RESUMO
Aldehyde dehydrogenase 2 (ALDH2) deficiency caused by genetic variant is present in more than 560 million people of East Asian descent, which can be identified by apparent facial flushing from acetaldehyde accumulation after consuming alcohol. Recent findings indicated that ALDH2 also played a critical role in detoxification of formaldehyde (FA). Our previous studies showed that FA could enhance macrophagic inflammatory responses through the induction of HIF-1α-dependent glycolysis. In the present study, pro-inflammatory responses and glycolysis promoted by 0.5 mg/m3 FA were found in mice with Aldh2 gene knockout, which was confirmed in the primary macrophages isolated from Aldh2 gene knockout mice treated with 50 µM FA. FA at 50 and 100 µM also induced stronger dose-dependent increases of pro-inflammatory responses and glycolysis in RAW264.7 murine macrophages with knock-down of ALDH2, and the enhanced effects induced by 50 µM FA was alleviated by inhibition of HIF-1α in RAW264.7 macrophages with ALDH2 knock-down. Collectively, these results clearly demonstrated that ALDH2 deficiency reinforced pro-inflammatory responses and glycolysis in macrophages potentiated by environmentally relevant concentration of FA, which may increase the susceptibility to inflammation and immunotoxicity induced by environmental FA exposure.
Assuntos
Acetaldeído , Etanol , Humanos , Camundongos , Animais , Aldeído-Desidrogenase Mitocondrial/genética , Etanol/toxicidade , Acetaldeído/toxicidade , Formaldeído/toxicidade , Camundongos Knockout , MacrófagosRESUMO
Arsenite (As+3) is a group one human carcinogen, which has been associated with many diseases. Previous studies indicated that As+3 could inhibit wound healing and repair. M2a cells are known as tissue remodeling macrophages, which play an important role in wound repair process. Peroxisome proliferator-activated receptor gamma (PPAR-γ), a key regulator of lipid and glucose metabolism, was found to mediate the IL-4-dependent M2a polarization of macrophages. In the present study, As+3 induced dose-dependent inhibition of M2a polarization starting from 0.1 µM in THP-1-derived macrophages stimulated with 20 ng/mL IL-4. Increased lipid accumulation and decreased PPAR-γ expression were also observed in As+3-treated M2a macrophages. Rosiglitazone (RSG), a potent PPAR-γ agonist, alleviated the suppressions of PPAR-γ and M2a polarization induced by 2 µM As+3. Collectively, these results not only demonstrated that As+3 was able to inhibit polarization of M2a cells through PPAR-γ suppression, but also indicated that PPAR-γ could be utilized as a target for the prevention and treatment of As+3-induced immunotoxicity.
Assuntos
Arsenitos , Tiazolidinedionas , Arsenitos/metabolismo , Arsenitos/toxicidade , Regulação para Baixo , Humanos , Interleucina-4/metabolismo , Lipídeos , Macrófagos/metabolismo , PPAR gama/metabolismoRESUMO
Environmental exposure to formaldehyde is known to be associated with cancers and many other diseases. Although formaldehyde has been classified as a group I carcinogen, the molecular mechanisms of its carcinogenicity are still not fully understood. Formaldehyde is also involved in the folate-driven onecarbon metabolism, and excess amount of formaldehyde was found to interfere with other metabolic pathways including glycolysis, which can enhance Warburg effect and induce immunosuppression in tumor microenvironment. Therefore, different tumor cells and THP-1 derived macrophages were utilized to explore the metabolism-related effects induced by formaldehyde at environmentally relevant concentrations. Significant increases of glucose uptake, glycolysis levels, HIF-1α signaling and methylglyoxal production were observed in tumor cells treated with 20 and 50 µM formaldehyde for 24 h, and the overproduced methylglyoxal in the conditioned medium collected from the tumor cells treated with formaldehyde triggered macrophage polarization towards M2 cells. Myricetin, a flavonol scavenging methylglyoxal, reversed the polarization of macrophages induced by methylglyoxal at 50 µM. These results not only provided essential evidences to reveal the molecular mechanisms of Warburg effect and metabolism-related immunosuppression related to formaldehyde exposure, but also indicated that methylglyoxal could be utilized as a target for therapeutic treatment or prevention of formaldehyde-induced immunotoxicity.
