RESUMO
This study focuses on the synthesis of functional allopurinol (ALP) imprinted biomaterials for a transdermal drug delivery using mung bean starch (MBS), polyvinyl alcohol (PVA), sodium benzoate (SB) as a crosslinking agent, and poloxamer (PX) as a thermo-sensitive polymer. Prepared functional biomaterials were characterized and evaluated by SEM, FT-IR analysis, and physical properties. Results of ALP recognition properties indicated that adsorbed amounts (Q) of ALP on functional ALP imprinted biomaterials were 3.8 to 4.9-fold higher than that of non-ALP imprinted biomaterial. Results of ALP release revealed that the ALP release rate for PX added biomaterials was 1.10 (36.5 °C) or 1.30 (45 °C) times faster than that at 25 °C. These results indicate that functional ALP imprinted biomaterials have thermo-sensitive properties due to the addition of PX. Results of ALP release using artificial skin indicated that ALP release was increased at a relatively steady-state rate for 3 h and that the ALP release behavior followed the non-Fickian diffusion mechanism.
Assuntos
Alopurinol/química , Sistemas de Liberação de Medicamentos/métodos , Amido/farmacologia , Administração Cutânea , Adsorção , Alopurinol/farmacologia , Materiais Biocompatíveis/farmacologia , Difusão/efeitos dos fármacos , Hidrogéis , Polímeros/química , Álcool de Polivinil/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Adesivo TransdérmicoRESUMO
Chemotherapy induced nausea and vomiting (CINV) is an issue, which usually occurs in cancer patient. Despite high bioavailability of oral and intravenous administration, these have some drawbacks. The oral route causes hepatic first pass metabolism and intravenous route is invasive in nature. Hence, antiemetic drug by means of transdermal route is necessary to administer in such cases. The aim of the present investigation is to develop suitable Transdermal Therapeutic System (TTS) with an objective to enhance solubility and skin permeability properties of metoclopramide base. Preformulation study begins with an approach to enhance solubility of 40 metoclopramide base by solid dispersion technique. transdermal films were prepared with 41 the solid dispersion as well as with pure drug. Phase solubility study at various temperatures reveals binding constants (Ka, 95-350 M-1 for PVP K30; 56-81 M-1 for HPßCD). Spontaneity of solubilization was justified by AL type linear profiles. The films showed satisfactory diffusion (%), permeation rate and flux after 8 h study. The transdermal patches as prepared were analyzed under FTIR, DSC and SEM. Both solubility and permeability rate in this investigation have been enhanced. So, it can be affirmed that this route would effectively enhance bioavailability
Assuntos
Solubilidade , Metoclopramida/antagonistas & inibidores , Pacientes/classificação , Preparações Farmacêuticas/administração & dosagem , Disponibilidade Biológica , Espectroscopia de Infravermelho com Transformada de Fourier , Difusão/efeitos dos fármacos , Tratamento Farmacológico , Administração Intravenosa/instrumentação , Filmes Cinematográficos , Neoplasias/patologiaRESUMO
D-borneolum is commonly used as a permeation enhancer in Traditional Chinese Medicine (TCM) formulas for transdermal application. Additionally, two other sources of borneolums were recorded in the 2015 edition of the Chinese Pharmacopoeia (ChP), including L-borneolum and borneolum syntheticum. To guide the selection and application of borneolum, the safety and enhancing effect of three sources of borneolums were investigated on transdermal permeation of compounds with different octanol-water partition coefficient (log P) values and molecular weights (MWs). Both the results of cellular cytotoxicity and in vitro transdermal permeation experiments showed that all three sources of borneolums could be applied in TDDS as permeation enhancers. Moreover, all three sources of borneolums achieved optimal permeation-enhancing performances on transdermal drugs with lower log P values as well as higher MWs. Further study was carried out to elucidate the potential molecular mechanism of borneolum enhancing transdermal drug delivery via transmission electron microscope (TEM) and coarse-grained molecular dynamic (CG-MD) simulation. Borneolum significantly promoted transdermal delivery of drugs via changing the dense morphology of the stratum corneum (SC), disturbing the ordered arrangement of ceramide (CER) and free fatty acid (FFA) molecules in lipid layers, and further increasing the diffusion rate of drugs in the lipid layers.
