RESUMO
The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.
Assuntos
Polímeros/química , Poli-Inos/química , Fenômenos Biofísicos , Biofísica , Polarização de Fluorescência , Células HL-60 , Humanos , Potenciais da Membrana , Polímero PoliacetilênicoRESUMO
Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing D-instead of L-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane.
Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Polimixina B/análogos & derivados , Polimixina B/química , Ligação Competitiva , Espectroscopia de Ressonância de Spin Eletrônica , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/química , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat. PURPOSE: Assessing the intracellular transport of exogenous PIP3 attached to a polymeric carrier in an attempt to overcome cellular insulin resistance. METHODS: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3 cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes. RESULTS AND DISCUSSION: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3 that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3 complexes are retarded at the cell perimeter. PEI-25/PIP3 complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin. CONCLUSIONS: Inducing insulin actions by intracellular PIP3 delivery (PEI-25/PIP3 complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3 in a "second-messenger agonist" approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3 delivery predicts biological function in cells.
Assuntos
Sistemas de Liberação de Medicamentos , Resistência à Insulina , Insulina/metabolismo , Fosfatos de Fosfatidilinositol/administração & dosagem , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células Cultivadas , Portadores de Fármacos/química , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Polietilenoimina/química , Polímeros/química , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
One of the most significant predictors of prompt rehospitalization following psychiatric hospital discharge is missing follow-up out-patient appointments. Previous studies have suggested that system responsiveness accounted for much of the variance in predicting compliance with aftercare. Collaborations established at our institution allowed us to partially control this variable, opening the way to explore other obstacles to aftercare. All severely mentally ill subjects discharged from our hospital are provided follow-up appointments within two weeks. We retrospectively evaluated compliance with aftercare appointment and investigated factors that were associated with compliance. Eighty-one subjects were evaluated. Twenty-seven (33.8 %) did not attend their first follow-up appointment. Subjects with a primary substance-related syndrome were the most likely to miss their appointment (83.3%, chi 2 = 17.02, p = .0045), as were uninsured patients (51.6%, chi 2 = 8.79, p = .003). There was a trend for individuals not previously involved with their aftercare providers to miss their appointment (48.9%, chi 2 = 3.35, p = .067). Despite partial control of the system responsiveness variable, compliance with aftercare was suboptimal. This was due to a combination of client vulnerability variables and uncontrollable system responsiveness factors.
Assuntos
Transtornos Mentais/diagnóstico , Transtornos Mentais/reabilitação , Adolescente , Adulto , Idoso , Feminino , Seguimentos , Hospitalização , Humanos , Masculino , Transtornos Mentais/psicologia , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Cooperação do Paciente/estatística & dados numéricosRESUMO
Objective: Vegetarians might be at nutritional risk due to their food preferences. The goal of this study was to compare nutritional status and food intake of vegetarians and omnivores that use restaurants at the Federal University of Parana in Brazil. Methods: Clinical cross-sectional observational study performed between May 2014 and March 2015, assessing 84 vegetarians and 131 omnivores, adults, of both sexes. Anthropometric and body composition characteristics were evaluated on the total sample. In a subsample of 38 vegetarians and 63 omnivores, food intake of macronutrients, fibers, vitamin B12, vitamin D, calcium, iron and zinc were evaluated. Blood tests for vitamin B12, iron, and ferritin were performed in a subsample of 40 individuals of each group. Results: The studied groups presented similar anthropometric data and body composition, although both had individuals classified as at risk for cardiovascular diseases according to body mass index (BMI) (16.7% and 24.4% above 25 kg/m2 among vegetarians and omnivores, respectively). The caloric intake did not differ as well, although the main sources of energy intake among vegetarians were carbohydrates as compared to omnivores that had lipids as the main source of energy in their meals. Vegetarians reached the recommended intake of fibers, and the omnivores, had a lower intake of this nutrient as expected. For vitamin B12 the prevalence of inadequate intake was 37.8% on the vegetarians group and 0.6% on the omnivores, and for calcium, 49% for both groups. Both presented vitamin D intake below the estimated average requirement. The intake of iron did not differ among groups, however, in the vegetarian group the inadequacy reached 50% for men and 100% for women; and in the omnivore group 93% for women. For zinc, the inadequacy risk was 100% for men and 90% for women in the vegetarian group and 25% of men and 4.5% for women on the omnivore group. Regarding the biochemical exams, the most evident deficiency was of serum vitamin B12 on vegetarians. Conclusions: The food choices among the investigated undergraduate vegetarians do not guarantee nutritional security as detected in this study. Except for calcium, the prevalence of inadequate intake of macro and micronutrients was higher among vegetarians as compared to omnivores, stablishing a nutritional risk status to this group regarding to intake of sources of vitamin B12, vitamin D, iron and zinc.