Assuntos
Formaldeído/efeitos adversos , Aldeído Pirúvico/metabolismo , Macrófagos Associados a Tumor/efeitos dos fármacos , Efeito Warburg em Oncologia/efeitos dos fármacos , Células A549 , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Células MCF-7 , Ativação de Macrófagos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Th1 , Microambiente Tumoral/efeitos dos fármacos , Macrófagos Associados a Tumor/metabolismoRESUMO
Orlistat is an FDA-approved over-the-counter drug to treat obesity through the inhibition of lipase activity. Macrophages, which express high levels of lipoprotein lipase (LPL), are important phagocytes in the innate immune system. Our previous studies indicated that environmentally relevant concentrations of arsenite (As+3) could inhibit the major immune functions of macrophages. As the down-regulation of LPL is known to increase the expression of ABCA1, the cholesterol exporter demonstrated to be related to the resistance of arsenic toxicity. We examined if orlistat could reverse the inhibitive effects of As+3 on macrophage functions. The results showed that 50 µM orlistat reversed As+3-induced suppressions on phagocytosis, NO production and cytokine secretion in THP-1 derived macrophages. The expression of ABCA1 was significantly increased by orlistat in As+3 co-treated macrophages, which was associated with decreased intracellular As+3 levels. Collectively, these results indicated that orlistat could reverse the suppressive effects induced by As+3 in macrophages through the increased expression of ABCA1, which has the potential to be developed as a therapeutic agent for arsenic-induced immunosuppression.
Assuntos
Arsenitos , Arsenitos/toxicidade , Colesterol , Macrófagos , Orlistate , Regulação para CimaRESUMO
The lack of effective chemical tools capable of dynamic tracking of labile glyoxal species (GOS) [e.g., methylglyoxal (MGO) and glyoxal (GO)] levels with high selectivity over other relevant electrophilic species, particularly, formaldehyde (FA) and nitric oxide (NO), has significantly hampered the understanding of their roles in a complex metabolic network and disease progressions. Herein, we report the rational design of the bioinspired 4-(2-guanidino)-1,8-naphthalimide fluorescent probes NAP-DCP-1 and NAP-DCP-3 from arginine-specific protein modifications. These probes undergo facile reversible fluorophore-promoted deprotonation-cyclization of a guanidium ion with labile GOS to form exocyclic five-membered dihydroxyimidazolidines. The probe NAP-DCP-1 can differentiate GOS levels in the serum of diabetic mice and patients from nondiabetic ones, which correlate very well with glucose levels, providing the GOS level as a potential new biomarker for diabetes diagnosis. Notably, the endoplasmic reticulum (ER)-targeting probe NAP-DCP-3 enabled the study of GOS perturbation in ER under various stress conditions and led to the discovery that formaldehyde (FA), either exogenously added or endogenously generated, could induce GOS level increases in ER. This finding reveals the previous unknown connection of FA with upregulated GOS levels and suggests that GOS is a key metabolite in bridging one-carbon metabolism with glycolysis and the downstream cell redox status. Moreover, the probes also showed potentials in separate quantification of MGO and GO via ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and unexpected selectivity modulation for GO over MGO via two-photon excitation. It is expected that probes reported herein provide powerful tools to study GOS level modulations in complex biological networks and would facilitate GOS-associated basic research and discovery.
