RESUMO
Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.
Assuntos
Cálcio , Receptores Acoplados a Proteínas G , Cálcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Microscopia Confocal/métodos , Transdução de Sinais , Miócitos de Músculo Liso/metabolismo , Sinalização do Cálcio , Humanos , Xantenos/metabolismo , Fura-2/metabolismo , Metabolismo dos Lipídeos , Esôfago/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Compostos de AnilinaRESUMO
Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.
Assuntos
Antifúngicos , Cromoblastomicose , Fonsecaea , Testes de Sensibilidade Microbiana , Oxazinas , Xantenos , Xantenos/metabolismo , Oxazinas/metabolismo , Antifúngicos/farmacologia , Humanos , Fonsecaea/efeitos dos fármacos , Fonsecaea/genética , Fonsecaea/metabolismo , Cromoblastomicose/microbiologia , Cromoblastomicose/tratamento farmacológico , Colorimetria/métodosRESUMO
Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.
Assuntos
Antineoplásicos , Xantenos , Animais , Antineoplásicos/farmacologia , Bioensaio , Sobrevivência Celular , Mamíferos/metabolismo , Oxazinas , Xantenos/metabolismo , Xantenos/farmacologiaRESUMO
Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 µm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.
Assuntos
Trifosfato de Adenosina/metabolismo , Miosina Tipo II/metabolismo , Organelas/metabolismo , Compostos de Anilina/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Jurkat , Xantenos/metabolismoRESUMO
Purpose: The presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. Methods: Three distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by Lys C and trypsin. Mass spectrometry-based proteomic analysis followed by gene ontology and protein interaction network analysis was performed. Results: Dye penetration showed that fiber cells first shrink the extracellular spaces of the broad sides followed by closure of the extracellular space between narrow sides at a normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected, including upregulation of several cell junctional proteins. AQP0 and its interacting partners, Ezrin and Radixin, were among a few proteins that were upregulated, accompanying the closure of extracellular space of the narrow sides, suggesting a particularly important role for AQP0 in controlling the narrowing of the extracellular spaces between fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling, and glutathione synthesis. Conclusions: The formation of a lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.
Assuntos
Membrana Celular/metabolismo , Cristalinas/metabolismo , Espaço Extracelular/metabolismo , Cápsula do Cristalino/metabolismo , Cristalino/metabolismo , Animais , Aquaporinas/metabolismo , Transporte Biológico , Bovinos , Cromatografia Líquida , Proteínas do Citoesqueleto/metabolismo , Difusão , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Mapas de Interação de Proteínas , Proteômica , Espectrometria de Massas em Tandem , Xantenos/metabolismoRESUMO
After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the G2M phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação 2 em Eucariotos/metabolismo , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Estrutura Molecular , Oxazinas/metabolismo , Xantenos/metabolismo , eIF-2 Quinase/genéticaRESUMO
Reduced glucose uptake into the skeletal muscle is an important pathophysiological abnormality in type 2 diabetes, and is caused by impaired translocation of glucose transporter 4 (GLUT4) to the skeletal muscle cell surface. Here, we show a xanthene derivative, DS20060511, induces GLUT4 translocation to the skeletal muscle cell surface, thereby stimulating glucose uptake into the tissue. DS20060511 induced GLUT4 translocation and stimulated glucose uptake into differentiated L6-myotubes and into the skeletal muscles in mice. These effects were completely abolished in GLUT4 knockout mice. Induction of GLUT4 translocation by DS20060511 was independent of the insulin signaling pathways including IRS1-Akt-AS160 phosphorylation and IRS1-Rac1-actin polymerization, eNOS pathway, and AMPK pathway. Acute and chronic DS20060511 treatment attenuated the glucose intolerance in obese diabetic mice. Taken together, DS20060511 acts as a skeletal muscle-specific GLUT4 translocation enhancer to facilitate glucose uptake. Further studies of DS20060511 may pave the way for the development of novel antidiabetic medicines.
Assuntos
Intolerância à Glucose/genética , Transportador de Glucose Tipo 4/genética , Músculo Esquelético/metabolismo , Translocação Genética , Xantenos/metabolismo , Animais , Intolerância à Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , CamundongosRESUMO
Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.
