RESUMO
RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.
Assuntos
Ficusina/química , RNA de Cadeia Dupla/química , Animais , Pareamento de Bases , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , RNA Longo não Codificante/químicaRESUMO
During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , N-Glicosil Hidrolases/metabolismo , Animais , Sistema Livre de Células/química , Reagentes de Ligações Cruzadas/química , DNA/biossíntese , DNA/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ficusina/química , N-Glicosil Hidrolases/química , Xenopus laevisRESUMO
Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.
Assuntos
Reagentes de Ligações Cruzadas , DNA , Conformação de Ácido Nucleico , Raios Ultravioleta , DNA/química , Reagentes de Ligações Cruzadas/química , Processos Fotoquímicos , Ficusina/químicaRESUMO
Characterization of RNA-protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.
Assuntos
Reagentes de Ligações Cruzadas/síntese química , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ficusina/química , Ligação Proteica , RNA Nucleolar Pequeno/química , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiaeRESUMO
The Psoralen (Pso) molecule finds extensive applications in photo-chemotherapy, courtesy of its triplet state forming ability. Sulfur and selenium replacement of exocyclic carbonyl oxygen of organic chromophores foster efficient triplet harvesting with near unity triplet quantum yield. These triplet-forming photosensitizers are useful in Photodynamic Therapy (PDT) applications for selective apoptosis of cancer cells. In this work, we have critically assessed the effect of the sulfur and selenium substitution at the exocyclic carbonyl (TPso and SePso, respectively) and endocyclic oxygen positions of Psoralen. It resulted in a significant redshifted absorption spectrum to access the PDT therapeutic window with increased oscillator strength. The reduction in singlet-triplet energy gap and enhancement in the spin-orbit coupling values increase the number of intersystem crossing (ISC) pathways to the triplet manifold, which shortens the ISC lifetime from 10-5â s for Pso to 10-8â s for TPso and 10-9â s for SePso. The intramolecular photo-induced electron transfer process, a competitive pathway to ISC, is also considerably curbed by exocyclic functionalizations. In addition, a maximum of 115â GM of two-photon absorption (2PA) with IR absorption (660-1050â nm) confirms that the Psoralen skeleton can be effectively tweaked via single chalcogen atom replacement to design a suitable PDT photosensitizer.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fotoquimioterapia/métodos , Humanos , Selênio/química , Ficusina/química , Ficusina/farmacologia , Enxofre/químicaRESUMO
Psoralens are eponymous for PUVA (psoralen plus UV-A radiation) therapy, which inter alia can be used to treat various skin diseases. Based on the same underlying mechanism of action, the synthetic psoralen amotosalen (AMO) is utilized in the pathogen reduction technology of the INTERCEPT® Blood System to inactivate pathogens in plasma and platelet components. The photophysical behavior of AMO in the absence of DNA is remarkably similar to that of the recently studied psoralen 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). By means of steady-state and time-resolved spectroscopy, intercalation and photochemistry of AMO and synthetic DNA were studied. AMO intercalates with a higher affinity into A,T-only DNA (KD = 8.9 × 10-5 M) than into G,C-only DNA (KD = 6.9 × 10-4 M). AMO covalently photobinds to A,T-only DNA with a reaction quantum yield of ΦR = 0.11. Like AMT, it does not photoreact following intercalation into G,C-only DNA. Femto- and nanosecond transient absorption spectroscopy reveals the characteristic pattern of photobinding to A,T-only DNA. For AMO and G,C-only DNA, signatures of a photoinduced electron transfer are recorded.
