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1.
Curr Issues Mol Biol ; 46(3): 2105-2118, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38534751

RESUMO

Astrocytes play an important role in the regulation of the inflammatory response in the CNS, e.g., in demyelinating diseases. Since the chemokine CXCL1 is known to be secreted by astrocytes and to have a pro-inflammatory effect on immune cells in the CNS, we verified the effect of testosterone on its secretion in vitro (in the astrocytic cell line DI TNC1). Testosterone reduced the increase in CXCL1 production caused by the pro-inflammatory agent lysophosphatidylcholine and restored the basal production level of CXCL1. The androgen receptor (present and functional in the studied cell line) was strongly suggested to mediate this effect-its non-steroid ligand flutamide exerted an agonist-like effect, mimicking the activity of testosterone itself on CXCL1 secretion. This novel mechanism has important implications for the known immunomodulatory effect of testosterone and potentially other androgenic hormones. It provides a potential explanation on the molecular level and shows that astrocytes are important players in inflammatory homeostasis in the CNS and its hormonal regulation. Therefore, it suggests new directions for the development of the therapeutic intervention.

2.
Int J Nanomedicine ; 18: 2109-2126, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37122501

RESUMO

Background: Mesalazine is one of the main drugs used to treat inflammatory bowel diseases. However, its applicability is limited by its rapid inactivation and removal from the organism, as well as the need for its membrane transporter-dependent cellular uptake to exert therapeutic effect. The present study involved the development of an innovative nanocarrier, based on poly(amidoamine) (PAMAM) dendrimer of the 4th generation, to obtain higher concentrations of the drug in the intestinal epithelial cells, thus increasing its anti-inflammatory potential. The work involved synthesis and in vitro characterization of covalent PAMAM-mesalazine conjugate with succinic linker. Results: PAMAM-mesalazine conjugate was synthesized and characterized by 1H NMR, 13C NMR, FTIR and MALDI-TOF MS. This allowed to confirm the purity of the obtained compound and intermediates. Based on the analyses, it was found that ~45 drug molecules were successfully attached to one molecule of PAMAM dendrimer. The conjugate was then characterized in terms of hydrodynamic diameter, zeta potential, spectral properties, drug release from the carrier, as well as cellular uptake and cytotoxicity in two in vitro models of gastrointestinal epithelium (CaCo-2 and HT-29 human cell lines). Analyzing cellular parameters related to the specific mechanism of action of mesalazine (inhibition of NF-κB signaling, decrease in interleukin and prostaglandin synthesis, and ROS scavenging), we showed that such a dendrimer-based carrier may enhance cellular uptake of the drug, which translated into its improved anti-inflammatory efficacy. Conclusion: The use of PAMAM macromolecule as a carrier for mesalazine increases the bioavailability of the drug, ensuring enhanced cellular uptake and bypassing the need to utilize mesalazine-specific membrane transporters. All these characteristics translate into an improved anti-inflammatory activity of mesalazine in vitro. In conjunction with appropriately designed in vivo studies, such a compound may prove to be a promising alternative to the therapeutics currently used in inflammatory bowel diseases.


Assuntos
Dendrímeros , Nanopartículas , Humanos , Dendrímeros/farmacologia , Mesalamina/farmacologia , Células CACO-2 , Proteínas de Membrana Transportadoras , Anti-Inflamatórios/farmacologia , Excipientes
3.
Am J Ophthalmol ; 236: 63-68, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34710353

