RESUMO
Multipotent, self-renewing neural stem cells and their progeny [collectively referred to as neural precursor cells (NPCs)] represent a population of cells with great promise for CNS repair. To effectively harness their potential for therapeutic applications, the factors that regulate NPC behavior and/or fate must be well understood. The ability of immunomodulatory molecules to affect NPC behavior is of interest because of recent work elucidating the complex interactions between the immune system and nervous system. Herein, we examined the effects of cyclosporin A, a commonly used immunosuppressive molecule, on NPC proliferation kinetics, survival, and fate using in vitro assays at the population level and at the single-cell level. The use of pure populations of NPCs revealed a direct effect of cyclosporin A on cell survival, resulting in increased numbers and larger colonies, with no effect on proliferation kinetics. Cyclosporin A did not alter the differentiation profile of NPC colonies, indicating that it did not promote selective survival of a particular neural lineage. Additionally, we observed decreased cell-cell adhesions in developing cyclosporin A-treated NPC colonies. Consistent with the in vitro observations, in vivo administration of cyclosporin A to adult animals increased the numbers of NPCs within the neurogenic niche lining the lateral ventricles. Together, our findings establish that cyclosporin A has direct effects on NPCs both in vitro and in vivo, making it a promising candidate molecule for developing clinically relevant strategies to stimulate NPCs for brain repair.
Assuntos
Ciclosporina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Telencéfalo/efeitos dos fármacos , Animais , Encefalopatias/terapia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Imunossupressores/farmacologia , Ventrículos Laterais/citologia , Masculino , Camundongos , Regeneração Nervosa/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Telencéfalo/citologia , Telencéfalo/fisiologiaRESUMO
Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long-term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self-renewal is dependent on colony-proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC-derived cells, leading to clonal selection of colony reestablishing cells. hESC-derived feeders co-transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self-renewing hESCs. Our findings demonstrate that hESC-derived feeders arise from a distinct subpopulation of hESCs that respond to paracrine cues at the colony periphery that are required to sustain and establish clonal hESC self-renewal.
Assuntos
Comunicação Celular , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/citologia , Nicho de Células-Tronco/citologia , Animais , Morte Celular , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Células Clonais , Fibroblastos/citologia , Humanos , Imageamento Tridimensional , Camundongos , Modelos BiológicosRESUMO
Precise information about the size, shape, temporal dynamics, and spatial distribution of cells is beneficial for the understanding of cell behavior and may play a key role in drug development, regenerative medicine, and disease research. The traditional method of manual observation and measurement of cells from microscopic images is tedious, expensive, and time consuming. Thus, automated methods are in high demand, especially given the increasing quantity of cell data being collected. In this article, an automated method to measure cell morphology from microscopic images is proposed to outline the boundaries of individual hematopoietic stem cells (HSCs). The proposed method outlines the cell regions using a constrained watershed method which is derived as an inverse problem. The experimental results generated by applying the proposed method to different HSC image sequences showed robust performance to detect and segment individual and dividing cells. The performance of the proposed method for individual cell segmentation for single frame high-resolution images was more than 97%, and decreased slightly to 90% for low-resolution multiframe stitched images.
Assuntos
Forma Celular , Células-Tronco Hematopoéticas/citologia , Microscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Animais , CamundongosRESUMO
The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.
Assuntos
Segregação de Cromossomos , Replicação do DNA , DNA/metabolismo , Neurônios/fisiologia , Células-Tronco/fisiologia , Animais , Antimetabólitos/metabolismo , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Divisão Celular/fisiologia , Proliferação de Células , DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Neurônios/citologia , Linhagem , Prosencéfalo/citologia , Células-Tronco/citologiaRESUMO
BACKGROUND: Methods of manual cell localization and outlining are so onerous that automated tracking methods would seem mandatory for handling huge image sequences, nevertheless manual tracking is, astonishingly, still widely practiced in areas such as cell biology which are outside the influence of most image processing research. The goal of our research is to address this gap by developing automated methods of cell tracking, localization, and segmentation. Since even an optimal frame-to-frame association method cannot compensate and recover from poor detection, it is clear that the quality of cell tracking depends on the quality of cell detection within each frame. METHODS: Cell detection performs poorly where the background is not uniform and includes temporal illumination variations, spatial non-uniformities, and stationary objects such as well boundaries (which confine the cells under study). To improve cell detection, the signal to noise ratio of the input image can be increased via accurate background estimation. In this paper we investigate background estimation, for the purpose of cell detection. We propose a cell model and a method for background estimation, driven by the proposed cell model, such that well structure can be identified, and explicitly rejected, when estimating the background. RESULTS: The resulting background-removed images have fewer artifacts and allow cells to be localized and detected more reliably. The experimental results generated by applying the proposed method to different Hematopoietic Stem Cell (HSC) image sequences are quite promising. CONCLUSION: The understanding of cell behavior relies on precise information about the temporal dynamics and spatial distribution of cells. Such information may play a key role in disease research and regenerative medicine, so automated methods for observation and measurement of cells from microscopic images are in high demand. The proposed method in this paper is capable of localizing single cells in microwells and can be adapted for the other cell types that may not have circular shape. This method can be potentially used for single cell analysis to study the temporal dynamics of cells.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Modelos Biológicos , Análise de Célula Única/métodos , Algoritmos , Animais , Células-Tronco Hematopoéticas/citologia , Camundongos , Probabilidade , Fatores de TempoRESUMO
Cell segmentation and/or localization is the first stage of a (semi)automatic tracking system. We addressed the cell localization problem in our previous work where we characterized a typical blood stem cell in a microscopic image as an approximately circular object with dark interior and bright boundary. We also addressed the modelling of adjacent and dividing cells in our previous work as a deconvolution method to model individual blood stem cell as well as adjacent and dividing blood stem cells where an optimization algorithm was combined with a template matching method to segment cell regions and locate the cell centers. Our previous cell deconvolution method is capable of modelling different cell types with changes in the model parameters. However in cases where either a complex parameterized shape is needed to model a specific cell type, or in place of cell center localization, an exact cell segmentation is needed, this method will not be effective. In this paper we propose a method to achieve cell boundary segmentation. Considering cell segmentation as an inverse problem, we assume that cell centers are located in advance. Then, the cell segmentation will be solved by finding cell regions for optimal representation of cell centers while a template matching method is effectively employed to localize cell centres.