Assuntos
Corantes Fluorescentes/química , Glioxal/química , Espectrometria de Massas/métodos , Animais , Arginina/química , Cromatografia Líquida de Alta Pressão , Ciclização , Diabetes Mellitus/sangue , Diabetes Mellitus/diagnóstico , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/diagnóstico , Retículo Endoplasmático/química , Corantes Fluorescentes/síntese química , Formaldeído/química , Glioxal/análise , Glioxal/sangue , Glioxal/metabolismo , Células HeLa , Humanos , Camundongos , Microscopia de FluorescênciaRESUMO
Mebendazole (MBZ) is a tubulin-suppressive antihelmintic agent with low toxicity, which has been repurposed to treat different types of tumors. Chemoresistance is quite common in refractory or relapsed T cell acute lymphoblastic leukemia (T-ALL), which leads to dismal chances of recovery. In this study, MBZ was found to suppress the proliferation and reduce the viability of T-ALL cell line, CCRF-CEM, and its chemoresistant derivative, CEM/C1, at nanomolar concentrations. The inhibitive effects were found to be dose-dependent and not to be affected by the chemoresistance of CEM/C1 cells. Cell cycle arrest, caspase 3/7 activation and tubulin disruption were found in the MBZ-treated T-ALL cells. Notch1 signaling, which is often aberrantly activated in T-ALL cells, was showed to be suppressed by MBZ treatments. MBZ administration in murine T-ALL models also suppressed the growth of CEM/C1 cells, indicating that MBZ may be developed as a therapeutic agent for chemoresistant T-ALLs. The mRNA levels of the Notch1 and Hes1 were also confirmed to be suppressed by MBZ in vivo, which was consistent with the in vitro observations. This study demonstrated, for the first time, that MBZ could inhibit chemoresistant T-ALL cells both in vitro and in vivo, and the Notch1 signaling pathway was suppressed by MBZ treatment.
Assuntos
Antineoplásicos/uso terapêutico , Mebendazol/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Moduladores de Tubulina/uso terapêutico , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Receptor Notch1/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The controversy surrounding the use of diphtheria toxin (DT) as a therapeutic agent against tumor cells arises mainly from its unexpected harmfulness to healthy tissues. We encoded the cytotoxic fragment A of DT (DTA) as an objective gene in the Light-On gene-expression system to construct plasmids pGAVPO (pG) and pU5-DTA (pDTA). Meanwhile, a cRGD-modified ternary complex comprising plasmids, chitosan, and liposome (pG&pDTA@cRGD-CL) was prepared as a nanocarrier to ensure transfection efficiency. Benefiting from spatiotemporal control of this light-switchable transgene system and the superior tumor targeting of the carrier, toxins were designed to be expressed selectively in illuminated lesions. In vitro studies suggested that pG&pDTA@cRGD-CL exerted arrest of the S phase in B16F10 cells upon blue light irradiation and, ultimately, induced the apoptosis and necrosis of tumor cells. Such DTA-based treatment exerted enhanced antitumor activity in mice bearing B16F10 xenografts and displayed prolonged survival time with minimal side effects. Hence, we described novel DTA-based therapy combined with nanotechnology and the Light-On gene-expression system: such treatment could be a promising strategy against melanoma.
Assuntos
Toxina Diftérica/genética , Expressão Gênica/efeitos da radiação , Terapia Genética , Lipossomos/química , Melanoma Experimental/terapia , Nanotecnologia/métodos , Fragmentos de Peptídeos/genética , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Quitosana/química , Expressão Gênica/genética , Lipossomos/ultraestrutura , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Peptídeos Cíclicos/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Esferoides Celulares/efeitos da radiação , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
An important challenge for reaction-based fluorescent probes is that they generally require analyte consumption for fluorescence signal generation, thus creating potential perturbation of native analyte homeostasis or change of local concentrations. Herein, we reported two formaldehyde (FA) regeneration fluorescent probes, NAP-FAP-1 and NAP-FAP-2. An unprecedented regiospecific FA-induced intramolecularity strategy is implemented in the probe design, which adopts 3-(benzylamino)-succinimide as the FA-selective reaction group. The probes are able to capture the analyte molecule, induce regiospecific imide bond cleavage, and then release the captured FA molecule with simultaneous fluorescence turn-on response via a unique dual PeT/ICT quenching mechanism. The probes have shown potentials in detection, comparison, and imaging of FA levels intracellularly and inside lysosomes. These features make them useful for the study of FA homeostasis and functions in biological systems with minimal perturbation.