RESUMO

PURPOSE: To evaluate corneal sensitivity and corneal nerve morphology among patients with Wolfram syndrome (WFS). DESIGN: An observational clinical case series with confirmatory experiments. METHODS: We included a group of 12 patients with biallelic mutations in the WFS1 gene and a control group composed of 30 individuals with type 1 diabetes (T1D). All participants (n = 42) underwent a complete ophthalmic examination, esthesiometry, and retinal nerve fiber layer assessment using optical coherence tomography. Morphologic assessment of corneal neuropathy by in vivo corneal confocal microscopy was conducted in 11 patients with WFS (both eyes) and 1 WFS patient (1 eye) as well as in 24 patients with T1D (both eyes in 6 patients and 1 eye in 18 patients). Additionally, corneas from Wfs1KO mice and their wild-type littermates were subjected to laser scanning confocal microscopy. RESULTS: Corneal sensitivity was significantly reduced in patients with WFS compared with patients with T1D (4.50 cm [interquartile range, 3.50-5.50 cm] vs 6.00 cm [interquartile range, 6.00-6.00 cm]; P < 10-5). Additionally, corneal nerve fiber and branch density as well as nerve fiber length were low among patients with WFS. Corneal sensitivity correlated with macular average thickness (R = 0.6928; P = .039) and best-corrected visual acuity (R = -0.61; P = .002) in the WFS group. Similarly, Wfs1 knockout mice also presented corneal neurodegeneration changes when corneal nerve fiber density and length were measured using laser scanning confocal microscopy. CONCLUSIONS: Decreased corneal sensitivity and corneal nerve degeneration are observed in WFS. Corneal sensitivity is linked with the degree of disease progression as measured by visual acuity and retinal thinning.


Assuntos
Distrofias Hereditárias da Córnea , Diabetes Mellitus Tipo 1 , Síndrome de Wolfram , Animais , Córnea/inervação , Humanos , Camundongos , Microscopia Confocal/métodos , Fibras Nervosas , Tomografia de Coerência Óptica/métodos , Síndrome de Wolfram/diagnóstico , Síndrome de Wolfram/genética
4.
Biomacromolecules ; 22(8): 3396-3407, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34286584

RESUMO

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles.


Assuntos
Dendrímeros , Nanopartículas , Preparações Farmacêuticas , Linhagem Celular , Dendrímeros/farmacologia , Humanos , Imunomodulação , Interleucina-8/genética , Manose , Células Mieloides
5.
Carbohydr Res ; 505: 108338, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34023694

RESUMO

Reductive cleavage of methyl 3,4-O-benzylidene-α-L-fucopyranosides with BH3·THF-TfOH and NaCNBH3-TfOH systems resulted in enhanced reaction rates and selectivity compared to BH3·THF-Bu2BOTf. With this latter system, the nature of the O-2 substituent exerted a clear control on the reactivity but practically did not affect the regioselectivity. With TfOH the direction of cleavage was determined, as expected, by the configuration of the acetal carbon atom, but slightly influenced by its competitive epimerization. Protic conditions provided higher regioselectivity in the openings of the exo isomers, affording a useful approach to the practical synthesis of 3-O-benzyl ethers.


Assuntos
Acetais , Compostos de Benzilideno , Oxirredução , Estereoisomerismo
6.
Molecules ; 25(19)2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32992824

RESUMO

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.


Assuntos
DNA , Dendrímeros , Corantes Fluorescentes , Transfecção , DNA/química , DNA/farmacologia , Dendrímeros/síntese química , Dendrímeros/química , Dendrímeros/farmacologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos
7.
Sci Rep ; 9(1): 18720, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822695

RESUMO

A new family of paratanaoidean Tanaidacea - Paranarthrurellidae fam. nov. - is erected to accommodate two genera without family classification (Paratanaoidea incertae sedis), namely Armatognathia Kudinova-Pasternak, 1987 and Paranarthrurella Lang, 1971. Seven new species of Paranarthrurella and two of Armatognathia are described from material taken in different deep-sea areas of the Atlantic and Pacific oceans. The type species of Paranarthrurella - P. caudata (Kudinova-Pasternak, 1965) - is redescribed based on the paratype. The genus Cheliasetosatanais Larsen and Araújo-Silva, 2014 originally classified within Colletteidae is synonymised with Paranarthrurella, and Arthrura shiinoi Kudinova-Pasternak, 1973 is transferred to Armatognathia. Amended diagnoses of Armatognathia and Paranarthrurella genera are given. Choosing characters for distinguishing and defining both genera was supported by Principal Component Analysis. Designation of the new family is supported by molecular phylogenetic analysis of COI and 18S datasets. The distribution of all species currently included in the new family was visualised and their bathymetric distribution analysed.


