RESUMO
The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand how nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI [(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one] that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that polyfluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of â¼40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual transition to a more defined structure for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that polyfluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.
Assuntos
Aptâmeros de Nucleotídeos , Brassica , Aptâmeros de Nucleotídeos/química , Ligantes , Conformação de Ácido Nucleico , RNA/químicaRESUMO
Pogostone, a compound with various pharmaceutical activities, is a major constituent of the essential oil preparation called Pogostemonis Herba, which is obtained from the plant Pogostemon cablin. The biosynthesis of pogostone has not been elucidated, but 4-methylvaleryl-CoA (4MVCoA) is a likely precursor. We analyzed the distribution of pogostone in P. cablin using gas chromatography-mass spectrometry (GC-MS) and found that pogostone accumulates at high levels in the main stems and leaves of young plants. A search for the acyl-activating enzyme (AAE) that catalyzes the formation of 4MVCoA from 4-methylvaleric acid was launched, using an RNAseq-based approach to identify 31 unigenes encoding putative AAEs including the PcAAE2, the transcript profile of which shows a strong positive correlation with the distribution pattern of pogostone. The protein encoded by PcAAE2 was biochemically characterized in vitro and shown to catalyze the formation of 4MVCoA from 4-methylvaleric acid. Phylogenetic analysis showed that PcAAE2 is closely related to other AAE proteins in P. cablin and other species that are localized to the peroxisomes. However, PcAAE2 lacks a peroxisome targeting sequence 1 (PTS1) and is localized in the cytosol.
Assuntos
Coenzima A Ligases/genética , Óleos Voláteis/metabolismo , Proteínas de Plantas/genética , Pogostemon/genética , Sequência de Aminoácidos , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Pogostemon/metabolismo , Alinhamento de SequênciaRESUMO
Dihydro eurotiumide B was prepared from a 5-phenylthiobutenolide via a tandem annulation/reductive thiolation reaction.
RESUMO
Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.
Assuntos
Asteraceae/enzimologia , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Pironas/metabolismo , Yarrowia/metabolismo , Asteraceae/genética , Meios de Cultura/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Yarrowia/genéticaRESUMO
Naphthacemycin A9 was synthesized in nine steps by a sequence featuring a Diels-Alder reaction of a hindered aryl butadiene and a Hauser-Kraus annulation reaction.
RESUMO
The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of "light-up" aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Células Epiteliais/ultraestrutura , Imagem Óptica/métodos , RNA/química , Saccharomyces cerevisiae/ultraestrutura , Transcrição Gênica , Animais , Aptâmeros de Nucleotídeos/síntese química , Compostos de Benzil/química , Células CHO , Carbocianinas/química , Linhagem Celular , Cricetulus , Células Epiteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Imidazolinas/química , Cinética , Ligantes , Pulmão/citologia , Pulmão/metabolismo , Vison , Sondas Moleculares/química , RNA/genética , RNA/metabolismo , Corantes de Rosanilina/química , Saccharomyces cerevisiae/metabolismo , Razão Sinal-RuídoRESUMO
The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.
Assuntos
Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Cocaína/urina , 2-Aminopurina/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Calorimetria , Cocaína/metabolismo , Eletroforese em Gel de Poliacrilamida , Framicetina/química , Framicetina/metabolismo , Humanos , Ligantes , Conformação de Ácido Nucleico , Cloreto de Sódio/químicaRESUMO
We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.
Assuntos
Aptâmeros de Nucleotídeos , Genes Reporter , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transcrição Gênica , Transferência Ressonante de Energia de Fluorescência , Saccharomyces cerevisiae/genéticaRESUMO
2-Pyrones, such as triacetic acid lactone, are a promising class of biorenewable platform chemicals that provide access to an array of chemical products and intermediates. We illustrate through the combination of results from experimental studies and first-principle density functional theory calculations that key structural features dictate the mechanisms underlying ring-opening and decarboxylation of 2-pyrones, including the degree of ring saturation, the presence of CâC bonds at the C4âC5 or C5âC6 positions within the ring, as well as the presence of a ß-keto group at the C4 position. Our results demonstrate that 2-pyrones undergo a range of reactions unique to their structure, such as retro-Diels-Alder reactions and nucleophilic addition of water. In addition, the reactivity of 2-pyrones and the final products formed is shown to depend on the solvent used and the acidity of the reaction environment. The mechanistic insights obtained here provide guidance for the selective conversion of 2-pyrones to targeted chemicals.
