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1.
Chem Biol Interact ; 310: 108731, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265827

RESUMO

Lung cancer is one of the most common and lethal types of oncological diseases. Despite the advanced therapeutic approaches, the prognosis for lung cancer still remains poor. Apparently, there is an imperative need for more efficient therapeutic strategies. In this work we report that concurrent treatment of human adenocarcinoma A549 cells with specific concentrations of two antitumor agents, the sphingosine kinase 1 inhibitor N, N dimethylsphingosine (DMS) and the alkylphosphocholine miltefosine, induced synergistic cytotoxic effect, which was confirmed by calculation of the combination index. The simultaneous action of these agents, induced significant decrease of A549 cell number, as well as pronounced morphological alterations. Combined drugs caused substantial apoptotic events, and significant reduction of the pro-survival marker sphingosine- 1-phosphate (S1P), when compared to the individual treatments with each of the anticancer drugs alone. Miltefosine is known to affect the synthesis of choline-containing phospholipids, including sphingomyelin, but we report for the first time that it also reduces S1P. Here we suggest a putative mechanism underlying the effect of miltefosine on sphingosine kinase 1, involving miltefosine-induced inhibition of protein kinase C. In conclusion, our findings provide a possibility for treatment of lung cancer cells with lower concentrations of the two antitumor drugs, DMS and miltefosine, which is favorable, regarding their potential cytotoxicity to normal cells.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Apoptose/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Células A549 , Adenocarcinoma de Pulmão/patologia , Protocolos de Quimioterapia Combinada Antineoplásica , Sinergismo Farmacológico , Humanos , Lisofosfolipídeos/análise , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Esfingosina/análogos & derivados , Esfingosina/análise
2.
Mater Sci Eng C Mater Biol Appl ; 100: 608-615, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948097

RESUMO

A series of Zn-doped hybrid materials based on silica from tetraethoxysilane (TEOS) and hydroxypropyl cellulose (HPC) were prepared by a sol-gel route. The structure, morphology and thermal behavior of synthesized hybrids were characterized by infrared (IR) spectroscopy, ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM) and differential thermal analysis with thermogravimetric analysis (DTA/TG). The obtained materials were investigated for a potential biomedical application. The antibacterial properties of hybrids were investigated by measuring the inhibition zones formed around the materials containing different zinc content in presence of reference strains of Gram-positive and Gram-negative bacteria. The biocompatibility tests showed no cytotoxicity and genotoxicity, as well as no changes in actin cytoskeleton organization for hybrids with Zn content below 5 wt%.


Assuntos
Tecnologia Biomédica/métodos , Celulose/análogos & derivados , Nanopartículas/química , Dióxido de Silício/química , Zinco/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Morte Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Celulose/química , Análise Diferencial Térmica , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Camundongos , Testes de Sensibilidade Microbiana , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta
3.
Biomacromolecules ; 19(7): 3040-3047, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29870244

RESUMO

Reversible self-folding actions of natural biomacromolecules play crucial roles for specific and unique biological functions in Nature. Hence, controlled folding of single polymer chains has attracted significant attention in recent years. Herein, reversible single-chain folded glycopolymer structures in α-shape with different density of sugar moieties in the knot were created. The influence of folding as well as the sugar density in the knot was investigated on the binding capability with lectins, such as ConA, DC-SIGN, and DC-SIGNR. The synthesis of triblock glycocopolymers bearing ß-CD and adamantane for the host-guest interaction and also mannose residues for the lectin interaction was achieved using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique. The reversible single-chain folding of glycopolymers was achieved under a high dilution of an aqueous solution and the self-assembled folding was monitored by 2D nuclear overhauser enhancement spectroscopy (NOESY) NMR and dynamic light scattering. The lectin binding profiles consistently provided an unprecedented effect of single chain folding as the single-chain folded structures enhanced greatly the binding ability in comparison to the unfolded linear structures.


Assuntos
Moléculas de Adesão Celular/química , Lectinas Tipo C/química , Manose/análogos & derivados , Receptores de Superfície Celular/química , Adamantano/análogos & derivados , Configuração de Carboidratos , Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo , beta-Ciclodextrinas/química
4.
Chem Commun (Camb) ; 54(52): 7211-7214, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29897060

RESUMO

The first total synthesis of phyllostictine A (PA) is reported, which confirms the structure of this fungal metabolite and its (6S,7R,8S)-stereochemistry. Both synthetic PA and an analogue containing the 5-methylene-1,5-dihydro-2H-pyrrol-2-one nucleus exhibit µM inhibitory activity in root growth assays against Arabidopsis thaliana, indicating that this heterocyclic subunit is key to the herbicidal activity of the natural product.