Assuntos
Crustáceos/classificação , Filogenia , Anatomia Comparada/métodos , Distribuição Animal , Grupos de População Animal/classificação , Animais , Oceano Atlântico , Ecologia/métodos , Oceano Pacífico , Fisiologia Comparada/métodos , Análise de Componente Principal , Especificidade da Espécie
8.
J Pharmacol Exp Ther ; 371(2): 309-319, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31501221

RESUMO

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Flavonoides/metabolismo , Corantes Fluorescentes/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/análise , Animais , Transporte Biológico/fisiologia , Cães , Relação Dose-Resposta a Droga , Flavonoides/análise , Corantes Fluorescentes/análise , Cinética , Células Madin Darby de Rim Canino
9.
Biomed Res Int ; 2019: 9616248, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032369

RESUMO

BACKGROUND: Plasma membrane Ca2+-ATPase (PMCA) is the most sensitive cellular calcium detector. It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. In the brain, PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases. These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. METHODS: To mimic the changes in aged neurons we used stable transfected differentiated PC12 cells with downregulated PMCA2 or PMCA3 and analyzed the effect of CCL5 on calcium transients with Fluo-4 reagent. Chemokine receptors were evaluated using Western blot, and IP3 receptors expression level was assessed using qRT-PCR and Western blot. RESULTS: In PMCA-reduced cell lines, CCL5 released more Ca2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer. Also, in these lines we detected altered expression level of CCR5 and IP3 receptors. CONCLUSION: Although modification of PMCAs composition could provide some protection against calcium overload, reduction of PMCA2 appeared to be more detrimental to the cells than deficiency of PMCA3. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons.


Assuntos
Encéfalo/metabolismo , Quimiocina CCL5/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Compostos de Anilina/farmacologia , Animais , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Morte Celular/genética , Diferenciação Celular/genética , Membrana Celular/genética , Homeostase/genética , Humanos , Neurônios/metabolismo , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacos , Xantenos/farmacologia
10.
Biomacromolecules ; 20(3): 1429-1442, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30707833

RESUMO

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form.


Assuntos
Antimetabólitos Antineoplásicos/química , Antineoplásicos/química , Clofarabina/química , Dendrímeros/química , Maltose/química , Polipropilenos/química , Vidarabina/análogos & derivados , Antimetabólitos Antineoplásicos/farmacocinética , Humanos , Ressonância de Plasmônio de Superfície , Células U937 , Vidarabina/química , Vidarabina/farmacocinética
11.
Front Biosci (Landmark Ed) ; 23(9): 1612-1627, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29293453

RESUMO

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.


Assuntos
Células Endoteliais/metabolismo , Espaço Extracelular/metabolismo , Integrina beta3/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Adesão Celular , Linhagem Celular , Dissulfetos/metabolismo , Fibronectinas/metabolismo , Humanos , Hibridomas , Immunoblotting , Microscopia Confocal , Polilisina/metabolismo
12.
Biomacromolecules ; 19(2): 531-543, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29323872

RESUMO

Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.


Assuntos
Dendrímeros , Portadores de Fármacos , Leucemia/tratamento farmacológico , Fosfato de Vidarabina/análogos & derivados , Dendrímeros/química , Dendrímeros/farmacocinética , Dendrímeros/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Leucemia/metabolismo , Leucemia/patologia , Células THP-1 , Células U937 , Fosfato de Vidarabina/química , Fosfato de Vidarabina/farmacocinética , Fosfato de Vidarabina/farmacologia
13.
Biochem Biophys Res Commun ; 495(2): 1635-1641, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29203246

RESUMO

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11ß1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.


Assuntos
Cadeias alfa de Integrinas/química , Cadeias alfa de Integrinas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Humanos , Imunoprecipitação , Integrina beta1/química , Integrina beta1/metabolismo , Microscopia Confocal , Domínios e Motivos de Interação entre Proteínas , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Ressonância de Plasmônio de Superfície
14.
Colloids Surf B Biointerfaces ; 159: 211-216, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28797971