Assuntos
Ficusina , Furocumarinas , Ficusina/farmacologia , Ficusina/química , Furocumarinas/farmacologia , Furocumarinas/química , DNA/química , Análise EspectralRESUMO
Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , Saccharomyces cerevisiae/genética , Transcriptoma , Sítios de Ligação , Diferenciação Celular , Biologia Computacional , Reagentes de Ligações Cruzadas/química , Bases de Dados Genéticas , Células-Tronco Embrionárias/metabolismo , Ficusina/química , Regulação Fúngica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Células HeLa , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Neoplásico/química , RNA Neoplásico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Dorstenia psilurus is a widely used plant spice in traditional African medicine to treat pain-related conditions. However, the anti-inflammatory mechanisms underlying this activity and the main active ingredients of D. psilurus have not yet been fully characterized. This study aimed to isolate and identify the main active anti-inflammatory constituents of the D. psilurus extract and to investigate the underlying anti-inflammatory mechanisms in murine macrophages. Chromatographic techniques and spectroscopic data were used for compound isolation and structure elucidation. The Griess reagent method and the ferrous oxidation-xylenol orange assay were used to evaluate the inhibition of NO production and 15-lipoxygenase activity, respectively. Cyclooxygenase activity was assessed using the fluorometric COX activity assay kit, and Th1/Th2 cytokine measurement was performed using a flow cytometer. The results indicated that the extract and fractions of D. psilurus inhibit NO production and proliferation of RAW 264.7 macrophage cells. Bioguided fractionation led to the identification of psoralen, a furocoumarin, as the main bioactive anti-inflammatory compound. Psoralen inhibited NO production and 15-lipoxygenase activity and reduced pro-inflammatory Th1 cytokines (IFN-γ, TNF-α, and IL-2) while increasing the secretion of anti-inflammatory cytokines (IL-4, IL-6, and IL-10) in activated RAW 264.7 macrophage cells. The encouraging results obtained in this study suggest that psoralen-based multiple modulation strategies could be a useful approach to address the treatment of inflammatory diseases.
Assuntos
Citocinas , Ficusina , Lipopolissacarídeos , Macrófagos , Raízes de Plantas , Animais , Camundongos , Células RAW 264.7 , Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Raízes de Plantas/química , Lipopolissacarídeos/farmacologia , Ficusina/farmacologia , Ficusina/química , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/metabolismo , Células Th2/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Óxido Nítrico/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/químicaRESUMO
A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.
Assuntos
Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Cumarínicos/farmacologia , Ficusina/farmacologia , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Ficusina/síntese química , Ficusina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Photo-controlled or photo-regulated molecules, especially biologically active and operating in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the alkaline solvolysis of psoralen led to furocoumaric acid, which was further Z â E photoisomerized (365 nm) to furocoumarinic acid. The kinetics of Z â E photoisomerization was monitored by HPLC and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids, as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic acid was found to be visibly fluorescent at pH 2.0-12.0, with the maxima of fluorescence emission spectra being pH-dependent. The reverse E â Z photoisomerization predicted by quantum chemistry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is particularly valuable in view of a wide range of pharmacological effects previously predicted for this compound.
Assuntos
Furocumarinas/química , Furocumarinas/efeitos da radiação , Luz , Ficusina/química , Fluorescência , Concentração de Íons de Hidrogênio , Isomerismo , Conformação Molecular , Pironas/química , Espectrofotometria UltravioletaRESUMO
DNA-encoded chemical library (DEL) has emerged as a powerful technology for ligand discovery in biomedical research. Recently, we have developed a DNA-encoded dynamic library (DEDL) approach by incorporating the concept of dynamic combinatorial library (DCL) with DELs. DEDL has shown excellent potential in ligand discovery towards a variety of protein targets. However, the requirement of having a pair of unnatural p-stilbazoles as the interstrand DNA crosslinker has limited the chemical diversity of DEDLs. Here, we replaced p-stilbazole with psoralen (PS) and tested the feasibility of psoralen as the crosslinker in DEDL selection. Since psoralen is commercially available and does not require any special crosslinking partner, existing DELs may be directly used to create high-diversity DEDLs. This study is expected to greatly facilitate the development of DEDLs as a versatile tool in drug discovery.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Ficusina/química , Bibliotecas de Moléculas Pequenas/química , Técnicas de Química Combinatória , Reagentes de Ligações Cruzadas/síntese química , DNA/síntese química , Descoberta de Drogas , Ficusina/síntese química , Processos Fotoquímicos , Bibliotecas de Moléculas Pequenas/síntese químicaRESUMO
Protein degradation is a fundamental process in all living organisms. An important part of this system is a multisubunit, barrel-shaped protease complex called the proteasome. This enzyme is directly responsible for the proteolysis of ubiquitin- or pup-tagged proteins to smaller peptides. In this study, we present a series of 92 psoralen derivatives, of which 15 displayed inhibitory potency against the Mycobacterium tuberculosis proteasome in low micromolar concentrations. The best inhibitors, i.e., 8, 11, 13 and 15, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays. N-(cyanomethyl)acetamide 8 (Ki = 5.6 µM) and carboxaldehyde-based derivative 15 (Ki = 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters 11 and 13 irreversibly inhibited the enzyme with Ki values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor, PR-957, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (Ki = 5.2 ± 1.9 µM, kinact/Ki = 96 ± 41 M-1·s-1). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.