Assuntos
Arabidopsis/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Raízes de Plantas/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/química , Estrutura Molecular , Raízes de Plantas/crescimento & desenvolvimento
5.
Fitoterapia ; 128: 233-241, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29800610

RESUMO

Dichloromethane extract of propolis (DCME) originating from Pitcairn Island demonstrated potent cytotoxicity against triple-negative MDA-MB-231 human breast carcinoma cells. The results from MTT assay showed that DCME inhibits the growth of the cancer cells in a dose- and time-dependent manner and upon the cell growth inhibition propolis extract provoked apoptotic changes in the cell nuclei. A detailed chemical investigation of DCME led to the isolation of four new cycloartane triterpenes (1-4), along with 17 known compounds (5-21). The structures of the new compounds were elucidated by means of extensive analysis of their spectroscopic data and comparison with those reported for their analogues. In vitro antimicrobial activity of new compounds (1-4) along with the DCME against four human pathogens was evaluated. All tested constituents except compound 2 were highly active against Escherichia coli with MIC 64 µg/ml. Compound 1 exhibited high antifungal activity against Candida albicans with potency close to that of the positive control (amphotericin B). The DCME showed very good antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. This is the first study on propolis from Pitcairn Island.


Assuntos
Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Própole/química , Triterpenos/isolamento & purificação , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Linhagem Celular Tumoral , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ilha Pitcairn , Staphylococcus aureus/efeitos dos fármacos , Triterpenos/farmacologia
6.
Biomacromolecules ; 18(6): 1928-1936, 2017 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-28460523

RESUMO

Glycopolypeptides with defined block sequences were prepared by sequential addition of two different N-carboxyanhydrides (NCAs), followed by selective deprotection and functionalization of predefined positions within the polypeptide backbone. The sequential arrangement of the galactose units and the block-sequence length have been systematically varied. All the glycopolypeptides have been obtained with a similar overall composition and comparable molecular weights. Circular dichroism measurements revealed some dependence of the secondary structure on the primary composition of the glycopolypeptides at physiological pH. While statistical, diblock, and tetrablock glycopolypeptides adopted a random coil conformation, the octablock glycopolypeptide was mostly α-helical. The ability to selectively bind to lectins was investigated by turbidity measurements as well as surface plasmon resonance (SPR) studies. It was found that the extent of binding was dependent on the position of the galactose units and thus the primary glycopolypeptide structure. The octablock glycopolypeptide favored interaction with lectin RCA120 while the tetrablock glycopolypeptide demonstrated the strongest binding activity to Galectin-3. The results suggest that different lectins are very sensitive to glyco coding and that precise control of carbohydrate units in synthetic polymeric glycopeptides will remain important.


Assuntos
Galactose/química , Galectina 3/química , Glicopeptídeos/química , Lectinas/química , Anidridos/química , Glicopeptídeos/síntese química , Concentração de Íons de Hidrogênio , Ligantes , Ligação Proteica , Estrutura Secundária de Proteína
7.
Methods Mol Biol ; 1497: 159-191, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27864766

RESUMO

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/metabolismo , Bioensaio/métodos , Regulação da Expressão Gênica de Plantas/fisiologia , Cinética , Ligantes , Transdução de Sinais/fisiologia
8.
Open Biol ; 6(10)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27805904

RESUMO

We study the binding of plant hormone IAA on its receptor TIR1, introducing a novel computational method that we call tomographic docking and that accounts for interactions occurring along the depth of the binding pocket. Our results suggest that selectivity is related to constraints that potential ligands encounter on their way from the surface of the protein to their final position at the pocket bottom. Tomographic docking helps develop specific hypotheses about ligand binding, distinguishing binders from non-binders, and suggests that binding is a three-step mechanism, consisting of engagement with a niche in the back wall of the pocket, interaction with a molecular filter which allows or precludes further descent of ligands, and binding on the pocket base. Only molecules that are able to descend the pocket and bind at its base allow the co-receptor IAA7 to bind on the complex, thus behaving as active auxins. Analysing the interactions at different depths, our new method helps in identifying critical residues that constitute preferred future study targets and in the quest for safe and effective herbicides. Also, it has the potential to extend the utility of docking from ligand searches to the study of processes contributing to selectivity.