RESUMO

Traditional amine terminated PAMAM dendrimers may be readily surface engineered by a facile one-pot conversion with dialkyl itaconate esters into 4-carbomethoxypyrrolidone terminated PAMAM (G=0-4) dendrimers. These terminated dendrimers are uniquely characterized by exhibiting blue fluorescence emissions (λexc=370nm, λmaxem=440nm). Thanks to this property they can be directly analyzed by confocal microscopy and flow cytometry without additional fluorescence labeling, treatment of dendrimers with chemicals or adjusting pH. These intrinsically fluorescent dendrimers were shown to be very effective for assessing important biological cell features such as cellular entry, intracellular trafficking/localization and efflux properties. For example, all tested cell lines (e.g., B14, BRL-3A, and mHippoE-18) rapidly accumulated PAMAM-pyrrolidone dendrimer. The BRL-3A cell line exhibited both cytoplasmic and nuclear localization patterns; whereas in B14 cells and mHippoE-18 cells, the blue dendrimer fluorescence could only be detected in intracellular endosome-like structures. The dendrimer was observed to be released from all three cell lines during the first 24h; however, efflux was substantially slower from the B-14 cell line. The highest efflux rate was observed for the mHippoE-18 cells. This first successful biological experiment opens a broad spectrum of using these dendrimers as new bioimaging agents for extensive biological cell characterizations.


Assuntos
Dendrímeros/química , Linhagem Celular , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal
15.
Front Cell Neurosci ; 11: 181, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28701926

RESUMO

Ketamine causes psychotic episodes and is often used as pharmacological model of psychotic-like behavior in animals. There is increasing evidence that molecular mechanism of its action is more complicated than just N-methyl-D-aspartic acid (NMDA) receptor antagonism and involves interaction with the components of calcium homeostatic machinery, in particular plasma membrane calcium pump (PMCA). Therefore, in this study we aimed to characterize brain region-specific effects of ketamine on PMCA activity, interaction with NMDA receptor through postsynaptic density protein 95 (PSD95) scaffolding proteins and glutamate release from nerve endings. In our study, ketamine induced behavioral changes in healthy male rats consistent with psychotic effects. In the same animals, we were able to demonstrate significant inhibition of plasma membrane calcium ATPase (PMCA) activity in cerebellum, hippocampus and striatum. The expression level and isoform composition of PMCAs were also affected in some of these brain compartments, with possible compensatory effects of PMCA1 substituting for decreased expression of PMCA3. Expression of the PDZ domain-containing scaffold protein PSD95 was induced and its association with PMCA4 was higher in most brain compartments upon ketamine treatment. Moreover, increased PSD95/NMDA receptor direct interaction was also reported, strongly suggesting the formation of multiprotein complexes potentially mediating the effect of ketamine on calcium signaling. We further support this molecular mechanism by showing brain region-specific changes in PSD95/PMCA4 spatial colocalization. We also show that ketamine significantly increases synaptic glutamate release in cortex and striatum (without affecting total tissue glutamate content), inducing the expression of vesicular glutamate transporters and decreasing the expression of membrane glutamate reuptake pump excitatory amino acid transporters 2 (EAAT2). Thus, ketamine-mediated PMCA inhibition, by decreasing total Ca2+ clearing potency, may locally raise cytosolic Ca2+ promoting excessive glutamate release. Regional alterations in glutamate secretion can be further driven by PSD95-mediated spatial recruitment of signaling complexes including glutamate receptors and calcium pumps, representing a novel mechanism of psychogenic action of ketamine.

16.
Biomacromolecules ; 18(5): 1509-1520, 2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28414464

RESUMO

Poly(propyleneimine) dendrimers of fourth generation partially modified with maltose (open shell structure, PPI-m OS) have been proposed as carriers for nucleotide anticancer drugs. The aim of this work was to provide basic insight into interactions between fluorescently labeled PPI-m dendrimer and two distinct leukemia cell models: CCRF-1301 lymphoid cell line and HL-60 myeloid cell line. We applied qualitative confocal imaging and quantitative flow cytometry, as well as trypan blue quenching and pharmacological inhibition, to investigate the course, kinetics, and molecular mechanisms of internalization of nanoparticles. CCRF-1301 cells take up glycodendrimer macromolecules via a relatively slow, adsorptive endocytosis process, which is cholesterol-dependent, clathrin- and caveolin-independent, and not followed by recycling or exocytosis. Morphological features of this phenomenon point to the involvement of aggregation-induced cell polarity changes (capping). In HL-60 cells, internalization is very fast, independent of binding to the cell surface, and proceeds from the fluid phase via a classical clathrin-dependent mechanism, ending up in an endolysosomal compartment from which it is not further released. This substantial difference in internalization rate and mechanism between two cell types has important repercussions for potential application of this class of glycodendrimers as drug delivery agents.