Assuntos
Ficusina/farmacologia , Inibidores de Proteassoma/farmacologia , Tuberculose/tratamento farmacológico , Ficusina/química , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Inibidores de Proteassoma/química , Proteólise/efeitos dos fármacos , Tuberculose/microbiologia , Ubiquitina/química , Ubiquitinas/químicaRESUMO
BACKGROUND: Psoralen is a coumarin-like and coumarin-related benzofuran glycoside, which is a commonly used traditional Chinese medicine to treat patients with kidney and spleen-yang deficiency symptom. Psoralen has been reported to show estrogen-like activity, antioxidant activity, osteoblastic proliferation accelerating activity, antitumor effects and antibacterial activity. However, the antitumor mechanism of psoralen is not fully understood. This study aimed to investigate the therapeutic efficacy of psoralen in human hepatoma cell line SMMC7721 and the mechanism of antitumor effects. RESULTS: Psoralen inhibited proliferation of SMMC7721 in a dose- and time-dependent manner, and promoted apoptosis. Further, psoralen activated the ER stress signal pathway, including the expansion of endoplasmic reticulum, increasing the mRNA levels of GRP78, DDIT3, ATF4, XBP1, GADD34 and the protein levels of GDF15, GRP78, IRE1α, XBP-1s in a time-dependent manner. Psoralen induces cell cycle arrest at G1 phase by enhancing CyclinD1 and reducing CyclinE1 expression. Moreover, TUDC couldn't inhibit the psoralen-induced ER stress in SMMC7721 cells. CONCLUSIONS: Psoralen can inhibit the proliferation of SMMC7721 cells and induce ER stress response to induce cell apoptosis, suggesting that psoralen may represent a novel therapeutic option for the prevention and treatment hepatocellular carcinoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ficusina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Ficusina/química , Ficusina/uso terapêutico , Humanos , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Furocoumarins are known for their phototoxic and potential carcinogenic effects. These types of compounds have previously been reported from fennel (Foeniculum vulgare Mill.), a widely used medicinal plant and spice; however, no reliable quantitative data are available on the occurrence of these compounds in fennel fruits. For the first time, we report a comprehensive analysis of fennel fruit samples of different origins, representing a wide range of accessions for their furocoumarin content. Psoralene, 5-methoxypsoralene (bergapten), and imperatorin contents of 33 fennel samples were analyzed using a sensitive liquid chromatography-mass spectrometry (LC-MS) method. When applied at the highest therapeutic dose described in the monograph issued by the European Medicines Agency, the furocoumarin content of the fruits ranged up to 1.22 µg/d, which is below the most restrictive recommendations. Based on our findings, fennel consumption can be considered as safe, at least based on its low furocoumarin content.
Assuntos
Foeniculum/química , Furocumarinas/farmacologia , Extratos Vegetais/farmacologia , Ficusina/química , Ficusina/farmacologia , Frutas/química , Furocumarinas/química , Cromatografia Gasosa-Espectrometria de Massas , Extratos Vegetais/química , SolventesRESUMO
Psoralen (P) and isopsoralen (IP) are the main active ingredients in the dried fruit of Psoralen corylifolia L. (PC), with a wide range of pharmacology activities. The intestinal bacteria biotransformation plays a central role in the metabolism of the complex ingredients in traditional Chinese medicine (TCM). Our study aimed to investigated the metabolic profile of P and IP in the intestinal condition, co-cultured with human fecal bacteria anaerobically. Four bio-transforming products were obtained, including 6,7-furano-hydrocoumaric acid (P-1) and 6,7-furano-hydro- coumaric acid methyl ester (P-2), which transformed from P, and 5,6-furano-hydrocoumaric acid (IP-1) and 5,6-furano-hydrocoumaric acid methyl ester (IP-2), which were transformed from IP. It is worth mentioning that IP-2 is a new compound that has not been published. Their structures were analyzed based on their spectroscopic data. Moreover, a highly sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was used to characterize the metabolic pathways of P, IP, and their bio-transforming products in the reaction samples. In addition, the dampening effects against the oxidative stress of P, IP, and their bio-transforming products by human intestinal flora were estimated in vitro via the human colorectal cells (HCT116) and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. The results showed that the metabolites have stronger activity than P and IP, which possibly provides a basis for elucidating the treating mechanisms of PC extract against inflammatory bowel disease.
