RESUMO
The increase in the incidence of neurodegenerative diseases linked to aging or injury needs to be addressed in research into neuroprotective or neuroregenerative therapies, and requires the development of specific biological models. To achieve this goal we propose (1) the use of the mouse olfactory epithelium as a biological support which specifically exhibits a regenerative or a self-renewing capacity and during the lifetime necessitates the presence of neural stem cells, and (2) the use of an intraperitoneal injection of 2,6-dichlorobenzonitrile (diclobenil) as a chemical inducer of neurodegeneration in olfactory epithelium by selectively killing mature cells. We developed a biological model to follow the processes of neurodegeneration (chemically induced) and neuroregeneration (self-renewal of olfactory epithelium). The purpose of this study was to develop a method to monitor quickly neurodegeneration/neuroregeneration processes in order to further screen protective and regenerative therapies. For this purpose, we used the sedimentation field flow fractionation elution of olfactory epithelium. We obtained specific elution profiles and retention parameters allowing the monitoring of the induction and kinetics of biological processes. The use of insulin-like growth factor 1α as a neuroprotective agent in an innovative nebulization protocol showed sedimentation field flow fractionation to be a simple, fast and low-cost method to monitor such a biological event on the scale of an entire organism.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Nitrilas , Mucosa Olfatória/inervação , Mucosa Olfatória/patologia , Animais , Apoptose , Modelos Animais de Doenças , Fator de Crescimento Insulin-Like I/uso terapêutico , Cinética , Masculino , Camundongos , Regeneração Nervosa/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Mucosa Olfatória/efeitos dos fármacosRESUMO
Recently, the use of SdFFF in cancer research has been studied in order to better understand major phenomena implicated in cancer development and therapy: apoptosis and differentiation. In this report, we used SdFFF as a monitoring and cell separation tool to study the kinetics of apoptosis. Incubation of K562 cells with diosgenin, used as cellular model, led to a surprising apoptotic process occurring in two phases (after 24 and 48 h incubation), associated with specific p-ERK expression. Based on the capacity to sort apoptotic cells, results showed that SdFFF cell separation was an effective analytical tool to obtain different subpopulations regardless of the kinetics and extent of apoptosis. Results also showed that, after proper biological calibration of elution profiles, SdFFF can be used to monitor either the induction or the kinetics of a biological event.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Diosgenina/farmacologia , Fracionamento por Campo e Fluxo , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células K562 , Cinética , Neoplasias/enzimologia , Neoplasias/patologiaRESUMO
Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.
Assuntos
Diferenciação Celular , Fracionamento Celular , Fracionamento por Campo e Fluxo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Bromodesoxiuridina , Adesão Celular , Linhagem da Célula , Proliferação de Células , Citometria de Fluxo , Imunofluorescência , Humanos , Cinética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Fenótipo , Ácidos Siálicos/metabolismo , Células Tumorais CultivadasRESUMO
Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.
Assuntos
Diferenciação Celular , Diosgenina/metabolismo , Leucemia Eritroblástica Aguda/patologia , Megacariócitos/patologia , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Primers do DNA , Fracionamento por Campo e Fluxo , Humanos , Ploidias , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrofotometria UltravioletaRESUMO
Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Amido/análise , Triticum/química , Amilases/metabolismo , Fracionamento por Campo e Fluxo/instrumentação , Hidrólise , Tamanho da Partícula , Reprodutibilidade dos Testes , Amido/metabolismoRESUMO
Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.
Assuntos
Apoptose , Separação Celular/métodos , Diosgenina/farmacologia , Fracionamento por Campo e Fluxo/métodos , Linhagem Celular Tumoral , Diosgenina/metabolismo , Fracionamento por Campo e Fluxo/instrumentação , Humanos , Técnicas In VitroRESUMO
The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using "Hyperlayer" elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a "pseudoplacodal" epithelioid cell type from which derived "floating cells". These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.
Assuntos
Separação Celular/métodos , Fracionamento por Campo e Fluxo/métodos , Mucosa Olfatória/embriologia , Células-Tronco/citologia , Animais , Células Cultivadas , Embrião de Galinha , Fator de Crescimento Neural/farmacologia , Mucosa Olfatória/citologia , Receptor trkA/biossíntese , Células-Tronco/efeitos dos fármacosRESUMO
Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.
