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1.
Anal Chem ; 93(17): 6629-6637, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33880916

RESUMO

A substantial fraction of common metabolites contains carboxyl functional groups. Their 13C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the 13C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. 15N1-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via 15N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how 13C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine 15N atom in the derivatives. We have applied this method for the determination of 13C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.


Assuntos
Ácidos Carboxílicos , Compostos de Trimetil Amônio , Animais , Carbono , Espectroscopia de Ressonância Magnética , Camundongos
2.
Macromol Rapid Commun ; 41(9): e2000021, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32212226

RESUMO

Aqueous palladium-catalyzed direct arylation polymerization (DArP) of 2-bromothiophene derivatives 6-(2-(2-bromothiophen-3-yl)ethoxy)hexyl trimethylammonium bromide (T1) and 4-(2-(2-bromothien-3-yl)ethoxy)butylsulfonate (T2) is achieved. The supporting ligand, triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt (m-TPPTs), facilitates DArP of both derivatives; however, its separation from the polymers by dialysis is difficult due to its strong aggregation in water and N,N-dimethylacetamide (DMAc). This is supported by dynamic light scattering, gel permeation chromatography (GPC), and single-crystal X-ray crystallography. Pyrimidine-Pd(OAc)2 is utilized in the DArP of T1 to afford PT1 without ligand contamination. Density functional theory calculations to determine the coordinating capability of the carboxylate/pivalic acid/water to palladium indicate the viability of implementing DArP in water. Finally, polyelectrolyte molecular-weight overestimation by GPC in water is attributed to the polyelectrolyte effect. Aggregation of the conjugated polyelectrolytes leads to a contracted hydrodynamic volume, and the molecular weight and dispersity assessed by GPC in DMAc significantly deviate from the actual values. An objective approach to evaluate the molecular weight for conjugated polyelectrolytes requires further development.


Assuntos
Paládio/química , Ácidos Sulfônicos/química , Compostos de Trimetil Amônio/química , Catálise , Estrutura Molecular , Polimerização , Água/química
3.
Sci Rep ; 10(1): 3959, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127558

RESUMO

The importance of graft copolymerization in the field of polymer science is analogous to the importance of alloying in the field of metals. This is attribute to the ability of the grafting method to regulate the properties of polymer 'tailor-made' according to specific needs. This paper described a novel plant-based coagulant, LE-g-DMC that synthesized through grafting of 2-methacryloyloxyethyl trimethyl ammonium chloride (DMC) onto the backbone of the lentil extract. The grafting process was optimized through the response surface methodology (RSM) using three-level Box-Behnken Design (BBD). Under optimum conditions, a promising grafting percentage of 120% was achieved. Besides, characterization study including SEM, zeta potential, TGA, FTIR and EDX were used to confirm the grafting of the DMC monomer chain onto the backbone of lentil extract. The grafted coagulant, LE-g-DMC outperformed lentil extract and alum in turbidity reduction and effective across a wide range of pH from pH 4 to pH 10. Besides, the use of LE-g-DMC as coagulant produced flocs with excellent settling ability (5.09 mL/g) and produced the least amount of sludge. Therefore, from an application and economic point of views, LE-g-DMC was superior to native lentil extract coagulant and commercial chemical coagulant, alum.


Assuntos
Lens (Planta)/química , Micro-Ondas , Extratos Vegetais/química , Compostos de Trimetil Amônio/química , Modelos Teóricos , Espectroscopia de Infravermelho com Transformada de Fourier
4.
Biomolecules ; 9(7)2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288435

RESUMO

The unique electrochemical properties of ionic liquids (ILs) have motivated their use as solvents for organic synthesis and green energy applications. More recently, their potential in pharmaceutical chemistry has prompted investigation into their effects on biomolecules. There is evidence that some ILs can destabilize proteins via a detergent-like manner; however, the mechanism still remains unknown. Our hypothesis is that if ILs are denaturing proteins via a detergent-like mechanism, detergent-mediated protein unfolding should be enhanced in the presence of ILs. The properties of myoglobin was examined in the presence of a zwitterionic (N,N-dimethyl-N-dodecylglycine betaine (Empigen BB®, EBB)), cationic (tetradecyltrimethylammonium bromide (TTAB)), and anionic (sodium dodecyl sulfate (SDS)) detergent as well as ILs based on alkylated imidazolium chlorides. Protein structure was measured through a combination of absorbance, fluorescence, and circular dichroism (CD) spectroscopy: absorbance and CD were used to monitor heme complexation to myoglobin, and tryptophan fluorescence quenching was used as an indicator for heme dissociation. Notably, the detergents tested did not fully denature the protein but instead resulted in loss of the heme group. At low IL concentrations, heme dissociation remained a traditional, cooperative process; at high concentrations, ILs with increased detergent-like character exhibited a more complex pattern, which is most likely attributable to micellization of the ionic liquids or direct denaturation or heme dissociation induced by the ILs. These trends were consistent across all species of detergents. 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence was further used to characterize micelle formation in aqueous solutions containing detergent and ionic liquid. The dissociation thermodynamics show that EBB- and TTAB-induced dissociation of heme is not significantly impacted by room temperature ionic liquids (RTILs), whereas SDS-induced dissociation is more dramatically impacted by all RTILs examined. Together, these results indicate a complex interaction of detergents, likely based on headgroup charge, and the active component of RTILs to influence heme dissociation and potentially protein denaturation.