Assuntos
Biotransformação , Ficusina/metabolismo , Furocumarinas/metabolismo , Microbioma Gastrointestinal , Cromatografia Líquida de Alta Pressão , Ficusina/química , Furocumarinas/química , Humanos , Limite de Detecção , Metabolômica/métodos , Estrutura Molecular , Estresse Oxidativo , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
Molecular dynamics simulations are employed to determine the inhibitory mechanisms of three drugs, 5-(4-phenoxybutoxy)psoralen (PAP-1), vernakalant, and flecainide, on the voltage-gated K+ channel Kv1.5, a target for the treatment of cardiac arrhythmia. At neutral pH, PAP-1 is neutral, whereas the other two molecules carry one positive charge. We show that PAP-1 forms stable dimers in water, primarily through hydrophobic interactions between aromatic rings. All three molecules bind to the cavity between the Ile508 and Val512 residues from the four subunits of the channel. Once bound, the drug molecules are flexible, with the average root-mean-square fluctuation being between 2 and 3 Å, which is larger than the radius of gyration of a bulky amino acid. The presence of a monomeric PAP-1 causes the permeating K+ ion to dehydrate, thereby creating a significant energy barrier. In contrast, vernakalant blocks the ion permeation primarily via an electrostatic mechanism and, therefore, must be in the protonated and charged form to be effective.
Assuntos
Antiarrítmicos/química , Arritmias Cardíacas/tratamento farmacológico , Canal de Potássio Kv1.5/química , Sequência de Aminoácidos/genética , Anisóis/química , Anisóis/farmacologia , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/genética , Sítios de Ligação , Cristalografia por Raios X , Ficusina/química , Ficusina/uso terapêutico , Flecainida/química , Flecainida/uso terapêutico , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Pirrolidinas/química , Pirrolidinas/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Tyrosinase (TYR) plays a vital role in melanin biosynthesis and is widely regarded as a relatively specific marker for melanocytic lesions which involve vitiligo, malignant cutaneous melanoma, Parkinson's disease (PD), etc. However, the detection of TYR in living cells with fluorescent probes is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS). Herein, we synthesized a melanosome-targeting near-infrared (NIR) fluorescent probe (HB-NP) with a large Stokes shift (195 nm), achieving a highly sensitive and selective in situ detection for intracellular TYR, by incorporating a m-hydroxybenzyl moiety that recognizes TYR specifically and the morpholine unit which facilitates the probe accumulating in the melanosome into a salicyladazine skeleton. When treated with TYR, the probe itself with weak fluorescence is lit up via an inhibited photoinduced electron-transfer (PET) effect and HB-NP shows a strong fluorescence signal (nearly 48-fold enhancement) with a low detection limit of 0.5 U mL-1. HB-NP has been successfully applied in visualizing and in situ quantification of the intracellular TYR activity. Moreover, owing to the different expression levels of TYR, two human uveal melanoma cells with different invasive behaviors are distinguished by means of bioimaging and the effects of the inhibitor, kojic acid, and the up-regulating treatment, psoralen/ultraviolet A, on TYR activity of the two melanoma cells are evaluated. HB-NP is expected to be a useful tool to monitor diseases associated with the abnormal level of melanin and screen medicines for TYR disorder more effectively.