Assuntos
Ácidos Carboxílicos/química , Furanos/química , Pironas/química , Água/química , Modelos Moleculares , Conformação Molecular , Teoria QuânticaRESUMO
Here, low-energy poly(ethylene terephthalate) (PET) chemical recycling in water: PET copolymers with diethyl 2,5-dihydroxyterephthalate (DHTE) undergo selective hydrolysis at DHTE sites, autocatalyzed by neighboring group participation, is demonstrated. Liberated oligomeric subchains further hydrolyze until only small molecules remain. Poly(ethylene terephthalate-stat-2,5-dihydroxyterephthalate) copolymers were synthesized via melt polycondensation and then hydrolyzed in 150-200 °C water with 0-1 wt% ZnCl2 , or alternatively in simulated sea water. Degradation progress follows pseudo-first order kinetics. With increasing DHTE loading, the rate constant increases monotonically while the thermal activation barrier decreases. The depolymerization products are ethylene glycol, terephthalic acid, 2,5-dihydroxyterephthalic acid, and bis(2-hydroxyethyl) terephthalate dimer, which could be used to regenerate virgin polymer. Composition-optimized copolymers show a decrease of nearly 50% in the Arrhenius activation energy, suggesting a 6-order reduction in depolymerization time under ambient conditions compared to that of PET homopolymer. This study provides new insight to the design of polymers for end-of-life while maintaining key properties like service temperature and mechanical properties. Moreover, this chemical recycling procedure is more environmentally friendly compared to traditional approaches since water is the only needed material, which is green, sustainable, and cheap.
RESUMO
The objective of the present study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would affect certain mitochondrial parameters, as well as systemic oxidative stress. A total of sixty-four mice were fed a high-fat (HF) diet for 5 weeks. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet, and administered orally with MitoVit E (40 mg MitoVit E/kg body weight) or drug vehicle (10 % (v/v) ethanol in 0·9 % (w/v) NaCl solution), every other day for 5 weeks. Mitochondrial ATP and H(2)O(2) production rates in both the liver and the gastrocnemius were not affected by MitoVit E administration in either LF or MF diet-fed mice. However, the number and average size of the subsarcolemmal mitochondria, but not the intermyofibrillar mitochondria, from the soleus muscle were significantly higher in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). After the mice were switched from the HF diet to the four dietary treatments (LF-C, LF-E, MF-C and MF-E), the decrease in urinary isoprostane concentration was significantly greater in the LF-E group than in the other three groups during the whole study (weeks 6-10). In addition, MitoVit E significantly increased plasma superoxide dismutase (SOD) activity in the MF diet-fed group without affecting plasma glutathione peroxidase activity or H(2)O(2) levels. Overall, these data suggest that MitoVit E affects subsarcolemmal mitochondrial density and systemic oxidative stress parameters such as plasma SOD activity and urinary isoprostane concentration.
Assuntos
Sistemas de Liberação de Medicamentos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Suplementos Nutricionais , Peróxido de Hidrogênio/metabolismo , Isoprostanos/urina , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Oxirredução , Ubiquinona/farmacologiaRESUMO
Chroman aldehydes bearing an acetyl group plus alkoxyl or hydroxyl groups inhibit HIV infectivity in HeLa37 cells.
Assuntos
Aldeídos/síntese química , Aldeídos/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Cromanos/síntese química , Cromanos/farmacologia , HIV/efeitos dos fármacos , Aldeídos/química , Antivirais/química , Linhagem Celular , Cromanos/química , Infecções por HIV/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Estrutura MolecularRESUMO
Organophosphate (OP) compounds, a family of highly hazardous chemical compounds included in nerve agents and pesticides, have been linked to more than 250,000 annual deaths connected to various chronic diseases. However, a solid-state sensing system that is able to be integrated into a clothing system is rare in the literature. This study aims to develop a nanofiber-based solid-state polymeric material as a soft sensor to detect OP compounds present in the environment. Esters of polydiacetylene were synthesized and incorporated into a cellulose acetate nanocomposite fibrous assembly developed with an electrospinning technique, which was then hydrolyzed to generate more hydroxyl groups for OP binding. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), Instron® tensile tester, contact angle analyzer, and UV-Vis spectroscopy were employed for characterizations. Upon hydrolysis, polydiacetylene esters in the cellulosic fiber matrix were found unaffected by hydrolysis treatment, which made the composites suitable for OP sensing. Furthermore, the nanofibrous (NF) composites exhibited tensile properties suitable to be used as a textile material. Finally, the NF composites exhibited colorimetric sensing of OP, which is visible to the naked eye. This research is a landmark study toward the development of OP sensing in a protective clothing system.