Assuntos
Algoritmos , Inteligência Artificial , Células-Tronco Hematopoéticas/citologia , Interpretação de Imagem Assistida por Computador/métodos , Microscopia/métodos , Reconhecimento Automatizado de Padrão/métodos , Células Cultivadas , Humanos , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Hematopoietic Stem Cells (HSCs) form blood and immune cells and are responsible for the constant renewal of blood. To produce new blood cells, HSCs proliferate and differentiate to different blood cell types continuously during their lifetime. Hence they are of substantial interest in stem cell therapy and cancer research. To classify HSCs to different groups, they must be observed/tracked over time and their key features including cell size, shape, and motility must be extracted. The manual tracking is an onerous task and automated methods are in high demand. The first stage of an semi-automatic/automatic tracking system is cell segmentation. In our previous work we addressed the cell segmentation/localization problem. Modelling adjacent or splitting cells is very challenging and our previous methods might fail to accurately model a group of adjacent cells or a splitting cell. In this paper we address this issue and propose a deconvolution method to precisely model individual HSCs as well as adjacent (splitting) HSCs. An optimization algorithm is combined with a template matching method to segment cell regions and locate the cell centers.
Assuntos
Algoritmos , Inteligência Artificial , Contagem de Células/métodos , Separação Celular/métodos , Células-Tronco Hematopoéticas/citologia , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , Células Cultivadas , Humanos , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.
Assuntos
Técnicas de Cultura de Células/instrumentação , Citometria de Fluxo/instrumentação , Glucose/metabolismo , Interpretação de Imagem Assistida por Computador/instrumentação , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Oxigênio/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Citometria de Fluxo/métodos , Humanos , Interpretação de Imagem Assistida por Computador/métodosRESUMO
Transgene variegation is caused by epigenetic switching between expressing and silent states. gamma-retrovirus vectors can be variegated in stem cells, but the dynamics of epigenetic remodeling during transgene variegation are unknown. Here, we measured variegated enhanced green fluorescent protein gamma-retrovirus expression over 4 days in individual embryonic stem cells while tracking cells in order to create expression lineage trees: 56 colony founder cells and their progeny were tracked over seven generations. Nineteen lineages produced synchronized inheritable trajectories of transgene silencing or reactivation, indicative of epigenetic remodeling with long-term stable inheritance. Short-term fluctuations in fluorescence intensity were also observed, which contributed low-amplitude variation to transgene expression level. These two processes have different frequencies and inheritability, but together contribute to variegated transgene expression. Inhibition of DNA methylation with 5-azacytidine eliminated long-term transgene silencing over 4 days, but short-term fluctuations continued. Our approach applies real-time imaging technology to track the long-term dynamics of transgene expression to investigate the timing and expression patterns leading to variegation.
Assuntos
Epigênese Genética , Vetores Genéticos , Retroviridae/genética , Animais , Azacitidina/farmacologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Camundongos , Microscopia de Fluorescência , Proteínas Recombinantes/genéticaRESUMO
The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.
Assuntos
Clonagem Molecular/métodos , Frutose-Bifosfato Aldolase/biossíntese , Magnaporthe/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Reatores Biológicos , Dimerização , Escherichia coli/enzimologia , Fermentação , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Zinco/análiseRESUMO
The study of cell behavior is of crucial importance in drug and disease research. The fields of bioinformatics and biotechnology rely on the collection, processing, and analysis of huge numbers of biocellular images, including cell features such as cell size, shape, and motility. However manual methods of inferring these values are so onerous that automated methods of cell tracking and segmentation are in high demand. In this paper, a novel model-based cell tracker is designed to locate and track individual cells. The proposed cell tracker has been successfully applied to track hematopoietic stem cells (HSCs) based on identified cell locations and probabilistic data association.