Assuntos
Líquidos Iônicos/química , Mioglobina/química , Dodecilsulfato de Sódio/química , Compostos de Trimetil Amônio/química , Técnicas Eletroquímicas , Modelos Moleculares , Estrutura Molecular , Compostos Orgânicos/química , Desdobramento de Proteína , Termodinâmica
5.
Sci Rep ; 9(1): 8666, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209282

RESUMO

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.


Assuntos
Ácido Aminolevulínico/farmacologia , Dinamina II/metabolismo , Exocitose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Linhagem Celular Tumoral , Dinamina II/antagonistas & inibidores , Dinamina II/genética , Exocitose/efeitos da radiação , Heme/antagonistas & inibidores , Heme/biossíntese , Humanos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Fotoquimioterapia , Compostos de Trimetil Amônio/farmacologia , Raios Ultravioleta
6.
FEBS Open Bio ; 9(2): 291-303, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30761254

RESUMO

Enucleation is the process whereby the nucleus is extruded from the erythroblast during late stage mammalian erythropoiesis. However, the specific signaling pathways involved in this process remain unclear. To better understand the mechanisms underlying erythroblast enucleation, we investigated erythroblast enucleation using both the spleens of adult mice with phenylhydrazine-induced anemia and mouse fetal livers. Our results indicated that both iron-bound transferrin (holo-Tf) and the small-molecule iron transporter hinokitiol with iron ions (hinokitiol plus iron) promote hemoglobin synthesis and the enucleation of mouse spleen-derived erythroblasts. Although an antitransferrin receptor 1 (TfR1) monoclonal antibody inhibited both enucleation and hemoglobin synthesis promoted by holo-Tf, it inhibited only enucleation, but not hemoglobin synthesis, promoted by hinokitiol plus iron. Furthermore, siRNA against mouse TfR1 were found to suppress the enucleation of mouse fetal liver-derived erythroblasts, and the endocytosis inhibitor MitMAB inhibited enucleation, hemoglobin synthesis, and the internalization of TfR1 promoted by both types of stimuli. Collectively, our results suggest that TfR1, iron ions, and endocytosis play important roles in mouse erythroblast enucleation.


Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Eritroblastos/citologia , Eritroblastos/metabolismo , Receptores da Transferrina/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Eritroblastos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Fenil-Hidrazinas/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores da Transferrina/antagonistas & inibidores , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Relação Estrutura-Atividade , Compostos de Trimetil Amônio/farmacologia
7.
J Am Soc Mass Spectrom ; 29(12): 2360-2367, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30151680

RESUMO

Prostaglandins (PGs) are biologically active metabolites of arachidonic acid containing 20 carbon atoms, a cyclic moiety, and two side chains (A and B) in common. The bioassay of PGs requires high sensitivity because of their low concentration in tissues and blood and has usually been carried out by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. Chemical derivatization of PG carboxylic acid groups to introduce positive charge-carrying groups is an established strategy to improve the sensitivity and selectivity of such assays. In this study, we exploited this approach for structural identification of a series of PGs using cholamine derivatization through an amidation reaction. However, we observed that collision-induced dissociation of these derivatives gave rise to unexpected product ions that we postulated were formed by unique long-range intramolecular reactions resulting in dehydration of the B chain accompanied by fragmentation of the A chain through an unusual Hofmann rearrangement. Evidence for the proposed mechanism is presented based on ESI-MS/MS and high resolution mass spectrometry studies of cholamine derivatives of PGE1, PGE2, PGD2, PGI2, and C-17 methyl deuterium-labeled limaprost. Graphical Abstract.