Assuntos
Canfanos/metabolismo , Absorção Cutânea/efeitos dos fármacos , Pele/metabolismo , Administração Cutânea , Animais , Linhagem Celular Tumoral , Difusão/efeitos dos fármacos , Lipídeos , Masculino , Medicina Tradicional Chinesa/métodos , Simulação de Dinâmica Molecular , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Resumen Los objetivos de este trabajo fueron estudiar la sensibilidad antibiótica de aislamientos de Corynebacterium pseudotuberculosis procedentes de pequeños rumiantes e investigar la presencia de integrones que contienen genes de resistencia. Se estudiaron 15 aislamientos de diferentes fuentes por los métodos de difusión y dilución. Por el método de difusión, amoxicilina-clavulánico, ampicilina, cefotaxima, cefoxitina, ciprofloxacina, cloranfenicol, eritromicina, estreptomicina, gentamicina, imipenem, kanamicina, norfloxacina, penicilina, rifampicina, tetraciclina, trimetroprima-sulfametoxazol y vancomicina fueron activos frente al 100% de los aislamientos, mientras que amicacina presentó resultados variables. En los aislamientos que desarrollaron frente a amicacina se investigó la presencia de integrones de clase 1. El resultado fue negativo, sugiriendo la ausencia del integrón. Utilizando el método de dilución, los antibióticos más activos correspondieron a los grupos de cefalosporinas, gluco-péptidos, macrólidos, quinolonas y tetraciclinas. Se demostró menor actividad de p-lactámicos y aminoglucósidos. No se registró variabilidad en los perfiles antibióticos en los aislamientos procedentes de diferentes fuentes.
Abstract The aims of this work were to study the antibiotic susceptibility in Corynebacterium pseudotuberculosis isolated from small ruminants and to determine the presence of integrons that contain resistance genes. Fifteen isolates of different sources were analysed using the diffusion and the dilution methods. When the diffusion method was performed, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were effective against the 100% of isolates, while amikacin showed variable results. The isolates that were able to grow with amikacin, were studied in relation to the presence of integron class 1. The result was negative, suggesting the absence of integron. Using dilution method, the antibiotics belonging to the cephalosporin, glycopeptide, macrolide, quinolone, and tetracycline groups were the most active ones for the C. pseudotuberculosis biovar ovis isolates. Less activity of p-lactam and aminoglycosides were observed. There was no observation of variability in the antibiotic patterns in the strains coming from different sources.
Assuntos
Animais , Ovinos/microbiologia , Corynebacterium pseudotuberculosis/efeitos dos fármacos , Integrons/efeitos dos fármacos , Antibacterianos/uso terapêutico , Técnicas In Vitro/métodos , Ruminantes/microbiologia , Diluição/análise , Difusão/efeitos dos fármacos , Linfadenite/prevenção & controleRESUMO
Water mobility in cancer cells could be a powerful parameter to predict the progression or remission of tumors. In the present descriptive work, new insight into this concept was achieved by combining neutron scattering and thermal analyses. The results provide the first step to untangle the role played by water dynamics in breast cancer cells (MCF-7) after treatment with a chemotherapy drug. By thermal analyses, the cells were probed as micrometric reservoirs of bulk-like and confined water populations. Under this perspective we showed that the drug clearly alters the properties of the confined water. We have independently validated this idea by accessing the cellular water dynamics using inelastic neutron scattering. Finally, analysis of the quasi-elastic neutron scattering data allows us to hypothesize that, in this particular cell line, diffusion increases in the intracellular water in response to the action of the drug on the nanosecond timescale.
Assuntos
Neoplasias da Mama/metabolismo , Hidrodinâmica , Espaço Intracelular/efeitos dos fármacos , Difração de Nêutrons/métodos , Paclitaxel/farmacologia , Água/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Varredura Diferencial de Calorimetria , Difusão/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Células MCF-7 , NêutronsRESUMO
Male-to-female transgender (MtF TG) individuals often report using illegal subcutaneous silicone injections for body feminisation. It leads to silicone dissemination and various dermatologic complications.We report the long-term complications of these feminisation procedures with blood smear examination and dermatologic examination.Between July 2015 and December 2015, 77 MtF TG consulting at Bichat Hospital (Paris, France) were included in this cross-sectional study. Blood smear examinations were performed by a trained haematologist to quantify the presence of silicone vacuoles in monocytes.All patients reported a history of massive amounts of silicone injections (mean 4 L, range 0.5-15 L). Most patients were South American (75/77, 97%). Fifty-nine (59/75, 79%) were HIV-seropositive, mostly with undetectable HIV RNA plasma levels (46/58, 80%). Clinical examinations reported dermatologic complications for all patients: lymphatic or subcutaneous migration of silicone (59%), inflammation (50%), varicose veins (39%), post-inflammatory pigmentation (20%), infection (14%) and abscesses (4%). Blood smear examination showed intracytoplasmic vacuoles containing silicone in monocytes in all patients.We did not chemically prove the silicone nature of the vacuoles. The design of this study does not allow evaluation of short-term complications that should not be minimized.Illicit massive silicone injections always induced chronic and definitive silicone blood diffusion with dermatologic complications. This study highlights the dangers and the inefficiency of clandestine esthetic surgery. There is a need for targeted information campaigns with transgender populations about silicone injections. Otherwise, these practices may persist.