Assuntos
Flores/fisiologia , Gentiana/fisiologia , Pigmentação/genética , Proteínas de Plantas/genética , Xantonas/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Antocianinas/genética , Antocianinas/metabolismo , Cromatografia Líquida de Alta Pressão , Flores/genética , Gentiana/genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Estrutura Molecular , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Proteínas de Plantas/metabolismo , Análise de Sequência de RNA , Xantenos/metabolismo , Xantonas/química , Xantonas/isolamento & purificaçãoRESUMO
Microglia, the resident brain phagocytes, likely play a key role in human immunodeficiency virus (HIV) infection of the central nervous system (CNS) and subsequent neuropathogenesis; however, the nature of the infection-induced changes that yield damaging CNS effects and the stimuli that provoke microglial activation remains elusive, especially in the current era of using antiretroviral (ARV) drugs for ARV therapy (ART). Altered microglial metabolism can modulate cellular functionality and pathogenicity in neurological disease. While HIV infection itself alters brain energy metabolism, the effect of ARV drugs, particularly those currently used in treatment, on metabolism is understudied. Dolutegravir (DTG) and emtricitabine (FTC) combination, together with tenofovir (TAF or TDF), is one of the recommended first line treatments for HIV. Despite the relatively good tolerability and safety profile of FTC, a nucleoside reverse transcriptase inhibitor, and DTG, an integrase inhibitor, adverse side effects have been reported and highlight a need to understand off-target effects of these medications. We hypothesized that similar to previous ART regimen drugs, DTG and FTC side effects involve mitochondrial dysfunction. To increase detection of ARV-induced mitochondrial effects, highly glycolytic HeLa epithelial cells were forced to rely on oxidative phosphorylation by substituting galactose for glucose in the growth media. We assessed ATP levels, resazurin oxidation-reduction (REDOX), and mitochondrial membrane potential following 24-hour exposure (to approximate effects of one dose equivalent) to DTG, FTC, and efavirenz (EFV, a known mitotoxic ARV drug). Further, since microglia support productive HIV infection, act as latent HIV cellular reservoirs, and when dysfunctional likely contribute to HIV-associated neurocognitive disorders, the experiments were repeated using BV2 microglial cells. In HeLa cells, FTC decreased mitochondrial REDOX activity, while DTG, similar to EFV, impaired both mitochondrial ATP generation and REDOX activity. In contrast to HeLa cells, DTG increased cellular ATP generation and mitochondrial REDOX activity in BV2 cells. Bioenergetic analysis revealed that DTG, FTC, and EFV elevated BV2 cell mitochondrial respiration. DTG and FTC exposure induced distinct mitochondrial functional changes in HeLa and BV2 cells. These findings suggest cell type-specific metabolic changes may contribute to the toxic side effects of these ARV drugs.
Assuntos
Alcinos/farmacologia , Fármacos Anti-HIV/farmacologia , Benzoxazinas/farmacologia , Ciclopropanos/farmacologia , Emtricitabina/farmacologia , Células Epiteliais/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Microglia/efeitos dos fármacos , Oxazinas/farmacologia , Piperazinas/farmacologia , Piridonas/farmacologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microglia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxazinas/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Latência Viral/efeitos dos fármacos , Xantenos/metabolismoRESUMO
Nowadays, natural dyes are expected by the cosmetic and food industries. In contrast to synthetic dyes, colorants derived from natural sources are more environmentally friendly and safer for human health. In this work, plant extracts from Gomphrena globasa L., Clitoria ternatea L., Carthamus tinctorius L., Punica granatum L. and Papaver rhoeas L. as the natural and functional dyes for the cosmetics industry were assessed. Cytotoxicity on keratinocyte and fibroblast cell lines was determined as well as antioxidant and anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the extracts was determined. The obtained extracts were also applied in face cream formulation and color analyses were performed. It has been shown that the obtained extracts were characterized by no cytotoxicity and a high antioxidant potential. The extracts also show strong ability to inhibit the activity of collagenase and moderate ability to inhibit elastase and provide effective and long-lasting hydration after their application on the skin. Application analyses showed that the extracts of P. rhoeas L., C. ternatea L. and C. tinctorius L. can be used as effective cosmetic dyes that allow for attainment of an intense and stable color during the storage of the product. The extracts of P. granatum L. and G. globasa L., despite their beneficial effects as active ingredients, did not work effectively as cosmetic dyes, because cosmetic emulsions with these extracts did not differ significantly in color from emulsions without the extract.