RESUMO
γ-Glutamyltranspetidase (GGT) is a cell-membrane-bound enzyme which selectively catalyzes cleavage of the γ-glutamyl bond of glutathione (GSH). It has been identified to be overexpressed in a number of malignant tumor cells. Therefore, fluorescent probes for fast and selective detection of GGT activities are greatly needed. However, the majority of currently available GGT fluorescent probes based on direct conjugation of a γ-glutamyl group to a specific fluorophore generally has slow enzymatic kinetics due to bulky fluorophore too close to the enzyme's active site. Moreover, the uncaged fluorophore with a free amine group might undergo oxidation or other enzymatic transformation and resulted in a complicated time-dependent fluorescence response. Herein, we reported design of a novel fluorescent GGT probe NM-GSH (2), which incorporated a fast intramolecular transcyclization cascade for rapid detection of GGT activities after enzymatic cleavage of the γ-glutamyl group. This design strategy allows introduction of bulky 1,8-naphthalimide fluorophore with improved enzymatic kinetics and lowered detection limit. The transcyclized product 4 gives more than 200-fold fluorescence increment. The probe NM-GSH showed both good selectivity and fast detection of GGT activities with the detection limit as low as 0.21 mU/mL. In addition, the fluorescent product 4 contains no free amine group and is more stable for detection. Most importantly, cell imaging studies showed that the transcyclized product 4 was enriched in lysosomes for selectively lighting up GGT-overexpressed ovarian cancer cells (OVCAR5) but not normal cells (HUVEC), indicating NM-GSH's potentials as an imaging agent in cancer diagnosis and treatment.
Assuntos
Fluorescência , Corantes Fluorescentes/química , gama-Glutamiltransferase/análise , gama-Glutamiltransferase/metabolismo , Linhagem Celular Tumoral , Ciclização , Corantes Fluorescentes/síntese química , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Microscopia Confocal , Estrutura MolecularRESUMO
Differentiation of biologically important thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) is still a challenging task. Herein, we present a novel fluorescent chemodosimeter capable of selectively detecting Cys over other biothiols including Hcy and GSH and other amino acids by a facile thiol-Michael addition/transcyclization rearrangement cascade click process. The unique transcyclization step is critical for the selectivity as a result of the kinetically favorable formation of a six-membered ring with the Cys Michael adduct. Moreover, the probe adopts a distinctive dual quenching mechanism-photoinduced electron transfer (PET) and photoinduced intramolecular charge transfer (ICT) to deliver a drastic turn-on fluorescence response only at the Cys-selective transcylization step. The judicious selection of strong electron-withdrawing naphthalimide fluorophore with maleimide group enhances the electrophilicity and thus reactivity for the cascade process leading to fast detection and ultrasensitivity with a detection limit of 2.0â nm (S/N=3). The probe has demonstrated its practical utility potential in Cys imaging in live cells.
Assuntos
Cisteína/química , Corantes Fluorescentes/química , Química Click , Ciclização , Cisteína/análise , Transporte de Elétrons , Corantes Fluorescentes/análise , Corantes Fluorescentes/síntese química , Glutationa/química , Células HeLa , Homocisteína/química , Humanos , Limite de Detecção , Maleimidas/química , Microscopia de Fluorescência , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
A triptycene-coumarin hybrid dye DCT-1 with a 1,4-dimethoxybenzene group as the electron donor and a coumarin fluorophore as the acceptor on the separated fins of a triptycene was synthesized. DCT-1 features a charge separated excited state with emissions sensitive to solvent polarities. Moreover, DCT-1 also exhibits aggregation-induced emission properties in water with excellent photostability and pH-stability for potential cell imaging applications.