Assuntos
Prostaglandinas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de Trimetil Amônio/química , Modelos Moleculares , Espectrometria de Massas em Tandem/métodos
8.
Langmuir ; 34(19): 5545-5550, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29715034

RESUMO

We studied the stabilities of short (4- and 3-bp) DNA duplexes within silica mesopores modified with a positively charged trimethyl aminopropyl (TMAP) monolayer (BJH pore diameter 1.6-7.4 nm). The DNA fragments with fluorescent dye were introduced into the pores, and their fluorescence resonance energy transfer (FRET) response was measured to estimate the structuring energies of the short DNA duplexes under cryogenic conditions (temperature 233-323 K). The results confirmed the enthalpic stability gain of the duplex within size-matched pores (1.6 and 2.3 nm). The hybridization equilibrium constants found for the size-matched pores were 2 orders of magnitude larger than those for large pores (≥3.5 nm), and this size-matching effect for the enhanced duplex stability was explained by a tight electrostatic interaction between the duplex and the surface TMAP groups. These results indicate the requirement of the precise regulation of mesopore size to ensure the stabilization of hydrogen-bonded supramolecular assemblies.


Assuntos
DNA/química , Dióxido de Silício/química , Corantes Fluorescentes , Congelamento , Hibridização de Ácido Nucleico , Porosidade , Termodinâmica , Compostos de Trimetil Amônio/química
9.
Langmuir ; 34(11): 3467-3484, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29482319

RESUMO

Oral bioavailability of a drug molecule requires its effective delivery to the target site. In general, majority of synthetically developed molecular entities have high hydrophobic nature as well as low bioavailability, therefore the need for suitable delivery vehicles arises. Self-assembled structures such as micelles, niosomes, and liposomes have been used as effective delivery vehicles and studied extensively. However, the information available in literature is mostly qualitative in nature. We have quantitatively investigated the partitioning of antibiotic drug streptomycin into cationic, nonionic, and a mixture of cationic and nonionic surfactant micelles and its interaction with the transport protein serum albumin upon subsequent delivery. A combination of calorimetry and spectroscopy has been used to obtain the thermodynamic signatures associated with partitioning and interaction with the protein and the resulting conformational changes in the latter. The results have been correlated with other class of drugs of different nature to understand the role of molecular features in the partitioning process. These studies are oriented toward understanding the physical chemistry of partitioning of a variety of drug molecules into suitable delivery vehicles and hence establishing structure-property-energetics relationships. Such studies provide general guidelines toward a broader goal of rational drug design.


Assuntos
Micelas , Octoxinol/química , Estreptomicina/química , Tensoativos/química , Compostos de Trimetil Amônio/química , Animais , Calorimetria/métodos , Bovinos , Desenho de Fármacos , Fluorescência , Tamanho da Partícula , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Pirenos/química , Soroalbumina Bovina/metabolismo , Estreptomicina/metabolismo , Temperatura , Termodinâmica
10.
J Biomol Struct Dyn ; 36(10): 2543-2557, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28768117

RESUMO

Protein aggregation into oligomers and mature fibrils are associated with more than 20 diseases in humans. The interactions between cationic surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) with varying alkyl chain lengths and bovine liver catalase (BLC) were examined by various biophysical approaches. The delicate coordination of electrostatic and hydrophobic interactions with protein, play imperative role in aggregation. In this article, we have reconnoitered the relation between charge, hydrophobicity and cationic surfactants DTAB and TTAB on BLC at pH 7.4 and 9.4 which are two and four units above pI, respectively. We have used techniques like turbidity, Rayleigh light scattering, far-UV CD, ThT, ANS, Congo red binding assay, DLS, and transmission electron microscopy. The low concentration ranges of DTAB (0-600 µM) and TTAB (0-250 µM) were observed to increase aggregation at pH 9.4. Nevertheless, at pH 7.4 only TTAB was capable of inducing aggregate. DTAB did not produce any significant change in secondary structure at pH 7.4 suggestive of the role of respective charges on surfactants and protein according to the pI and alkyl chain length. The morphology of aggregates was further determined by TEM, which proved the existence of a fibrillar structure. The surfactants interaction with BLC was primarily electrostatic as examined by ITC. Our work demystifies the critical role of charge as well as hydrophobicity in amyloid formation.