Assuntos
Antioxidantes/farmacologia , Corantes/farmacologia , Cosméticos/farmacologia , Citoproteção , Dessecação , Flores/química , Extratos Vegetais/farmacologia , Benzotiazóis/química , Compostos de Bifenilo/química , Morte Celular/efeitos dos fármacos , Colagenases/metabolismo , Cor , Citoproteção/efeitos dos fármacos , Células HaCaT , Humanos , Cinética , Inibidores de Metaloproteinases de Matriz/farmacologia , Oxazinas/metabolismo , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Picratos/química , Plantas/química , Creme para a Pele/farmacologia , Ácidos Sulfônicos/química , Raios Ultravioleta , Perda Insensível de Água/efeitos dos fármacos , Xantenos/metabolismoRESUMO
A resazurin micro-assay was developed to quantify acidifying bacteria. The resorufin fluorescent signal was measured over time and the determined time to reach the max slope (TMS) was plotted against CFU (colony forming unit) counts. This dynamic assay enabled to quantify nine lactic acid bacteria and a Bacillus licheniformis strain despite the increasing acidity of the medium.
Assuntos
Ácidos/metabolismo , Técnicas Bacteriológicas/métodos , Lactobacillales/crescimento & desenvolvimento , Oxazinas/química , Xantenos/química , Bacillus licheniformis/química , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus licheniformis/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Fluorescência , Lactobacillales/química , Lactobacillales/metabolismo , Oxazinas/metabolismo , Xantenos/metabolismoAssuntos
Apoproteínas/química , Receptores de Glutamato Metabotrópico/química , Apoproteínas/genética , Apoproteínas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Carbamatos/química , Carbamatos/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ácido Quisquálico/química , Ácido Quisquálico/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Xantenos/química , Xantenos/metabolismoRESUMO
Kombucha, also known as the Manchurian mushroom, is a symbiotic culture of bacteria and yeast, the so-called SCOBY. This paper presents a comprehensive evaluation of the ferments obtained from green coffee beans after different fermentation times with kombucha. Results for the ferments were compared to the green coffee extract that was not fermented. In this study, the antioxidant potential of obtained ferments was analyzed by assessing the scavenging of external and intracellular free radicals and the assessment of superoxide dismutase activity. Cytotoxicity of ferments on keratinocyte and fibroblast cell lines was assessed as well as anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the obtained ferments and the extract was determined, as well as their influence on skin hydration and transepidermal water loss (TEWL) after application of samples on the skin. It has been shown that the fermentation time has a positive effect on the content of bioactive compounds and antioxidant properties. The highest values were recorded for the tested samples after 28 days of fermentation. After 14 days of the fermentation process, it was observed that the analyzed ferments were characterized by low cytotoxicity to keratinocytes and fibroblasts. On the other hand, the short fermentation time of 7 days had a negative effect on the properties of the analyzed ferments. The obtained results indicate that both green coffee extracts and ferments can be an innovative ingredient of cosmetic products.
Assuntos
Antioxidantes/farmacologia , Café/química , Fermentação , Chá de Kombucha , Compostos de Bifenilo/química , Sobrevivência Celular/efeitos dos fármacos , Colagenases/metabolismo , Fermentação/efeitos dos fármacos , Fibroblastos/metabolismo , Flavonoides/análise , Fluorescência , Células HaCaT , Humanos , Espaço Intracelular/metabolismo , Cinética , Limite de Detecção , Inibidores de Metaloproteinases de Matriz/farmacologia , Oxazinas/metabolismo , Elastase Pancreática/metabolismo , Fenóis/análise , Picratos/química , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Tempo , Perda Insensível de Água/efeitos dos fármacos , Xantenos/metabolismoRESUMO
There is a great need for novel in vitro methods to predict human developmental toxicity to comply with the 3R principles and to improve human safety. Human-induced pluripotent stem cells (hiPSC) are ideal for the development of such methods, because they are easy to retrieve by conversion of adult somatic cells and can differentiate into most cell types of the body. Advanced three-dimensional (3D) cultures of these cells, so-called embryoid bodies (EBs), moreover mimic the early developing embryo. We took advantage of this to develop a novel human toxicity assay to predict chemically induced developmental toxicity, which we termed the PluriBeat assay. We employed three different hiPSC lines from male and female donors and a robust microtiter plate-based method to produce EBs. We differentiated the cells into cardiomyocytes and introduced a scoring system for a quantitative readout of the assay-cardiomyocyte contractions in the EBs observed on day 7. Finally, we tested the three compounds thalidomide (2.3-36 µM), valproic acid (25-300 µM), and epoxiconazole (1.3-20 µM) on beating and size of the EBs. We were able to detect the human-specific teratogenicity of thalidomide and found the rodent toxicant epoxiconazole as more potent than thalidomide in our assay. We conclude that the PluriBeat assay is a novel method for predicting chemicals' adverse effects on embryonic development.