Assuntos
Reguladores de Crescimento de Planta/química , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Simulação de Acoplamento Molecular , Plantas/química , Plantas/metabolismo , Ligação Proteica , Conformação Proteica
9.
Clin Hemorheol Microcirc ; 64(4): 941-949, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27792001

RESUMO

Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP's production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5-fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In contrast, for MDA-MB-231 the production of MMPs on gelatin materials was lower compared to control materials. With the application of EF the levels of MMP-9 decreased but MMP-2 expression raised significantly for gelatin materials. Overall, the results showed that studied gelatin materials suppressed attachment of cancerous cells, as well as suppressed their angiogenic potential revealed by decreased VEGF and MMP production. Thus, this study approved gelatin-based hydrogels with proper elasticity characteristics and different degradation behavior as useful matrices for use in vascular tissue regeneration or in restriction of tumor growth after tumor resection.


Assuntos
Células Endoteliais/metabolismo , Hidrogéis/metabolismo , Estimulação Elétrica , Células Endoteliais/citologia , Fibronectinas/síntese química , Gelatina , Humanos , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular
10.
Carbohydr Polym ; 140: 349-55, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26876861

RESUMO

Novel electrically conducting 2-hydroxyethylcellulose/polyaniline (HEC/PANI) nanocomposite cryogels were fabricated via the combination of cryogenic treatment and photochemical crosslinking. PANI nanofillers (one-dimentional tubes and three-dimentional particles) were synthesized via oxidative polymerization of aniline in aqueous media and, then, embedded in the HEC matrix. The effect of PANI content and morphology on the gel fraction yield and electrical conductivity of material was studied. Nanocomposite cryogels of high gel fraction yield (65-95%) and rather high electrical conductivity (0.02-0.1S/m) were obtained by using a relatively small amount (0.5-3wt.% to HEC) of pre-formed PANI nanofillers. The behavior of L929 cells adhered on HEC/PANI cryogels in the presence of electric field were also investigated. Cytotoxicity test showed very good survival and proliferation of cells on cryogels, while the electrical stimulation triggered changes in cell morphology as well as a specific alignment of cells in parallel to the electrical field.


Assuntos
Compostos de Anilina/química , Materiais Biocompatíveis/química , Celulose/análogos & derivados , Criogéis/química , Condutividade Elétrica , Nanocompostos/química , Engenharia Tecidual , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Celulose/química , Técnicas de Química Sintética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Teste de Materiais , Camundongos
11.
Int J Biol Macromol ; 82: 798-805, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26478091

RESUMO

This is the first study on the surface modification of a hemocyanin from marine snail Rapana thomasiana (RtH) with series of imidazolium-based amino acid ionic liquids [emim][AA]. We monitored the induced by [emim][AA] conformational changes in RtH molecule and evaluated the effect of these ionic liquids (ILs) on the protein thermal stability. The cytotoxicity of all obtained RtH-[emim][AA] complexes was assessed toward breast cancer cells (MCF-7) and murine fibroblasts (3T3). As a whole, even small amounts of the tested ILs altered the secondary structure of RtH. The thermal denaturation of RtH in presence of [emim][AA] displayed multi-component transitions, which were shifted toward lower temperatures in comparison to those estimated for the native RtH. The profiles of the RtH-IL calorimetric curves show a clear dependence on the structure of the added salts. In addition, all RtH-[emim][AA] complexes exhibited an enhanced antiprofilerative activity of toward MCF-7 cells in comparison to that of the native RtH. The best results are observed for RtH-[emim][Leu], RtH-[emim][Trp] or RtH-[emim][Ile], which applied in concentration of 700 µg/mL inhibited the MCF-7 cell viability (for 24h) by 66, 63 and 53%, respectively. In addition, these IL-RtH complexes were less cytotoxic to 3T3 cells, i.e. they exhibited some cell specificity.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Gastrópodes , Hemocianinas/química , Hemocianinas/farmacologia , Líquidos Iônicos/química , Células 3T3 , Animais , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Líquidos Iônicos/síntese química , Células MCF-7 , Camundongos , Estabilidade Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica
12.
Angew Chem Int Ed Engl ; 54(22): 6501-5, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25882792

RESUMO

We report a synthetic biology-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free-standing lipid membranes, mimicking the biological activity of coat-forming proteins, for example, from the I-/F-BAR family.