Assuntos
Fenômenos Biofísicos , Catalase/química , Tensoativos/química , Animais , Benzotiazóis/metabolismo , Calorimetria , Catalase/ultraestrutura , Cátions , Bovinos , Dicroísmo Circular , Difusão Dinâmica da Luz , Hidrodinâmica , Concentração de Íons de Hidrogênio , Nefelometria e Turbidimetria , Compostos de Amônio Quaternário/química , Espectrometria de Fluorescência , Termodinâmica , Compostos de Trimetil Amônio/química
11.
J Labelled Comp Radiopharm ; 61(4): 391-394, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29278650

RESUMO

[15 N]-Cholamine is an isotope tag for metabolomics research, because it possesses 2 important properties: an NMR active isotope and a permanent charge for MS sensitivity. Here, we present a scalable synthesis of [15 N]-cholamine.


Assuntos
Isótopos de Nitrogênio/química , Compostos de Trimetil Amônio/química , Brometos/química , Ácidos Carboxílicos/química , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos
12.
J Oleo Sci ; 66(11): 1229-1238, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29021491

RESUMO

The incorporation of additive in lipid bilayers is one of the ordinary approaches for modulating their properties. Additive effect on phase transition of ion-pair amphiphile (IPA) bilayers, however, is not known. In this work, four double-chained IPAs with different hydrocarbon chain lengths and symmetry were designed and synthesized from single-chained cationic and anionic surfactants by the precipitation method. By using differential scanning calorimetry (DSC), the thermotropic transition behavior from gel phase (Lß) through rippled phase (Pß') if any to liquid-crystalline phase (Lα) was studied for bilayers of these lipid-like IPAs in excess water. The effects of three sterol-like additives (cholesterol, α-tocopherol, and α-tocopheryl acetate) in IPA bilayers on thermal phase behavior were then systematically investigated. The experimental results revealed that with increasing concentration of additive, the phase transition temperatures were unaffected on the one hand and the enthalpies of phase transition were decreased on the other hand. When the addition of additive exceeded a specific amount, the phase transition disappeared. More hasty disappearance of phase transition was found for IPAs with lower total number of carbon atoms in the hydrocarbon chains. For IPAs with the same total number of carbon atoms in the hydrocarbon chains, the disappearance of phase transition is more hasty for the asymmetric one than for the symmetric one. Similar effects on thermal phase behavior of four IPA bilayers were exhibited by the three additives with similar chemical structures. Possible mechanism of additive effects on phase transition of IPA bilayers was then proposed in line with that of lipid bilayers.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Transição de Fase , alfa-Tocoferol/química , Varredura Diferencial de Calorimetria , Bicamadas Lipídicas/síntese química , Modelos Químicos , Estrutura Molecular , Dodecilsulfato de Sódio/química , Tetradecilsulfato de Sódio/química , Tensoativos/química , Compostos de Trimetil Amônio/química
13.
Water Res ; 126: 189-196, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28957695

RESUMO

Myristyltrimethylammonium bromide (MTAB) is a cationic surfactant used to improve biomass harvesting and pigment extraction form microalgae, but the mechanisms underlying its effectiveness are poorly defined. We document the mechanisms for enhanced harvesting and pigment extraction for the cyanobacterium Synechocystis sp. PCC 6803 using measurements from flow cytometer, zeta potential, release of soluble components, and microscopy. Harvesting efficiency increased as the MTAB/Biomass dose increased from 0 to 40%. A low MTAB dose (≤ 8%) mainly brought about coagulation and flocculation, which led to aggregation that improved harvesting, but 40% MTAB had the highest harvesting efficiency, 62%. Adding MTAB above a MTAB/Biomass dose of 8% also increased cell-membrane permeability, which allowed the solvent (ethyl acetate) to pass into the cells and resulted in a large increase in extraction efficiency of pigments: An MTAB/Biomass ratio of 60% for 180 min achieved the highest extraction efficiencies of chlorophyll and carotenoids, 95% and 91%, respectively. Combining harvesting and extraction performances with results from flow cytometry, zeta potential, release of soluble components, and microscopy lead to the following mechanistic understandings. MTAB dose from 8% to 40% solubilized EPS, which lowered the biomass's negative charge, but caused breakup of the large aggregates. An increase of cell permeability also in this stage allowed ethyl acetate to pass into the cells and achieve better pigment extraction. MTAB >40% led to cell lysis and a large increase in soluble organics, but complete cell lysis was not required to achieve the maximum extraction efficiency. The MTAB/Biomass % ratio for optimizing harvest efficiency and pigment extraction lay in the range of 40%-60%.