Objetivo: Os vegetarianos podem estar em risco nutricional devido às suas preferências alimentares. O objetivo deste estudo foi comparar o estado nutricional e a ingestão alimentar de vegetarianos e onívoros que utilizam restaurantes da Universidade Federal do Paraná, no Brasil. Método: Estudo clínico observacional de corte transversal realizado entre maio de 2014 e março de 2015, avaliando 84 vegetarianos e 131 onívoros, adultos, de ambos os sexos. As características antropométricas e de composição corporal foram avaliadas na amostra total. Em uma subamostra de 38 vegetarianos e 63 onívoros, a ingestão alimentar de macronutrientes, fibras, vitamina B12, vitamina D, cálcio, ferro e zinco foram avaliados. Exames de sangue para vitamina B12, ferro e ferritina foram realizados em uma subamostra de 40 indivíduos de cada grupo. Resultados: Os grupos estudados apresentaram dados antropométricos e composição corporal semelhantes, embora ambos apresentassem indivíduos classificados como de risco para doenças cardiovasculares de acordo com o índice de massa corporal (IMC) (16,7% e 24,4% acima de 25 kg/m2 entre vegetarianos e onívoros, respectivamente). A ingestão calórica também não diferiu, embora as principais fontes de ingestão de energia entre os vegetarianos fossem carboidratos em comparação aos onívoros que tinham lipídios como principal fonte de energia em suas refeições. Os vegetarianos atingiram a ingestão recomendada de fibras, e os onívoros tiveram uma ingestão menor desse nutriente como esperado. Para a vitamina B12, a prevalência de ingestão inadequada foi de 37,8% no grupo dos vegetarianos e de 0,6% nos onívoros, e para o cálcio de 49% nos dois grupos. Ambos apresentaram ingestão de vitamina D abaixo do requisito médio estimado. A ingestão de ferro não diferiu entre os grupos, no entanto, no grupo vegetariano a inadequação chegou a 50% para os homens e 100% para as mulheres; e no grupo onívoro, 93% para as mulheres. Para o zinco, o risco de inadequação foi de 100% para homens e 90% para mulheres no grupo vegetariano e 25% de homens e 4,5% para mulheres no grupo onívoro. Em relação aos exames bioquímicos, a deficiência mais evidente foi de vitamina B12 sérica nos vegetarianos. Conclusões: As escolhas alimentares entre os vegetarianos de graduação investigados não garantem a segurança nutricional, como detectado neste estudo. Com exceção do cálcio, a prevalência de ingestão inadequada de macro e micronutrientes foi maior entre os vegetarianos em relação aos onívoros, estabelecendo um risco nutricional para esse grupo em relação à ingestão de fontes de vitamina B12, vitamina D, ferro e zinco.
Assuntos
Humanos , Masculino , Feminino , Adulto , Composição Corporal , Dieta Vegetariana , Ingestão de Alimentos , Avaliação NutricionalRESUMO
PURPOSE: To develop and demonstrate a rapid and simple colorimetric film assay for evaluating lipid interactions of pharmaceutical compounds and gel formulations. METHODS: The colorimetric assay comprises glass-supported films of phospholipids and polydiacetylene, which undergo visible and quantifiable blue-red transformations induced by interactions with amphiphilic molecules applied in very small volumes on the film surface. The color transitions are recorded by scanning of the films, and quantified through a simple image analysis algorithm. RESULTS: We show that pharmaceutical molecules and gel formulations induce blue-red transformations after short incubation with the lipid/polydiacetylene (PDA) films. Colorimetric dose-response curves exhibit dependence upon the lipid affinity and extent of membrane binding of the pharmaceutical compounds examined. The colorimetric lipid/PDA film assay was employed for distinguishing the contributions of individual molecular components within gel formulations. CONCLUSIONS: The colorimetric data yield insight into the degree of lipid binding of the molecules tested. The film assay is particularly advantageous for analysis of semi-solid (gel or lotion) formulations, elucidating the lipid interaction characteristics of specific molecular components within the mixtures. The new colorimetric film assay constitutes a generic, rapid, and easily applicable platform for predicting and screening interactions of pharmaceutical compounds and complex formulations with lipid barriers.
Assuntos
Lipídeos/química , Polímeros/química , Poli-Inos/química , Absorção Cutânea , Adsorção , Química Farmacêutica , Colorimetria , Géis , Polímero PoliacetilênicoRESUMO
PURPOSE: The aims of this study are to develop a rapid colorimetric assay for evaluating membrane interactions and penetration through lipid barriers and to create a platform, amenable to high-throughput screening formats, for predicting the extent of penetration of pharmaceutical compounds through lipid layers. METHODS: The colorimetric platform comprises vesicles of phospholipids and the chromatic lipid-mimetic polymer polydiacetylene. The polymer undergoes visible, concentration-dependent blue-red transformations induced through interactions of the vesicles with the molecules examined. RESULTS: We observe rapid colorimetric transitions induced by addition of pharmaceutical compounds to the chromatic vesicle solutions. We find that the concentration ranges for which the color transitions are induced in the lipid/polymer vesicles are correlated with the degree of lipid interactions and bilayer penetration of the tested compounds. The colorimetric platform could distinguish between three primary types of membrane-permeation profiles: bilayer-surface attachment, membrane penetration, and absence of lipid interactions. Application of complementary bioanalytical techniques corroborated the interpretation of the colorimetric data. Different pharmaceutical compounds were tested by the new assay. The results indicated clearly distinct membrane interaction profiles for molecules expected by conventional methods to have similar membrane-insertion properties (i.e., close log D/log P values). In addition, the new colorimetric assay pointed to similar membrane activities for molecules having highly divergent log Ds. CONCLUSIONS: The colorimetric assay facilitates "color coding" that could distinguish among different membrane permeation profiles. The data point to the usefulness of the platform for characterization of drug compound interactions with lipid assemblies. The new colorimetric technology constitutes a generic, extremely fast, and easily applicable approach for predicting and screening interactions of pharmaceutical compounds with lipid barriers.