Assuntos
Ácidos Graxos Essenciais/metabolismo , Fígado/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Esterificação , Ácidos Graxos não Esterificados/metabolismo , Ácido Linoleico , Ácidos Linoleicos/metabolismo , Masculino , Ácido Oleico , Ácidos Oleicos/metabolismo , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Albumina Sérica/metabolismo , Triglicerídeos/metabolismoRESUMO
As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.
Assuntos
Separação Celular/métodos , Córtex Cerebral/citologia , Neurônios/citologia , Animais , Sobrevivência Celular , Córtex Cerebral/embriologia , Idade Gestacional , Neuroglia/citologia , Ratos , Coloração e RotulagemRESUMO
A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.
Assuntos
Apolipoproteínas E/metabolismo , Escherichia coli/enzimologia , Receptores de LDL/metabolismo , Apolipoproteínas E/genética , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Códon , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Humanos , Hipercolesterolemia/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por RestriçãoRESUMO
Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.
Assuntos
Amilases/metabolismo , Oryza/metabolismo , Amido/metabolismo , Fracionamento por Campo e Fluxo , HidróliseRESUMO
Field flow fractionation (FFF) is a new methodology described as being well-suited for the separation and characterization of biopolymers and particles. On theoretical grounds, cells may be separated with FFF if they differ in size, density or deformability. In the present study, we first tried to determine optimal separation conditions for red blood cells; thereafter we used FFF to examine red cell changes during a phenylhydrazine-induced hemolytic anemia. It has been shown that in less than 30 minutes, FFF is able to separate normal red blood cells from Heinz body-rich cells or reticulocytes that differ in size or density. The successive steps of hemolysis and regeneration appear clearly on the fractograms. Advantages and drawbacks of the method are discussed.
Assuntos
Separação Celular/métodos , Eritrócitos/citologia , Anemia Hemolítica/sangue , Anemia Hemolítica/induzido quimicamente , Animais , Fracionamento Químico/métodos , Eritrócitos/patologia , Fenil-Hidrazinas , CoelhosRESUMO
The effect of the dietary fiber on apo B mRNA level was studied in the intestine of rats that were fed either fiber-free or high-fiber (30% sugar-beet fiber) low-fat diets for 3 weeks. The fiber diet studied does not affect jejunal apo B mRNA levels but decreases the level of ileal apo B mRNA. In the rat cecum, in both fiber-free and fiber groups, we failed to detect the apo B mRNA. The test fiber diet feeding markedly increased fecal bile salt and cholesterol excretions. We suggest that dietary fiber can modify apo B expression in the intestine. The increased fecal bile salt excretion might be involved in such a modification.
Assuntos
Apolipoproteínas B/genética , Fibras na Dieta/administração & dosagem , Intestino Delgado/metabolismo , Animais , Bile/metabolismo , Northern Blotting , Expressão Gênica , Íleo/metabolismo , Jejuno/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Sedimentation field flow fractionation (SdFFF) operated at multi gravitational field is used to analyse a highly polydisperse TiO2 colloidal suspension. From the initial sample, time dependent eluted fractions are collected and submitted to electron microscopy (EM) shape and size analysis. To assess the accuracy of FFF in determining the average size of the different fractions, these are re-introduced into the channel by means of two different procedures, the on-channel concentration of the fractions and the direct re-injection of pre-concentrated fractions (DRI). Both methods appear accurate to determine the average size of every fraction, associated to a lower recovery in the case of DRI. The fractogram band spreading characteristics of the re-introduced fractions are correlated to the particle size distribution measured by EM. After density determination of fractionated particles, the fractogram is calibrated in terms of size and size distribution using data obtained from EM for each fraction. Quantitative analyses, based on particle counting showed high recovery (80-90%) of the eluted species. However, this loss limited the possibility to extend signal information to a quantitative one.