Assuntos
Biotecnologia/métodos , Microalgas/química , Tensoativos/química , Synechocystis/química , Compostos de Trimetil Amônio/química , Biomassa , Carotenoides/isolamento & purificação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Clorofila/isolamento & purificação , Relação Dose-Resposta a Droga , Floculação , Citometria de Fluxo , Microalgas/efeitos dos fármacos , Microalgas/crescimento & desenvolvimento , Solventes/química , Synechocystis/efeitos dos fármacos , Synechocystis/crescimento & desenvolvimento , Compostos de Trimetil Amônio/farmacologia
14.
J Comput Aided Mol Des ; 31(1): 21-28, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27432339

RESUMO

In participation of the fifth statistical assessment of modeling of proteins and ligands (SAMPL5), the strength of association of six guests (3-8) to two hosts (1 and 2) were measured by 1H NMR and ITC. Each host possessed a unique and well-defined binding pocket, whilst the wide array of amphiphilic guests possessed binding moieties that included: a terminal alkyne, nitro-arene, alkyl halide and cyano-arene groups. Solubilizing head groups for the guests included both positively charged trimethylammonium and negatively charged carboxylate functionality. Measured association constants (K a ) covered five orders of magnitude, ranging from 56 M-1 for guest 6 binding with host 2 up to 7.43 × 106 M-1 for guest 6 binding to host 1.


Assuntos
Ácidos Carboxílicos/química , Compostos Macrocíclicos/química , Proteínas/química , Compostos de Trimetil Amônio/química , Sítios de Ligação , Desenho de Fármacos , Ligantes , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Termodinâmica
15.
J Oleo Sci ; 65(9): 749-58, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581491

RESUMO

The present study is focused on the effect of the TTABr/MX/H2O-nanoparticles on the rate of piperidinolysis of ionized phenyl salicylate where TTABr represents tetradecyltrimethylammonium bromide and MX = NaCl, NaBr and CnH2n+1CO2Na with n = 4, 5, 6 and 7. Pseudo-first-order rate constant for the piperidinolysis of ionized phenyl salicylate at 35°C and constant concentration [PSa(-)]T = 0.2 mM, [Pip]T = 0.1 M, [NaOH] = 30 mM, [TTABr]T and different [MX] follow an empirical relationship which gives two empirical constant, (X)kcat and K(X/S). The value of relative counterion (X) binding constant, RX(Br) were calculated from the relationship; RX(Br) = (X)kcat/(Br)kcat. The values of RX(Br) for X = C4H9CO2(-), C5H11CO2(-), C6H13CO2(-), and C7H15CO2(-) are increasing with increase in the number of alkyl chain of counterion X.


Assuntos
Bromatos/química , Nanopartículas/química , Salicilatos/química , Cloreto de Sódio/química , Compostos de Sódio/química , Compostos de Trimetil Amônio/química , Ânions/química , Catálise , Cátions/química , Cinética
16.
Biochem Biophys Res Commun ; 478(4): 1609-16, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27586274

RESUMO

Treatment of Jurkat T cells with the dynamin inhibitor, myristyl trimethyl ammonium bromides (MiTMAB) caused cytokinesis impairment and apoptotic DNA fragmentation along with down-regulation of anti-apoptotic BAG3 and Mcl-1 levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9 and -3, and PARP cleavage, without accompanying necrosis. Bcl-xL overexpression completely abrogated these MiTMAB-induced mitochondrial damage and resultant caspase cascade activation, except for impaired cytokinesis and down-regulated BAG3 and Mcl-1 levels. Additionally, autophagic responses including Akt-mTOR pathway inhibition, formation of acridine orange-stainable acidic vesicular organelles, LC3-I/II conversion, and p62/SQSTM1 down-regulation were detected regardless of Bcl-xL overexpression. The autophagy inhibitors 3-methyladenine and LY294002 enhanced MiTMAB-induced apoptotic sub-G1 peak, BAG3 and Mcl-1 down-regulation, Bak activation, Δψm loss, and caspase activation. These results indicate that MiTMAB-caused cytokinesis failure leads to concomitant induction of apoptosis and cytoprotective autophagy, and suggest that inhibition of autophagy is a promising strategy to augment antitumor activity of MiTMAB.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Dinaminas/antagonistas & inibidores , Mitocôndrias/metabolismo , Compostos de Trimetil Amônio/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Anexina A5/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Dinaminas/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Morfolinas/farmacologia
17.
J Appl Microbiol ; 121(4): 1004-14, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27442261