Assuntos
Dendrímeros/química , Endocitose , Linfócitos/metabolismo , Maltose/análogos & derivados , Polipropilenos/química , Linhagem Celular Tumoral , Dendrímeros/farmacologia , Humanos , Linfócitos/efeitos dos fármacos
17.
Pharm Res ; 34(1): 136-147, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27766462

RESUMO

PURPOSE: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. METHODS: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. RESULTS: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 µM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25-100 µM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. CONCLUSION: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).


Assuntos
Dendrímeros/administração & dosagem , Células Mieloides/efeitos dos fármacos , NF-kappa B/imunologia , Polipropilenos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Açúcares/administração & dosagem , Linhagem Celular , Dendrímeros/química , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interleucina-4/imunologia , Maltose/química , Maltose/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Células Mieloides/imunologia , Polipropilenos/química , Polipropilenos/imunologia , RNA Mensageiro/imunologia , Transdução de Sinais/imunologia , Açúcares/química , Açúcares/imunologia , Fator de Transcrição RelA/imunologia
18.
Life Sci ; 162: 138-44, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27534909

RESUMO

AIMS: Cancer cells, due to the Warburg effect, are more dependent on glycolysis than normal cells, so glycolytic inhibitor 3-bromopyruvic acid (3-BP) was proposed as a promising candidate for anticancer therapy. Overexpression of multidrug transporters is the main reason of resistance of cancer cells to chemotherapy. As the activity of multidrug transporters imposes an energetic burden on the cells, it can be expected that inhibition of ATP generation may exert a selective cytotoxicity to cells overexpressing multidrug transporters. The aim of this study was to compare the effect of 3-BP on the survival and ATP level in MDCK-II cells and MDCK-II cells overexpressing ABCB1 (Pgp) or ABCG2 (BCRP). MAIN METHODS: Cell survival was measured with resazurin and with neutral red. ATP level was assayed with luciferin/luciferase kit. Luteolin transport was measured by an original method described in the paper. KEY FINDINGS: 3-BP (10-200µM) induced a decrease of ATP level after 1-h incubation in all cell lines studied, more drastically in ABCB1-overexpressing cells. 50 and 200µM 3-BP significantly decreased cell viability; the effect was more pronounced for ABCB1-overexpressing cells. PSC833, inhibitor of ABCB1, ameliorated the toxic effect of 3-BP on MDCK-II ABCB1 cells and MDCK-II cells. 3-BP inhibited luteolin transport in MDCK-II ABCG2 cells. SIGNIFICANCE: These results indicate that 3-BP shows selective toxicity against ABCB1- but not ABCG2-overexpressing cells, apparently due to enhanced ATP depletion but in a manner independent of the transport activity of Pgp, suggesting a novel mechanism of hypersensitivity of ABCB1-overexpressing cells to 3-BP.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Piruvatos/farmacologia , Animais , Cães , Células Madin Darby de Rim Canino
19.
Biochim Biophys Acta ; 1858(11): 2617-2624, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27349731

RESUMO

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Vetores Genéticos/química , Leishmania/genética , Fosfatos/química , Transportadores de Cassetes de Ligação de ATP/biossíntese , Sequência de Bases , Transporte Biológico , Primers do DNA/química , Ensaios Enzimáticos , Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Leishmania/citologia , Leishmania/metabolismo , Microssomos/química , Microssomos/metabolismo , Mutação , Organismos Geneticamente Modificados , Fosfatos/metabolismo , Porfirinas/química , Porfirinas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato
20.
Biochim Biophys Acta ; 1853(8): 1759-71, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25918011

RESUMO

ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticorpos Monoclonais/metabolismo , Endocitose , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Caveolina 1/metabolismo , Células Cultivadas , Colesterol/farmacologia , Clatrina/metabolismo , Cães , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células HEK293 , Células HT29 , Humanos , Células MCF-7 , Redes e Vias Metabólicas/efeitos dos fármacos , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo
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