Assuntos
DNA/química , Lipídeos de Membrana/química , Nanopartículas/química , Lipossomas Unilamelares/química , Colesterol/química , Difusão , Corantes Fluorescentes/química , Microscopia de Fluorescência , Nanopartículas/ultraestrutura , Oligonucleotídeos/química
13.
Anal Chem ; 87(2): 864-8, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25496140

RESUMO

DNA-encoded chemical library (DECL) technology has emerged as a new avenue in the field of drug discovery. Combined with high-throughput sequencing, DECL selection experiments can provide not only many lead compounds but also insights into the structure-affinity relationship. However, the counts of individual DNA codes reflect, but cannot be used to precisely rank, the binding affinities of the corresponding compounds to protein targets. Herein, we describe a chip-based approach to realize an automated high-throughput assay for the kinetic characterization of the interaction between DNA-conjugated small organic compounds and protein targets. Importantly, this method can be applied to both single-pharmacophore DECLs and self-assembled dual-pharmacophore DECLs.


Assuntos
Ciclofilinas/química , Ciclosporina/química , DNA/química , Imunossupressores/química , Bibliotecas de Moléculas Pequenas/química , Técnicas Biossensoriais , Técnicas de Química Combinatória , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , DNA/metabolismo , Desenho de Drogas , Descoberta de Drogas , Biblioteca Gênica , Humanos , Imunossupressores/metabolismo , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade
14.
Faraday Discuss ; 161: 31-43; discussion 113-50, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805736

RESUMO

Recently, DNA origami became a powerful tool for custom-shaped functional biomolecules. In this paper, we present the first approach towards assembling amphipathic three-dimensional DNA origami nanostructures and assessing their dynamics on the surface of freestanding phospholipid membranes. Our nanostructures were stiff DNA origami rods comprising six DNA helices. They were functionalized with hydrophobic cholesteryl-ethylene glycol anchors and fluorescently labeled at defined positions. Having these tools in hand, we could demonstrate not only the capability of the amphipathic nanorods to coat membranes of various phospholipid compositions, but also their switchable liquid-ordered/liquid-disordered partitioning on phase separated membranes. The observed translocation of our nanostructures between different domains was controlled by divalent ions. Moreover, selective fluorescent labeling enabled us to distinguish between the translational and rotational diffusion of our six helix bundles on the membranes by fluorescence correlation spectroscopy. The obtained data reveal how DNA origami can be employed as a valuable tool in membrane biophysics.


Assuntos
Membrana Celular/química , DNA/química , Nanoestruturas/química , Biofísica , Colesterol/análogos & derivados , Colesterol/química , Difusão , Etilenoglicóis/química , Interações Hidrofóbicas e Hidrofílicas , Conformação de Ácido Nucleico , Fosfatidilcolinas/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Espectrometria de Fluorescência/métodos
15.
Biophys J ; 99(6): 1976-85, 2010 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-20858444

RESUMO

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 µM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 µM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.


Assuntos
Heme/metabolismo , Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Multimerização Proteica , Eritrócitos/metabolismo , Humanos , Cinética , Estrutura Quaternária de Proteína , Soluções , Temperatura Ambiente
16.
J Phys Chem B ; 114(13): 4529-35, 2010 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-20235510

RESUMO

To address the interactions of hemin with phospholipid bilayers, we introduce hemin to a solution of dimyristoylphosphatidylcholine (DMPC), a long chain phospholipid, and 3-(cholamidopropyl)(dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), a detergent, in which vesicles form at around 37 degrees C. We show that vesicles composed of DMPC/CHAPSO form and grow, following a mechanism that does not trap solution and excludes larger solutes, such as hemin, from the vesicle interior. The existence of a limited number of patches of likely 2D crystalline hemin embedded in the phospholipid bilayer suggests that this layer is saturated with hemin molecules. We show that despite this saturation, even after prolonged contact with hemin-containing solution outside the vesicles, hemin is not released on the other side of the membrane; i.e., the phospholipid bilayer is impermeable to hemin. Comparison of the properties of the model membrane to those of the erythrocyte membrane suggests that the latter might also be impermeable to hemin and, given the absence of pores suitable to hemin in the erythrocyte membranes, that hemin might accumulate in erythrocytes after its release due to hemoglobin instability.