Assuntos
Silicones/efeitos adversos , Dermatopatias/induzido quimicamente , Transexualidade , Adulto , Idoso , Comportamento Criminoso , Estudos Transversais , Difusão/efeitos dos fármacos , Feminino , Testes Hematológicos , Humanos , Injeções Subcutâneas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Silicones/administração & dosagem , Dermatopatias/sangue , Dermatopatias/diagnóstico , Transexualidade/sangue , Adulto JovemRESUMO
Fisetin is a natural flavonoid with promising antitumor activity, whereas its clinical application is limited by its hydrophobic property. In this study, we aimed to load fisetin into poly(lactic acid) (PLA) nanoparticles to increase fisetin's solubility and therapeutic efficacy. Based on spontaneous emulsification solvent diffusion (SESD) method, the formulation of PLA nanoparticles was optimized by two successive experimental designs. One-factor-at-a-time variation experiments were first applied to investigate the effects of four process variables on three responses, including drug encapsulation efficiency, average particles size and cumulative drug release ratio, followed by determining the possible ranges of these variables. Subsequently, the combinations of four variables at best levels were evaluated using a Taguchi orthogonal array design with regard to the same three responses. Eventually, the nanoparticle prepared by optimized procedure showed a narrow size distribution around 226.85⯱â¯4.78â¯nm with a high encapsulation efficiency of 90.35%. The incorporation of fisetin in nanoparticles was subsequently confirmed by FT-IR and DSC spectroscopy. Furthermore, cytotoxicity assay against HCT116 colon cancer cells in vitro and antitumor test in a xenograft 4T1 breast cancer model in vivo demonstrated that the antitumor effect of drug-loaded nanoparticles was superior to that of free drug solution.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Nanopartículas/química , Poliésteres/química , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Difusão/efeitos dos fármacos , Portadores de Fármacos/química , Liberação Controlada de Fármacos/efeitos dos fármacos , Feminino , Flavonóis , Células HCT116 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Polímeros/química , Ratos , Ratos Sprague-Dawley , Solubilidade/efeitos dos fármacos , Distribuição TecidualRESUMO
Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Colesterol/química , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Lipossomos/química , Meliteno/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Difusão/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fluoresceínas/química , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Meliteno/química , Meliteno/genética , Mutação , Peptídeos/química , Peptídeos/genética , Agregados Proteicos , Eletricidade Estática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
AIMS: Keeping in view the therapeutic and pharmaceutical applications of sterculia gum polysaccharide in consideration, its modification has been carried out through grafting and crosslinking to develop the hydrogels for enhanced biomedical applications. Radiation method was used for formation of sterile network of sterculia gum, carbopol and graphene oxide (GO). These polymers were characterized by Cryo-SEMs, AFM, 13C NMR solid state, swelling studies. Some biomedical properties of hydrogels like thrombogenicity, haemolytic potential, antioxidant activity, mucoadhesion and gel strength were determined along with the drug delivery studies. SCOPE: In the present work, sterile polysaccharide gum based drug delivery system was developed for the slow delivery of gemcitabine, an anti-cancer drug, to overcome its side. CONCLUSIONS: The release profile of anti-cancer drug "gemcitabine" followed non-Fickian diffusion mechanism and release profile was best fitted in Korsmeyer-Peppas kinetic model of drug release. The hydrogels were found to be non-thrombogenic, non-haemolytic, mucoadhesive and antioxidant in nature. Incorporation of the GO nano-sheets in the composite hydrogel matrix has improved its mechanical and drug delivery properties and also exerted strong influence on the network density and mesh size of the hydrogels.
Assuntos
Goma de Karaya/química , Polimerização/efeitos dos fármacos , Polissacarídeos/química , Sterculia/química , Antioxidantes/química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Difusão/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos/efeitos dos fármacos , Grafite/química , Hidrogéis/química , Óxidos/química , Polímeros/química , GencitabinaRESUMO
Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.
