubicina/farmacologia , Naftoquinonas/farmacologia , Polietilenoimina/química , Células A549 , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Doxorrubicina/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Difusão Dinâmica da Luz , Ácido Fólico/farmacologia , Humanos , Hidrodinâmica , Cinética , Nanoconjugados/química , Naftoquinonas/química , Tamanho da Partícula , Polímeros/química , Espectroscopia de Prótons por Ressonância Magnética , Técnicas de Microbalança de Cristal de Quartzo , Espectrofotometria UltravioletaRESUMO
Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, ß-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.
Assuntos
Secas , Congelamento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Haploidia , Hordeum/fisiologia , Proteoma , Estresse Fisiológico , Adaptação Biológica , Fases de Leitura Aberta , Proteômica/métodosRESUMO
A key mechanism behind preferential processing of self-related information might be an early and automatic capture of attention. Therefore, the present study tested a hypothesis that one's own face will attract bottom-up attention even without conscious identification. To test this, we used a dot-probe paradigm with electrophysiological recordings, in which participants ( N = 18) viewed masked and unmasked pairs of faces (other, self) presented laterally. Analysis of the sensitivity measure d ' indicated that faces were not consciously identified in the masked condition. A clear N2 posterior-contralateral (N2pc) component (a neural marker of attention shifts) was found in both the masked and unmasked conditions, revealing that one's own face automatically captures attention when processed unconsciously. Therefore, our study (a) demonstrates that self-related information is boosted at an early (preconscious) stage of processing, (b) identifies further features (beyond simple physical ones) that cause automatic attention capture, and (c) provides further evidence for the dissociative nature of attention and consciousness.
Assuntos
Atenção , Reconhecimento Facial , Imaginação , Reconhecimento Visual de Modelos , Adulto , Feminino , Humanos , Masculino , Mascaramento Perceptivo , Tempo de Reação , Adulto JovemRESUMO
Novel conjugates of ferrocene with uracil, 5-fluorouracil, tegafur, or acyclovir are reported. Their synthesis involved (i) the azide-alkyne 1,3-dipolar cycloaddition or (ii) the formation of the ester linkage. For the first time, we present an in-depth insight into the supramolecular interactions between ß-cyclodextrin and ferrocene-nucleobase derivatives. Spectroscopic and voltammetric analyses performed within this work suggested that the ferrocene or adamantane unit of the conjugates interacted with the ß-cyclodextrin's inner cavity. The methods applied for the supramolecular studies included 1H-1H ROESY NMR, 1H NMR titration, Fourier-transform infrared spectroscopy, cyclic voltammetry, fluorescence spectra titration, and 1H DOSY NMR. 1H DOSY NMR was also employed to evaluate the apparent binding constants for all the complexes. The ferrocene-acyclovir conjugate Fc-5 featured the highest apparent binding constant value among all the complexes tested.
RESUMO
Here, we report the metabolic profile and the results of associated metabolic studies of 2-hydroxy-acridinone (2-OH-AC), the reference compound for antitumor-active imidazo- and triazoloacridinones. Electrochemistry coupled with mass spectrometry was applied to simulate the general oxidative metabolism of 2-OH-AC for the first time. The reactivity of 2-OH-AC products to biomolecules was also examined. The usefulness of the electrochemistry for studying the reactive drug metabolite trapping (conjugation reactions) was evaluated by the comparison with conventional electrochemical (controlled-potential electrolysis) and enzymatic (microsomal incubation) approaches. 2-OH-AC oxidation products were generated in an electrochemical thin-layer cell. Their tentative structures were assigned based on tandem mass spectrometry in combination with accurate mass measurements. Moreover, the electrochemical conversion of 2-OH-AC in the presence of reduced glutathione and/or N-acetylcysteine unveiled the formation of reactive metabolite-nucleophilic trapping agent conjugates (m/z 517 and m/z 373, respectively) through the thiol group. This glutathione S-conjugate was also identified after electrolysis experiment as well as was detected in liver microsomes. Summing up, the present work illustrates that the electrochemical simulation of metabolic reactions successfully supports the results of classical electrochemical and enzymatic studies. Therefore, it can be a useful tool for synthesis of drug metabolites, including reactive metabolites.
Assuntos
Acridinas/metabolismo , Antineoplásicos/metabolismo , Eletroquímica , Espectrometria de Massas , Desintoxicação Metabólica Fase II , Desintoxicação Metabólica Fase I , Acridinas/química , Animais , Eletrólise , Feminino , Glutationa/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos Sprague-DawleyRESUMO
The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented ß-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and ß-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting ß-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.
Assuntos
Fosfatos de Dinucleosídeos/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Compostos de Sulfidrila/farmacologia , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Relação Dose-Resposta a Droga , Endorribonucleases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
Information regarding the past-self may be viewed as information referring to other people. However, evidence supporting this notion at the neural level is rather sparse and it remains unclear whether the past-self is processed like any 'other' or like the close-other only. The aim of this event-related potential study was to investigate this issue. A reflection task requiring evaluation of positive and negative trait adjectives with respect to present- and past-self, a close-other and a famous person was applied. We hypothesized that the past-self and close-other conditions would share their neural underpinnings. The process of reflection on the past-self and close-other was indeed associated with similar mean amplitudes of the late positive component (LPC), whereas in the case of the past-self vs. famous person comparison LPC was significantly enhanced for the past-self. Analogous effects were observed when LPC was calculated for trials with traits judged as either suitable or unsuitable to describe a person who was the target of reflection. Thus, these findings suggest that the processing of information related to the past-self resembles processing of information related to a personally relevant other. Moreover, sex-differences were observed in reaction times and LPC amplitudes for responses reflecting the positivity bias.