Assuntos
Membrana Eritrocítica/química , Hemina/química , Modelos Biológicos , Ácidos Cólicos/química , Dimiristoilfosfatidilcolina/química , Espalhamento de Radiação
17.
Biopolymers ; 91(12): 1108-16, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19322821

RESUMO

We probe the role of free heme in the interactions between sickle cell hemoglobin (HbS) molecules in simulated physiological solutions: polymerization of deoxy-HbS is the primary pathogenic event of sickle cell anemia, and HbS releases heme after autoxidation more readily than normal adult hemoglobin. We characterize these interactions in terms of osmotic virial coefficients, which we determine by static light scattering. We analyze the results in the heme-hemoglobin system using the Kirkwood-Goldberg model. We show that in the absence of heme, the HbS molecules weakly attract and the attraction is not due to the lowered-as a result of the sickle cell mutation-molecular charge. We show that the part of the interface between the two alphabeta dimers, exposed in the deoxy-state, plays a crucial role in this attraction. We show that heme at micromolar concentrations induces strong attraction between the hemoglobin molecules. We show that the high efficacy of the heme results from the statistics of electrostatic and hydrophobic interactions between the heme and hemoglobin molecules.


Assuntos
Heme/química , Hemoglobina Falciforme/química , Algoritmos , Humanos , Cinética , Luz , Espalhamento de Radiação , Soluções , Termodinâmica
18.
Phys Rev Lett ; 102(5): 058101, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19257559

RESUMO

We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean-squared displacement of probe particles in the time range 10;{-3}-10 s in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.


Assuntos
Proteínas/química , Substâncias Viscoelásticas/química , Citosol/química , Citosol/metabolismo , Elasticidade , Cinética , Modelos Químicos , Muramidase/química , Muramidase/metabolismo , Proteínas/metabolismo , Soroalbumina Bovina/química , Soluções , Substâncias Viscoelásticas/metabolismo , Viscosidade
19.
Med Hypotheses ; 69(2): 361-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17336461

RESUMO

Alpha amanitin is a powerful natural hepatotoxin that belongs to the amatoxins isolated from deadly poisonous Amanita phalloides mushroom. The basic molecular mechanism of their toxicity was attributed to inhibition of RNA polymerase II of the eukaryotic cells. At present, the most effective clinical antidote to acute Amanita phalloides mushroom poisoning is silybin, an antioxidant possessing free radical scavenger activity and inhibiting lipid peroxidation, stabilizing membrane structure and protecting enzymes under conditions of oxidative stress. Bearing in mind the biological mechanism of silybin action and the fact that for different amatoxins (alpha, beta, and est. amanitins) does not established straight correlation between their in vivo LD50 and inhibitory constants (Ki) toward RNA polymerase III in vitro determined we supposed some additional toxic effects of these toxins might contribute to their severe hepatotoxicity. Our formerly in vitro experiments demonstrated that alpha amanitin could act either as an antioxidant or as a prooxidant depending on the treatment conditions and toxin concentration. By UV-visible spectroscopy we also shown that alpha amanitin was sensitive to oxidation by a system of lactoperoxidase/H(2)O(2) and assumed formation of free radical toxin intermediates. Having in mind some exogenic compounds including natural toxins can induce increased production of reactive oxygen species (ROS) we suggested similar generation of ROS provoked by alpha amanitin. Our recently in vitro studies have demonstrated that the alpha amanitin could increase superoxide dismutase (SOD) activity and inhibit catalase (CAT) activity to a considerable degree after together incubation of the toxin with any of enzymes. We have also shown that in vitro increased SOD activity was due to superoxide anion radical scavenging activity (SSA) of the toxin. This therefore informed the decision to study the in vivo effect of alpha amanitin on SOD and CAT activity and the level of lipid peroxidation (LPO) products in liver homogenates isolated from mice treated with the toxin. Statistical significant increased level of LPO products was found at the 6th day comparing to the 20th hour after mice treatment with a subletal dose of the toxin. Based on our previous in vitro and present in vivo studies we have made a hypothesize that in vivo during liver accumulation of the toxin it might be transformed to free radical intermediates causing increase in ROS levels. As a result a peroxidative process in hepatocytes might contribute to the severe alpha amanitin hepatotoxicity.


Assuntos
Amanitinas/metabolismo , Amanitinas/toxicidade , Radicais Livres/metabolismo , Radicais Livres/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Catalase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Dose Letal Mediana , Fígado/enzimologia , Masculino , Camundongos , Superóxido Dismutase/metabolismo
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