Assuntos
Membrana Celular/metabolismo , Microscopia Intravital/métodos , Profilinas/metabolismo , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Citocalasina D/metabolismo , Difusão/efeitos dos fármacos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Microscopia Intravital/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Profilinas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/instrumentação , Espectrometria de Fluorescência/instrumentaçãoRESUMO
AIM: The aim of the study was to develop novel zeta potential changing self-emulsifying drug delivery systems (SEDDS) containing phosphorylated polysaccharides. METHODS: Starch and hydroxypropyl starch (HPS) were phosphorylated by utilizing phosphorus pentoxide. The modified starches, starch phosphate (SP) and hydroxypropyl starch phosphate (HPSP), were loaded into SEDDS and investigated regarding particle size, zeta potential, stability and cell viability. The release of immobilized phosphate by intestinal alkaline phosphatase (IAP) was analyzed via malachite green assay. In parallel, the resulting shift in zeta potential of SEDDS was determined. Furthermore, Transwell chambers were applied in order to evaluate the mucus diffusion behavior of SEDDS utilizing fluorescein diacetate (FDA) as marker. RESULTS: The amount of attached phosphate for SP and HPSP revealed to be 119µmol/g and 259µmol/g, respectively. SEDDS consisting of 10% glycerol, 30% Capmul MCM, 30% Cremophor EL and 30% Captex 355 showed a droplet size of 39±12nm, stability over 240min and no significant decrease in cell viability within the applied concentrations. SEDDS containing 3mg/ml HPSP with a phosphate release of 204µmol/g, demonstrated a shift in zeta potential from -6.3mV to +1.0mV applying isolated IAP. Zeta potential changing SEDDS achieved a 2.5-fold and 5.4-fold higher amount of diffused FDA compared to the references within mucus permeation studies. CONCLUSION: SEDDS containing HPSP represent comparable high mucus diffusion properties emphasized by a highly significant change in zeta potential.
Assuntos
Emulsões/química , Polissacarídeos/química , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica/métodos , Difusão/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Emulsificantes/química , Emulsões/farmacologia , Humanos , Muco/metabolismo , Tamanho da Partícula , Permeabilidade , SolubilidadeRESUMO
Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P < 0.01). In experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger, and residence times were reduced for receptors in fish oil-treated cells (P < 0.05). In experiment 3, single particle tracking was used to determine the effects of PGF2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil-treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF2α.
Assuntos
Bovinos , Membrana Celular/efeitos dos fármacos , Óleos de Peixe/farmacologia , Lipídeos/análise , Células Lúteas/ultraestrutura , Receptores de Prostaglandina/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Difusão/efeitos dos fármacos , Dinoprosta/farmacologia , FemininoRESUMO
Over the past 60 years, hyaluronidase has been successfully utilized in ophthalmic surgery and is now being implemented in dermatosurgery as well as in other surgical disciplines. The enzyme is considered a "spreading factor" as it decomplexes hyaluronic acid (also called hyaluronan, HA), an essential component of the extracellular matrix (ECM). When applied as an adjuvant, hyaluronidase enhances the diffusion capacity and bioavailability of injected drugs. Therefore, the enzyme has been used as a local adjuvant to increase the diffusion capacity of local anesthetics, increasing the analgesic efficacy, and the anesthetized area particularly in the first minutes following injection, resulting in diminished intra- and postoperative pain. In aesthetic medicine, the off-label use of hyaluronidase is considered the gold standard for the management of HA-filler-associated complications. Here, we review the clinical use, underlying biological mechanisms, and future directions for the application of hyaluronidase in surgical and aesthetic medicine.
Assuntos
Anestésicos Locais/farmacocinética , Procedimentos Cirúrgicos Dermatológicos/métodos , Difusão/efeitos dos fármacos , Hialuronoglucosaminidase/uso terapêutico , Procedimentos Cirúrgicos Oftalmológicos/métodos , Anestésicos Locais/metabolismo , Disponibilidade Biológica , Derme/efeitos dos fármacos , Derme/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Modelos BiológicosRESUMO
One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg(2+) and Ca(2+) after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg(2+) or Ca(2+), indicating that the divalent cations accelerate the sliding likely by weakening the DNA-p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg(2+) and Ca(2+), which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.