Assuntos
Corantes Fluorescentes/química , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanossomas/química , Monofenol Mono-Oxigenase/análise , Invasividade Neoplásica , Ficusina/química , Ficusina/farmacologia , Corantes Fluorescentes/síntese química , Humanos , Raios Infravermelhos , Melanoma/metabolismo , Melanoma/patologia , Monofenol Mono-Oxigenase/metabolismo , Imagem Óptica , Pironas/química , Pironas/farmacologia , Raios UltravioletaRESUMO
Drug photosensitivity is a relatively common occurrence and a range of mechanisms may be involved. Some of these mechanisms will be discussed, including the most common, that of drug phototoxicity. Different types of photosensitivity are addressed with respect to clinical presentation, mechanisms and additionally the contribution to our understanding through clinically directed investigations and regulatory requirements. Repeated controlled therapeutic use of drug phototoxicity, with psoralen-UVA (PUVA) photochemotherapy and photodynamic therapy (PDT) will also be discussed. Finally, the potential for drug-induced photocarcinogenesis will also be covered.
Assuntos
Ficusina/química , Transtornos de Fotossensibilidade/induzido quimicamente , Fármacos Fotossensibilizantes/química , Raios Ultravioleta , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/patologia , Eritema/induzido quimicamente , Eritema/patologia , Ficusina/efeitos adversos , Ficusina/uso terapêutico , Humanos , Transtornos de Fotossensibilidade/prevenção & controle , Fármacos Fotossensibilizantes/efeitos adversos , Fármacos Fotossensibilizantes/uso terapêutico , Psoríase/tratamento farmacológico , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Protetores Solares/uso terapêuticoRESUMO
Prostate cancer is the most common male reproductive system cancer. The prevalence of prostate cancer in Europe and the United States is higher than that in the Asian region. However, the treatment of prostate cancer remains unsatisfactory. Psoralea corylifolia has been used to cure this disease as Chinese medicine in the Asian region. In this study, we analyzed the components of ethanol extraction of unprepared and prepared P. corylifolia by HPLC. Psoralen and isopsoralen content from the prepared P. corylifolia is twofold higher than that from unprepared, so we use the prepared extraction in this study. However, the effects of the ethanol extraction of P. corylifolia (PCE) on PC-3 human prostate cancer cells remain unclear. PC-3 cells were treated with PCE for different time periods and cells were examined for cell morphological change and total viable cells by using contrast phase microscopy and flow cytometer, respectively. Results indicated that PCE induced cell morphological changes and cytotoxic effect in PC-3 cells in dose-dependent manners. PCE induced chromatin condensation of PC-3 cells dose-dependently. PCE also induced apoptosis and autophagy in PC-3 by western blotting and acridine orange (AO) staining, respectively. Furthermore, a complementary DNA microarray analysis demonstrated that PCE treatment led to 944 genes upregulation and 872 genes downregulation. For example, the DNA damage-associated gene DNA-damage-inducible transcript 3 (DDIT 3) had a 62.1-fold upregulation and CDK1 2.68-fold downregulation. The differential genes were classified according to the Gene Ontology. Furthermore, GeneGo software was used for the key genes involved and their possible interaction pathways. Those genes were affected by P. corylifolia, which provided information for the understanding of the antiprostate cancer mechanism at the genetic level and provide additional targets for the treatments of human prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Extratos Vegetais/farmacologia , Psoralea/química , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Etanol/química , Ficusina/química , Ficusina/isolamento & purificação , Ficusina/farmacologia , Furocumarinas/química , Furocumarinas/isolamento & purificação , Furocumarinas/farmacologia , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Extratos Vegetais/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Psoralea/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Reversible immobilization of DNA and RNA is of great interest to researchers who seek to manipulate DNA or RNA in applications such as microarrays, DNA hydrogels, and gene therapeutics. However, there is no existing system that can rapidly capture and release intact nucleic acids. To meet this unmet need, we developed a functional hydrogel for rapid DNA/RNA capture and release based on the reversible photo-cycloaddition of psoralen and pyrimidines. The functional hydrogel can be easily fabricated through copolymerization of acrylamide with the synthesized allylated psoralen. The psoralen-functionalized hydrogel exhibits effective capture and release of nucleic acids spanning a wide range of lengths in a rapid fashion; over 90 % of the capture process is completed within 1â min, and circa 100 % of the release process is completed within 2â min. We observe no deleterious effects on the hybridization to the captured targets.