Assuntos
Antracenos/química , Cumarínicos/química , Corantes Fluorescentes/química , Linhagem Celular , Transporte de Elétrons , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Conformação Molecular , Imagem Óptica , Espectrometria de FluorescênciaRESUMO
Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of (15)N-enriched ions from metabolically labeled (15)N-sphingolipids in the plasma membrane, using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids--both in living cells and during fixation of living cells--exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous (15)N-sphingolipid microdomains with mean diameters of â¼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long-range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.
Assuntos
Fibroblastos/química , Lipídeos de Membrana/química , Esfingolipídeos/química , Membrana Celular/química , Hemaglutininas/química , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Espectrometria de Massa de Íon SecundárioRESUMO
The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.
Assuntos
Colesterol/metabolismo , Fibroblastos/metabolismo , Microdomínios da Membrana/metabolismo , Esfingolipídeos/metabolismo , Animais , Citoesqueleto/metabolismo , Fibroblastos/citologia , Camundongos , Células NIH 3T3RESUMO
Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.
Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Acilação , Animais , Transporte Biológico , Compostos de Boro/síntese química , Compostos de Boro/metabolismo , Sobrevivência Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Esfingosina/síntese química , Esfingosina/química , Esfingosina/metabolismo , Especificidade por SubstratoRESUMO
Macrophages are important innate immune cells which can be polarized into heterogeneous populations. The inflammatory-activated M1 cells are known to be involved in all kinds of inflammatory diseases, which were also found to be associated with dysregulation of iron metabolism. While iron overload is known to induce M1 polarization, the valence states of iron and its intracellular dynamics during macrophage inflammatory activation have not been identified. In this study, THP-1-derived macrophages were polarized into M1, M2a, M2b, M2c, and M2d cells, and intracellular ferrous iron (Fe(II)) was measured by our previously developed ultrasensitive Fe(II) fluorescent probe. Significant accumulation of Fe(II) was only observed in M1 cells, which was different from the alterations of total iron. Time-dependent change of intracellular Fe(II) during the inflammatory activation was also consistent with the expression shifts of transferrin receptor CD71, ferrireductase Steap3, and Fe(II) exporter Slc40a1. In addition, accumulation of Fe(II) was also found in the colon macrophages of mice with ulcerative colitis, which was positively correlated to inflammatory phenotypes, including the productions of NO, IL-1ß, TNF-α, and IL-6. Collectively, these results demonstrated the specific accumulation of Fe(II) in inflammatory-activated macrophages, which not only enriched our understanding of iron homeostasis in macrophages, but also indicated that Fe(II) could be further developed as a potential biomarker for inflammatory-activated macrophages.
Assuntos
Sobrecarga de Ferro , Ferro , Animais , Camundongos , Ferro/metabolismo , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sobrecarga de Ferro/metabolismo , Compostos Ferrosos/metabolismoRESUMO
The increased risk of breast cancer metastasis is closely linked to the effects of platelets. Our previously light-switchable diphtheria toxin A fragment (DTA) gene system, known as the LightOn system, has demonstrated significant therapeutic potential; it lacks antimetastatic capabilities. In this study, we devised an innovative system by combining cell membrane fusion liposomes (CML) loaded with the light-switchable transgene DTA (pDTA) and a ticagrelor (Tig) prodrug. This innovative system, named the sequential rocket-mode bioactivating drug delivery system (pDTA-Tig@CML), aims to achieve targeted pDTA delivery while concurrently inhibiting platelet activity through the sequential release of Tig triggered by reactive oxygen species with the tumor microenvironment. In vitro investigations have indicated that pDTA-Tig@CML, with its ability to sequentially release Tig and pDTA, effectively suppresses platelet activity, resulting in improved therapeutic outcomes and the mitigation of platelet driven metastasis in breast cancer. Furthermore, pDTA-Tig@CML exhibits enhanced tumor aggregation and successfully restrains tumor growth and metastasis. It also reduces the levels of ADP, ATP, TGF-ß, and P-selectin both in vitro and in vivo, underscoring the advantages of combining the bioactivating Tig prodrug nanoplatform with the LightOn system. Consequently, pDTA-Tig@CML emerges as a promising light-switchable DTA transgene system, offering a novel bioactivating prodrug platform for breast cancer treatment.