Assuntos
Cálcio/química , DNA/metabolismo , Magnésio/química , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Difusão/efeitos dos fármacos , Polarização de Fluorescência , Humanos , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteína Supressora de Tumor p53/genéticaRESUMO
Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (Dc,60S = 0.311 µm(2)/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by "hitchhiking" on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that "off- and reloading" distributes the protein translation machinery.
Assuntos
Endossomos/metabolismo , Polirribossomos/metabolismo , Ustilago/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Difusão/efeitos dos fármacos , Dineínas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hifas/metabolismo , Hifas/ultraestrutura , Cinesinas/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Mutação/genética , Polirribossomos/efeitos dos fármacos , Polirribossomos/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ustilago/citologia , Ustilago/efeitos dos fármacosRESUMO
The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1-311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with (383)DVELL(387) replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Hipocampo/citologia , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Receptor A2A de Adenosina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Difusão/efeitos dos fármacos , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Cadeias de Markov , Mutação , Ratos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genéticaRESUMO
Growing evidence shows that GM1 ganglioside is involved in amyloid deposition and toxicity. By means of real-time single particle tracking, we show that amyloid oligomers and aggregates formed by Aß1-42 and amylin, two peptides associated, respectively, with the development of Alzheimer's disease and type II diabetes, interact with GM1 and decrease dramatically its lateral diffusion on the plasma membrane of living neuroblastoma cells. The confinement of GM1, a constituent of membrane rafts involved in neuroprotection, at the level of both types of amyloid aggregates can interfere with cell signaling pathways and contribute to the loss of neuroprotection.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Gangliosídeo G(M1)/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Microdomínios da Membrana/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Multimerização Proteica , Peptídeos beta-Amiloides/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Difusão/efeitos dos fármacos , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Fatores de TempoRESUMO
Mathematical analytical model of the processes running in individual radical clusters during the chemical phase (under the presence of radiomodifiers) proposed by us earlier has been further developed and improved. It has been applied to the data presented by Blok and Loman characterizing the oxygen effect in SSB and DSB formation (in water solution and at low-LET radiation) also in the region of very small oxygen concentrations, which cannot be studied with the help of experiments done with living cells. In this new analysis the values of all reaction rates and diffusion parameters known from literature have been made use of. The great increase of SSB and DSB at zero oxygen concentration may follow from the fact that at small oxygen concentrations the oxygen absorbs other radicals while at higher concentrations the formation of oxygen radicals prevails. It explains the double oxygen effect found already earlier by Ewing. The model may be easily extended to include also the effects of other radiomodifiers present in medium during irradiation.
Assuntos
Cromossomos/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Modelos Biológicos , Radiação Ionizante , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/efeitos da radiação , Cromossomos/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Difusão/efeitos dos fármacos , Difusão/efeitos da radiação , Oxigênio/farmacologiaRESUMO
The potential for the use of in-silico models of disease in progression monitoring is becoming increasingly recognised, as well as its contribution to the development of complete curative processes. In this paper we report the development of a hybrid cellular automaton model to mimic the growth of avascular tumours, including the infusion of a bioreductive drug to study the effects of protein binding on drug transportation. The growth model is operated within an extracellular tumour microenvironment. An artificial Neural Network based scheme was implemented that modelled the behaviours of each cell (proliferation, quiescence, apoptosis and/or movement) based on the complex heterogeneous microenvironment; consisting of oxygen, glucose, hydrogen ions, inhibitory factors and growth factors. To validate the growth model results, we conducted experiments with multicellular tumour spheroids. These results showed good agreement with the predicted growth dynamics. The outcome of the avascular tumour growth model suggested that tumour microenvironments have a strong impact on cell behaviour. To address the problem of cellular proteins acting as resistive factors preventing efficient drug penetration, a bioreactive drug (tirapazamine) was added to the system. This allowed us to study the drug penetration through multicellular layers of tissue after its binding to cellular proteins. The results of the in vitro model suggested that the proteins reduce the toxicity of the drug, reducing its efficacy for the most severely hypoxic fractions furthest from a functional blood vessel. Finally this research provides a unique comparison of in vitro tumour growth with an intelligent in silico model to measure bioreductive drug availability inside tumour tissue through a set of experiments.
Assuntos
Antineoplásicos/metabolismo , Modelos Biológicos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Difusão/efeitos dos fármacos , Células HT29 , Humanos , Redes Neurais de Computação , Ligação Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Tirapazamina , Triazinas/metabolismo , Triazinas/farmacologiaRESUMO
Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (-3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.