RESUMO
BACKGROUND: There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo-like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates "lineage informatics," a lineage-centric approach to cytomics. METHODS: We cultured cells in a confined monolayer using a novel "gap chamber" that produces images with confocal-like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. RESULTS: Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo-like than traditional culture systems. CONCLUSIONS: The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long-term cell imaging.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem da Célula , Informática/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Informática/instrumentação , Masculino , Camundongos , Células-Tronco/fisiologiaRESUMO
To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Hematopoéticas/citologia , Microscopia de Vídeo/métodos , Animais , Linhagem da Célula , Proliferação de Células , Citometria de Fluxo , Cinética , Antígenos Comuns de Leucócito/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de TempoRESUMO
The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Replicação do DNA/fisiologia , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/fisiologia , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Citocinese/fisiologia , Fase G1/fisiologia , Proteína 1 de Manutenção de Minicromossomo , Proteínas de Manutenção de Minicromossomo , Modelos Biológicos , Complexo de Reconhecimento de Origem/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismoRESUMO
The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.
Assuntos
Técnicas de Cultura de Células , Celulase/química , Celulose/química , Enzimas Imobilizadas/química , Fator de Células-Tronco/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Celulase/genética , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Camundongos , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Fator de Células-Tronco/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Engenharia TecidualRESUMO
The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.
Assuntos
Inibidores Enzimáticos/análise , Enzimas/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Modelos Químicos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fatores de TempoRESUMO
Over the past decade, multiphoton microscopy has progressed from a photonic novelty to a technique whose application is currently experiencing exponential growth in the biological sciences. A novel application of this technology with significant therapeutic potential is the control of drug activity by multiphoton photolysis of caged therapeutics. As an initial case study, the potent isoform selective inhibitor N-(3-(aminomethyl)benzyl) acetamidine (1400W) of inducible nitric oxide synthase (iNOS) has been conjugated to a caging molecule 6-bromo-7-hydroxy-4-hydroxyquinoline-2-ylmethyl acetyl ester (Bhc). Here we present the first report of a bulk therapeutic effect, inhibition of nitric oxide production, in mammalian cell culture by multiphoton photolysis of a caged drug, Bhc-1400W. Mouse macrophage RAW 264.7 cells induced with bacterial lipopolysaccharides to express iNOS were used to assess the therapeutic value of the conjugated inhibitor. Both 1400W and Bhc-1400W are stable in metabolically active cells and an optimal time interval for the photorelease of the inhibitor was determined. The ratios of the IC(50) values of Bhc-1400W over 1400W calculated in the presence of iNOS enzyme and in RAW 264.7 cell culture are 19 and 100, respectively, indicating that a broad therapeutic range exists in cell culture. Multiphoton uncaging protocols and therapeutic doses of inhibitors were not cytotoxic. Photocontrol of LPS induced nitric oxide production was achieved in mammalian cell culture using a single laser focal volume. This technology has the potential to control active drug concentrations in vivo, a lack of which is one of the main problems currently associated with systemic drug administration.
Assuntos
Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Inibidores Enzimáticos/química , Camundongos , Óxido Nítrico Sintase Tipo II , FótonsRESUMO
Several systematic errors may occur during the analysis of uninhibited enzyme kinetic data using commercially available multiwell plate reader software. A MATLAB program is developed to remove these systematic errors from the data analysis process for a single substrate-enzyme system conforming to Michaelis-Menten kinetics. Three experimental designs that may be used to validate a new enzyme preparation or assay methodology and to characterize an enzyme-substrate system, while capitalizing on the ability of multiwell plate readers to perform multiple reactions simultaneously, are also proposed. These experimental designs are used to (i) test for enzyme inactivation and the quality of data obtained from an enzyme assay using Selwyn's test, (ii) calculate the limit of detection of the enzyme assay, and (iii) calculate Km and Vm values. If replicates that reflect the overall error in performing a measurement are used, the latter two experiments may be performed with internal estimation of the error structure. The need to correct for the systematic errors discussed and the utility of the proposed experimental designs were confirmed by numerical simulation. The proposed experiments were conducted using recombinant inducible nitric oxide synthase preparations and the oxyhemoglobin assay.
Assuntos
Bioquímica/métodos , Óxido Nítrico Sintase/metabolismo , Interpretação Estatística de Dados , Humanos , Cinética , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Fatores de TempoRESUMO
Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.
Assuntos
Calmodulina/metabolismo , Óxido Nítrico Sintase/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/genética , Galinhas/metabolismo , Ativação Enzimática/fisiologia , Metionina/metabolismo , Dados de Sequência Molecular , Mutação , OxirreduçãoRESUMO
Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.