RESUMO

AIMS: To establish the role of cardiolipin (CL) of the membrane in response to the presence of tetradecyltrimethylammonium in Pseudomonas putida A (ATCC 12633). METHODS AND RESULTS: Two ORFs of Ps. putida A (ATCC 12633), which in Ps. putida KT2440 encode the putative CL synthase genes cls and cls2, were cloned, sequenced and mutated. Only the double mutant lacking cls and cls2 showed a reduction of the CL content, 83% lower than the amount produced by the wild-type. Accompanying this change was a 40% decrease in the content of unsaturated fatty acid. Consequently, the membrane of the mutant was more rigid than the one of the parental strain, as observed using fluorescence polarization techniques. The mutant strain showed reduced viability in the presence of tetradecyltrimethylammonium. The incorporation of exogenous CL into its membrane relieved sensitivity to the cationic detergent. CONCLUSIONS: Pseudomonas Putida cells with low levels of CL die in the presence of tetradecyltrimethylammonium, because they cannot counter the fluidizing effect of the cationic surfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: The modification in the membrane phospholipids composition allows knowing the adaptation strategy of Ps. putida when these bacteria are exposed to cationic surfactant.


Assuntos
Antibacterianos/farmacologia , Pseudomonas putida/efeitos dos fármacos , Tensoativos/farmacologia , Compostos de Trimetil Amônio/farmacologia , Cardiolipinas/análise , Cardiolipinas/metabolismo , Clonagem Molecular , Polarização de Fluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fases de Leitura Aberta , Fosfolipídeos/metabolismo , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética
18.
Colloids Surf B Biointerfaces ; 145: 607-616, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27285732

RESUMO

Cholamine surface-modified gelatin nanoparticles prepared by the double desolvation method using acetone as a dehydrating agent were selected and potentially evaluated as non viral vectors of siRNA targeting a metastatic gene AEG-1 in MCF-7 breast carcinoma cells. The ability of modified gelatin nanoparticle to complex and deliver siRNA for gene silencing was investigated. Hence, Particle size, surface charge (zeta potential) and morphology of siRNA/Gelatin nanoparticles (siGNPs) were characterized via dynamic light scattering (DLS), scanning electron microscopy (SEM) and transmission electron microscope (TEM). Moreover, the nanoparticles cytotoxicity, loading efficiency and interaction with MCF-7 human breast carcinoma cells were evaluated. Cationized GNPs of mean size range of 174nm and PDI of 0.101 were produced. The loading efficiency of siGNPs at a Nitrogen/Phosphate (N/P) ratio (w/w) of 200:1 was approximately 96%. Cellular uptake was evaluated after FITC conjugation where the particles produced high transfection efficiency. Finally, ELISA analysis of AEG-1/MTDH expression demonstrated the gene silencing effect of siGNPs, as more than 75% MTDH protein were inhibited. Our data indicate that cholamine modified GNPs pose a promising non-viral siRNA carrier for altering gene expression in MCF-7 breast cancer cells with many advantages such as relatively high gene transfection efficiency and efficient silencing ability.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Gelatina/química , Inativação Gênica , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Compostos de Trimetil Amônio/farmacologia , Animais , Cátions , Moléculas de Adesão Celular/metabolismo , Morte Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescência , Inativação Gênica/efeitos dos fármacos , Glutaral/química , Humanos , Células MCF-7 , Proteínas de Membrana , Nanopartículas/ultraestrutura , Metástase Neoplásica , Tamanho da Partícula , Proteínas de Ligação a RNA , Sus scrofa , Transfecção
19.
Microbiology (Reading) ; 162(5): 813-822, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26925774

RESUMO

Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 µg (mg cell envelope protein)- 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)- 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.


Assuntos
Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Pseudomonas putida/metabolismo , Estresse Fisiológico/fisiologia , Tensoativos/farmacologia , Compostos de Trimetil Amônio/farmacologia , Animais , Cardiolipinas/metabolismo , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Coelhos
20.
Bioanalysis ; 8(6): 557-73, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26915807

RESUMO

This review discusses strategies for the identification of metabolites in complex biological mixtures, as encountered in metabolomics, which have emerged in the recent past. These include NMR database-assisted approaches for the identification of commonly known metabolites as well as novel combinations of NMR and MS analysis methods for the identification of unknown metabolites. The use of certain chemical additives to the NMR tube can permit identification of metabolites with specific physical chemical properties.


Assuntos
Metabolômica , Preparações Farmacêuticas/metabolismo , Bases de Dados Factuais , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metaboloma , Compostos de Trimetil Amônio/análise , Compostos de Trimetil Amônio/metabolismo
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