Assuntos
Neoplasias da Mama , Pró-Fármacos , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ticagrelor/farmacologia , Linhagem Celular Tumoral , Lipossomos , Transgenes , Microambiente Tumoral , Melanoma Maligno CutâneoRESUMO
Cholesterol is believed to be an important component in compositionally distinct lipid domains in the cellular plasma membrane, which are referred to as lipid rafts. Insight into how cholesterol influences the interactions that contribute to plasma membrane organization can be acquired from model lipid membranes. Here we characterize the lipid mixing and phase behavior exhibited by (15)N-dilaurolyphosphatidycholine ((15)N-DLPC)/deuterated distearoylphosphatiylcholine (D(70)-DSPC) membranes with various amounts of cholesterol (0, 3, 7, 15 or 19mol%) at room temperature. The microstructures and compositions of individual membrane domains were determined by imaging the same membrane locations with both atomic force microscopy (AFM) and high-resolution secondary ion mass spectrometry (SIMS) performed with a Cameca NanoSIMS 50. As the cholesterol composition increased from 0 to 19mol%, the circular ordered domains became more elongated, and the amount of (15)N-DLPC in the gel-phase domains remained constant at 6-7mol%. Individual and micron-sized clusters of nanoscopic domains enriched in D(70)-DSPC were abundant in the 19mol% cholesterol membrane. AFM imaging showed that these lipid domains had irregular borders, indicating that they were gel-phase domains, and not non-ideally mixed lipid clusters or nanoscopic liquid-ordered domains.
Assuntos
Colesterol/química , Lipídeos de Membrana/química , Microscopia de Força Atômica/métodos , Géis , Bicamadas Lipídicas/química , Fluidez de Membrana , Microdomínios da Membrana/química , Membranas/química , Peptídeos/química , Fosfatidilcolinas/química , TemperaturaRESUMO
An anthracenecarboximide-guanidine based turn-on fluorescent probe ANC-DCP-1 for selective detection of glyoxals (methylglyoxal and glyoxal, GOS) over formaldehyde under weak acidic conditions around pH 6.0 was reported. The probe showed great potential in studying relative GOS levels in weak acidic biological fluids such as in urine for diabetic diagnosis and prognosis, and also found application in the food industry such as for fast unique manuka factor (UMF) scale determination of Manuka honey.
RESUMO
A fluorescent probe (COU-LIP-1) for monitoring labile Fe(II) pools (LIP) with high selectivity and sensitivity was developed utilizing coumarin 343 as the fluorophore and 3-nitrophenylazanyl ester as both the reactive group and the fluorescence quenching group. Fe(II)-induced reductive cleavage of the N-O bond results in a turn-on response via a photo-induced photon transfer (PET) mechanism. The probe was applied for monitoring labile iron(II) changes in M1 and M2a macrophage activations and also erastin-induced ferroptosis, providing a powerful tool for selectively sensing LIP under both physiological and stressed conditions.
Assuntos
Ferroptose , Compostos Ferrosos/metabolismo , Corantes Fluorescentes/química , Ativação de Macrófagos , Tomografia por Emissão de Pósitrons/métodos , FluorescênciaRESUMO
An "AND"-logic-gate-based fluorescent probe NAP-DCP-4 with dual reactive sites is reported, which has improved selectivity for methylglyoxal over glyoxal, featuring formaldehyde-enhanced methylglyoxal detection and irreversible and reversible turn-on fluorescence responses at different excitation wavelengths. Its cell-impermeability enables facile monitoring of extracellular methylglyoxal level changes in the supernatant of activated macrophages.