Assuntos
Titânio/química , Fracionamento Químico , Microscopia EletrônicaRESUMO
The stoichiometric coefficients and apparent formation constants (Kf) of alpha-terpineol, thymol, geraniol and linalool complexes with beta-cyclodextrin (beta-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol-water mobile phases. All the terpene derivatives under study form 1:1 guest-CD complexes. Graphs of Kf as a function of the mobile phase composition appeared different from those classically described for RP-C18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute-CD inclusion and solute-stationary phase interactions may be involved in this specific behavior.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ciclodextrinas/química , Terpenos/química , beta-Ciclodextrinas , Cromatografia Líquida de Alta Pressão/instrumentação , Grafite/químicaRESUMO
Specific programming of automated HPLC systems allows total on-line qualification, validation and stability monitoring using the concept of deferred standards. Setting up such a process for routine analyses in an automated HPLC system requires specific autosampler programming as well as specific monitoring software. With an autosampler, a double injection procedure is programmed, the first introducing the sample, and the second, a few minutes deferred, the deferred control standard. Two additional compounds are therefore added to the sample before and during the chromatographic process: the intemal standard for sample quantification and the deferred standard for system control. Specific methodologies are described of how to obtain classical quantitative analysis information as well as system qualification validation stability information. Experiments were performed to develop specified methodologies to monitor the quality of quantitative analysis during the life of the column by using the deferred standard concept to probe the effects of column ageing on separation characteristics.
Assuntos
Cromatografia Líquida de Alta Pressão/normas , Automação , Cromatografia Líquida de Alta Pressão/métodos , Compostos Policíclicos/análise , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.
Assuntos
Fracionamento por Campo e Fluxo , Neuroblastoma/patologia , Células-Tronco/citologia , Linhagem Celular Tumoral , HumanosRESUMO
Apoptosis is one of the most important phenomena of cellular biology. Sedimentation field flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape or rigidity), we investigated the capacity of SdFFF in monitoring the early and specific biophysical modifications which occurred during cellular apoptosis induction. Then, we used, as an in vitro cellular apoptosis model, the association between human 1547 osteosarcoma cells and diosgenin, a plant steroid known to induce apoptosis. Four other molecules were studied: hecogenin, tigogenin, staurosporine and MG132. Our results demonstrated a correlation between SdFFF elution profile changes (peak shape modification and retention ratio evolution) and effective apoptosis induction. For the first time, we demonstrated that SdFFF could be used to monitor apoptosis induction as early as 6 h incubation, suggesting different applications such as screening series of molecules to evaluate their ability to induce apoptosis, or sorting apoptotic cells to study apoptosis pathway.
Assuntos
Apoptose , Neoplasias Ósseas/patologia , Diosgenina/farmacologia , Osteossarcoma/patologia , Linhagem Celular Tumoral , HumanosRESUMO
Serum lipids and apolipoprotein (apo) B and A-I concentrations were determined in 164 dairy cows which had undergone liver biopsy in early lactation. The animals were divided into groups according to fatty liver severity on the basis of hepatic triglyceride content. The serum free fatty acid (FFA) concentration was higher in cows that developed fatty livers than in normal cows, and it correlated highly with liver triglycerides. Serum total cholesterol and triglyceride levels did not correlate with hepatic triglycerides. Both apo B and apo A-I levels were significantly decreased in fatty liver cows. In particular, apo B levels showed a strongly negative correlation with liver triglycerides. The present results suggest that hepatic apolipoprotein synthesis is impeded in fatty liver cows.
Assuntos
Apolipoproteínas A/sangue , Apolipoproteínas B/sangue , Doenças dos Bovinos/sangue , Fígado Gorduroso/veterinária , Lipoproteínas HDL/sangue , Animais , Apolipoproteína A-I , Biópsia , Bovinos , Doenças dos Bovinos/patologia , Fígado Gorduroso/sangue , Feminino , Lactação/sangue , Fígado/química , Fígado/patologiaRESUMO
The effects of long term (8-14 wk) essential fatty acid (EFA)-deprived diets in rats are well documented. In the present study, we compared, in weanling rats, the effect of a short term (two wk) hydrogenated coconut oil, EFA-deprived, diet (D) with that of a corn oil, EFA-adequate, diet (A), using either sucrose (SU) or starch (ST) as carbohydrate. After two wk, rats fed the sucrose/hydrogenated coconut oil diet developed some characteristic features of EFA deprivation: slower growth rate, decreases in linoleic and arachidonic acid of plasma phospholipids and an increase in n-9 eicosatrienoic acid of plasma phospholipids. When rats ate the starch/hydrogenated coconut oil diet, there was a similar decrease in linoleic acid of plasma phospholipids, but only a small effect on growth rate and no change in the arachidonic acid content of plasma phospholipids. EFA deprivation and sucrose had opposite effects on plasma triglyceride (TG) levels: deprivation induced a decrease, whereas the sucrose induced an increase in very low density lipoprotein (VLDL) triglycerides. The observed decrease in plasma triglyceride during EFA deprivation might result from an activation of lipoprotein lipase during the early stages of deprivation.