Assuntos
Bioensaio/métodos , Corpos Embrioides/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Teratogênicos/toxicidade , Testes de Toxicidade/métodos , Linhagem Celular , Biologia do Desenvolvimento , Corpos Embrioides/fisiologia , Compostos de Epóxi/toxicidade , Feminino , Humanos , Masculino , Miócitos Cardíacos/fisiologia , Oxazinas/metabolismo , Células-Tronco Pluripotentes/fisiologia , Teratogênese , Talidomida/toxicidade , Triazóis/toxicidade , Ácido Valproico/toxicidade , Xantenos/metabolismoRESUMO
Ommochromes are widespread pigments that mediate multiple functions in invertebrates. The two main families of ommochromes are ommatins and ommins, which both originate from the kynurenine pathway but differ in their backbone, thereby in their coloration and function. Despite its broad significance, how the structural diversity of ommochromes arises in vivo has remained an open question since their first description. In this study, we combined organic synthesis, analytical chemistry and organelle purification to address this issue. From a set of synthesized ommatins, we derived a fragmentation pattern that helped elucidating the structure of new ommochromes. We identified uncyclized xanthommatin as the elusive biological intermediate that links the kynurenine pathway to the ommatin pathway within ommochromasomes, the ommochrome-producing organelles. Due to its unique structure, we propose that uncyclized xanthommatin functions as a key branching metabolite in the biosynthesis and structural diversification of ommatins and ommins, from insects to cephalopods.
Assuntos
Invertebrados/metabolismo , Oxazinas , Fenotiazinas , Pigmentação , Xantenos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Dípteros/metabolismo , Olho/metabolismo , Insetos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Oxazinas/química , Oxazinas/isolamento & purificação , Oxazinas/metabolismo , Fenotiazinas/química , Fenotiazinas/isolamento & purificação , Fenotiazinas/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/isolamento & purificação , Pigmentos Biológicos/metabolismo , Xantenos/química , Xantenos/isolamento & purificação , Xantenos/metabolismoRESUMO
Synthetic Ca2+ indicators are widely used to report changes in free [Ca2+], usually in the cytosol but also within organelles. Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [Ca2+] within the ER, where the free [Ca2+] is estimated to be between 100 µM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca2+] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca2+ measured in vitro (KDCa â¼22 µM). Using an antibody to quench the fluorescence of indicator that leaked from the ER, we established that the affinity of Mag-Fluo-4 within the ER is much lower (KDCa â¼1 mM) than that measured in vitro. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for Ca2+, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca2+ that are both appropriate for the ER lumen and capable of reporting a wide range of free [Ca2+].
Assuntos
Compostos de Anilina/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Xantenos/metabolismo , Compostos de Anilina/química , Animais , Anticorpos/metabolismo , Linhagem Celular , Galinhas , Esterificação , Fluorescência , Humanos , Xantenos/químicaRESUMO
The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.
Assuntos
Magaininas/metabolismo , Nanoporos , Lipossomas Unilamelares/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Simulação por Computador , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Magaininas/farmacologia , Lipossomas Unilamelares/metabolismo , Xantenos/metabolismoRESUMO
Microglia are implicated in the neuropathogenesis of HIV. Tetraspanin 2 (Tspan2) is closely related to CD9 and CD81 proteins, and are expressed on microglia cells. They have been implicated in cell fusion and adhesion and in the immune response, and neuroinflammation. Developing therapeutics that target microglia remains a challenge as these therapeutics must cross the Blood-Brain Barrier (BBB). Our goal was to use microglia derived exosomes as a vehicle to deliver siRNA across the BBB to target human telomerase reverse transcriptase immortalized human microglial cells (HTHU) latently infected by HIV (HTHU-HIV) and to evaluate if the knockdown of Tspan2 gene expression in changes the activation state of microglia cells, thereby modulating the neuroinflammatory response. A blood brain barrier (BBB) model that closely mimics and accurately reflects the characteristics and functional properties of the in vivo BBB was used to examine HTHU microglia exosome effects on BBB permeability, and their ability to migrate across the and delivery small interfering RNA (siRNA) to cells on the CNS side of the BBB model. Exosomes were loaded with Texas-Red control siRNA (20 pmol) or Cy5-Tspan2 siRNA and then placed in the apical side of the BBB model, 24 h after incubation, HTHU-HIV cells microglial cells on the lower chamber were either imaged for siRNA uptake or analyzed for gene expression induced modifications. HTHU exosomes transmigrate from the apical side of the BBB to deliver Texas-Red control siRNA or Cy5-Tspan2 siRNA to HTHU-HIV microglia cells on the CNS side of the BBB model. A dose dependent (5-40 pmol) increase in Cy5-Tspan2 uptake with a corresponding decrease in gene expression for Tspan2 occurred in HTHU-HIV microglia. A decrease in Tspan2 gene expression as a consequence of knockdown with Tspan2 siRNA at both 20 and 40 pmol concentrations resulted in a significant decrease in C-X-C motif chemokine 12 (CXCL12) and C-X-C chemokine receptor type 4 (CXCR4) gene expression in HTHU-HIV microglia. Furthermore, a decrease in the gene expression levels of the Interleukins, IL-13 and IL-10 and an increase in the gene expression levels for the Fc gamma receptor 2A(FCGR2A) and TNF-α occurred in HTHU-HIV microglial cells These data demonstrate that HTHU exosomes cross the BBB and are efficient delivery vehicles to the CNS. Moreover, modifying the expression levels of Tspan2, has downstream consequences that includes alterations in cytokines and microglia biomarkers. Graphical Abstract Microglia-derived exosomes loaded with Tspan2 siRNA transmigrate across the BBB and knockdown Tspan2 gene expression in human microglial cells latently infected by HIV. This knockdown increases CXCL12, CXCR4, FCGR2A and TNF-α while decreasing IL-13 and IL-10 gene expression in HTHU-HIV microglial cells. Modulating Tspan2 modulates microglia cytokines and phenotype biomarkers.