RESUMO
Our objective in this study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would decrease oxidative stress and associated obesity by preventing a previously proposed aconitase inhibition cascade. Sixty-four mice were fed a high-fat (HF) diet for 5 wk. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet and gavaged with MitoVit E (40 mg MitoVit E x kg body weight(-1)) or drug vehicle (10% ethanol in 0.9% NaCl solution) every other day for 5 wk. Epididymal fat weight, as well as liver lipid and remaining carcass lipid, were significantly lower in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). Liver mitochondrial H(2)O(2) production and the protein carbonyl level were also significantly lower in MF-E than in MF-C mice. In contrast, none of the biochemical variables (aconitase activity, ATP and H(2)O(2) production, and protein carbonyl level) in the muscle mitochondria were modified by MitoVit E in either MF or LF groups. Expression of acetyl-CoA carboxylase and fatty acid synthase in both liver and adipose tissue of MF groups was not affected by MitoVit E. However, expression of carnitine palmitoyltransferase 1a in the liver and uncoupling protein 2 in adipose tissue were significantly enhanced by MitoVit E in both LF and MF groups. In conclusion, MitoVit E attenuates hepatic oxidative stress and inhibits fat deposition in mice but not through alleviation of the aconitase inhibition cascade.
Assuntos
Tecido Adiposo/metabolismo , Fígado/metabolismo , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/genética , Trifosfato de Adenosina/metabolismo , Tecido Adiposo/anatomia & histologia , Animais , Composição Corporal , Peso Corporal , Dieta , Gorduras na Dieta/administração & dosagem , Ingestão de Alimentos , Ácido Graxo Sintases/genética , Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Lipídeos/análise , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/ultraestrutura , Obesidade/prevenção & controle , Tamanho do Órgão/efeitos dos fármacos , Compostos Organofosforados/administração & dosagem , RNA Mensageiro/análise , Ubiquinona/administração & dosagem , Ubiquinona/farmacologiaRESUMO
The effects of substituents on the aryl ring were studied by the preparation and testing of several PD173955 analogs. Inserting a single carbon atom into the C-N bond in the aniline subunit (PDC) reduced the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955, PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs with amino groups in place of the methylthio group are 10 times more inhibitory than the parent molecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for Imatinib.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Benzamidas , Humanos , Mesilato de Imatinib , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/química , Piridonas/química , Piridonas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
A flexible synthesis of the azafluorenone alkaloids 1, 2, 3, and 4 is described.
Assuntos
Alcaloides/síntese química , Alcaloides/farmacologia , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Compostos Aza/síntese química , Compostos Aza/farmacologia , Fluorenos/síntese química , Fluorenos/farmacologia , Alcaloides/química , Anti-Infecciosos/química , Compostos Aza/química , Catálise , Fluorenos/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
A variety of nucleophiles react efficiently with in situ generated nitroquinones. The reaction with substituted resorcinols led to the direct synthesis of the phenanthroviridinone and lagumycin skeleton via a highly regioselective Diels-Alder reaction.
RESUMO
A mild procedure for the low-temperature conversion of alkynes to diketones has been developed and employed in the synthesis of AI-2.
RESUMO
BACKGROUND: Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. RESULTS: Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. CONCLUSION: Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Hypericum/química , Ácidos Láuricos/isolamento & purificação , Ácidos Láuricos/farmacologia , Extratos Vegetais/química , Fármacos Anti-HIV/toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Células HeLa , Humanos , Ácidos Láuricos/toxicidade , Ácidos Mirísticos/isolamento & purificação , Ácidos Mirísticos/farmacologia , Ácidos Mirísticos/toxicidadeRESUMO
In a previous study of a naproxen dyad in a pair of N-methylimidazoliummethyl menthylether-NTf(2) chiral ionic liquids (J. Phys. Chem. B 2008, 112, 7555), we observed that though intramolecular electron transfer was impeded, a consistent small stereodifferentiation in the fluorescence lifetime of the dyad was obtained. We proposed that this discrimination was purely electronic in nature and did not arise from geometrical effects, which can influence nonradiative rate processes, such as intramolecular electron transfer. In our present work, we have studied the interaction of the same chiral naproxen dyad molecule in both the previously studied menthyl-based NTf(2) ionic liquids and also in bis(tertrabutylphosphonium) (TBP) d-,l-tartrate ionic liquids. Unlike in the menthyl-based IL pair, the amount of quenching is different in the bis(TBP) tartrate enantiomeric liquids and the tartrate enantiomers have a different temperature dependence on the nonradiative rate of the dyad. This chiral discrimination most likely arises from the steric effects of the different conformations of the chiral molecules. We have shown that the viscosity and polarity of the solvents can influence the rate of electron transfer. On the other hand, no such electron transfer quenching is observed in the menthyl-based NTf(2) IL solvents. To our knowledge, this is the first example of chiral ionic liquids inducing a stereoselective fluorescence quenching by photoinduced, intramolecular electron transfer.