Assuntos
Barreira Hematoencefálica/metabolismo , Exossomos/metabolismo , Imunidade Celular , Microglia/imunologia , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , Tetraspaninas/metabolismo , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/genética , Citocinas/biossíntese , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas do Tecido Nervoso/genética , Cultura Primária de Células , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Tetraspaninas/genética , Xantenos/metabolismoRESUMO
During aging and ischemic and hemorrhagic stroke, elastin molecules are degraded and elastin-derived peptides are released into the brain microenvironment. Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a repeating hexapeptide in the elastin molecule. It is well documented that the peptide sequence binds with high affinity to elastin-binding protein (EBP) located on the cell surface, thereby transducing a molecular signal into the cell. The aim of our study was to investigate whether EBP, aryl hydrocarbon receptor (Ahr), and peroxisome proliferator-activated receptor gamma (Pparγ) are involved in VGVAPG-stimulated proliferation. Primary astrocytes were maintained in DMEM/F12 medium without phenol red, supplemented with 10 or 1% charcoal/dextran-treated fetal bovine serum (FBS). The cells were exposed to increasing concentrations of VGVAPG peptide, and resazurin reduction was measured. In addition, Glb1, Pparγ, and Ahr genes were silenced. After 48 h of exposure to 10 nM and 1 µM of VGVAPG peptide, the level of estradiol (E2) and the expression of Ki67 and S100B proteins were measured. The results showed that at a wide range of concentrations, VGVAPG peptide increased the metabolism of astrocytes depending on the concentration of FBS. After silencing of Glb1, Pparγ, and Ahr genes, VGVAPG peptide did not affect the cell metabolism which suggests the involvement of all the mentioned receptors in its mechanism of action. Interestingly, in the low-FBS medium, the silencing of Glb1 gene did not result in complete inhibition of VGVAPG-stimulated proliferation. On the other hand, in the medium with 10% FBS VGVAPG increased Ki67 expression after Pparγ silencing, whereas in the medium with 1% FBS VGVAPG decreased Ki67 expression. Following the application of Ahr siRNA, VGVAPG peptide decreased the production of E2 and increased the expression of Ki67 and S100B proteins.
Assuntos
Astrócitos/metabolismo , Elastina/metabolismo , Oligopeptídeos/metabolismo , PPAR gama/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Estradiol/sangue , Feminino , Antígeno Ki-67/metabolismo , Camundongos , Oxazinas/metabolismo , PPAR gama/genética , Gravidez , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Hidrocarboneto Arílico/genética , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Xantenos/metabolismoRESUMO
Four new xanthene derivatives, penicixanthenes A-D (1-4), and one known compound 5 were isolated from a marine mangrove endophytic fungus Penicillium sp. JY246 that was obtained from the stem of Ceriops tagal. Their structures were determined by detailed NMR, MS spectroscopic data, modified Mosher's method, and calculated electronic circular dichroism data. All of the isolated compounds were examined for insecticidal activity. Compounds 2 and 3 showed growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner with the IC50 values 100 and 200 µg/mL, respectively, and compounds 1, 3, and 4 showed insecticidal activity against newly hatched larvae of Culex quinquefasciatus with LC50 values of 38.5 (±1.16), 11.6 (±0.58), and 20.5 (±1) µg/mL, respectively. The four xanthene derivatives have the potential to be developed as new biopesticides.