RESUMO
Botanicals are widely used as dietary supplements and for the prevention and treatment of disease. Despite a long history of use, there is generally little evidence supporting the efficacy and safety of these preparations. Curcumin has been used to treat a myriad of human diseases and is widely advertised and marketed for its ability to improve health, but there is no clear understanding how curcumin interacts with cells and affects cell physiology. D. discoideum is a simple eukaryotic lead system that allows both tractable genetic and biochemical studies. The studies reported here show novel effects of curcumin on cell proliferation and physiology, and a pleiotropic effect on gene transcription. Transcriptome analysis showed that the effect is two-phased with an early transient effect on the transcription of approximately 5% of the genome, and demonstrates that cells respond to curcumin through a variety of previously unknown molecular pathways. This is followed by later unique transcriptional changes and a protein kinase A dependent decrease in catalase A and three superoxide dismutase enzymes. Although this results in an increase in reactive oxygen species (ROS; superoxide and H2O2), the effects of curcumin on transcription do not appear to be the direct result of oxidation. This study opens the door to future explorations of the effect of curcumin on cell physiology.
Assuntos
Curcumina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Catalase/metabolismo , Dictyostelium/enzimologia , Dictyostelium/metabolismo , Humanos , Superóxido Dismutase/metabolismo , TranscriptomaRESUMO
Theaflavins, flavonoids found in black tea, exhibit a variety of health-promoting activities, but the mechanisms by which they act are not clear. Here, we assess the effects of black tea extract and isolated theaflavins on Dictyostelium discoideum, a model organism exhibiting an unusual life cycle relying on conserved pathways involved in human disease. Dictyostelium has been used to characterize the activities of numerous bioactive small molecules, including catechins, from which theaflavins are produced during the preparation of black tea. We show that theaflavins block growth, development, and motility in Dictyostelium, results that suggest catechins and theaflavins exert similar activities in this organism.
Assuntos
Biflavonoides/farmacologia , Camellia sinensis/química , Catequina/farmacologia , Catecóis/farmacologia , Dictyostelium/efeitos dos fármacos , Cultura Axênica , Biflavonoides/química , Biflavonoides/isolamento & purificação , Catequina/química , Catequina/isolamento & purificação , Catecóis/química , Catecóis/isolamento & purificação , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Dictyostelium/crescimento & desenvolvimento , Extratos Vegetais/química , Relação Estrutura-AtividadeRESUMO
Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Dictyostelium/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Dictyostelium/fisiologiaRESUMO
Starting with the established antiepileptic drug, valproic acid, we have taken a novel approach to develop new antiseizure drugs that may be effective in status epilepticus. We first identified that valproic acid has a potent effect on a biochemical pathway, the phosphoinositide pathway, in Dictyostelium discoideum, and we demonstrated that this may relate to its mechanism of action against seizures in mammalian systems. Through screening in this pathway, we have identified a large array of fatty acids and fatty acid derivatives with antiseizure potential. These were then evaluated in an in vitro mammalian system. One compound that we identified through this process is a major constituent of the ketogenic diet, strongly arguing that it may be the fatty acids that are mediating the antiseizure effect of this diet. We further tested two of the more potent compounds in an in vivo model of status epilepticus and demonstrated that they were more effective than valproic acid in treating the status epilepticus. This article is part of a Special Issue entitled "Status Epilepticus".
Assuntos
Anticonvulsivantes/uso terapêutico , Estado Epiléptico/terapia , Animais , Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo , Dieta Cetogênica , Avaliação Pré-Clínica de Medicamentos , Humanos , Fosfatidilinositóis/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estado Epiléptico/dietoterapia , Estado Epiléptico/tratamento farmacológico , Terapias em Estudo , Ácido Valproico/uso terapêuticoRESUMO
Lanestosa is a chronically polluted site (derelict mine) where the soil (Lanestosa (LA) soil) exceeds screening values (SVs) of regulatory policies in force (Basque Country; Europe) for Zn, Pb and Cd. A scenario-targeted toxicity assessment was carried out on the basis of a multi-endpoint bioassay approach. Acute and chronic toxicity bioassays were conducted with selected test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates and with bioaccumulation studies in earthworms. Besides, the toxicity profile was compared with that of the mine runoff (RO) soil and of a fresh artificially polluted soil (LAAPS) resembling LA soil pollutant profile. Extractability studies in LA soil revealed that Pb, Zn and Cd were highly available for exchange and/or release into the environment. Indeed, Pb and Zn were accumulated in earthworms and LA soil resulted to be toxic. Soil respiration, V. fischeri, vegetative and developmental cycles of D. discoideum and survival and juvenile production of E. fetida were severely affected. These results confirmed that LA soil had unacceptable environmental risk and demanded intervention. In contrast, although Pb and Zn concentrations in RO soil revealed also unacceptable risk, both metal extractability and toxicity were much lower than in LA soil. Thus, within the polluted site, the need for intervention varied between areas that posed dissimilar risk. Besides, since LAAPS, with a high exchangeable metal fraction, was the most toxic, ageing under in situ natural conditions seemingly contributed to attenuate LA soil risk. As a whole, combining multi-endpoint bioassays with scenario-targeted analysis (including leaching and ageing) provides reliable risk assessment in soils posing unacceptable environmental risk according to SVs, which is useful to optimise the required intervention measures.
Assuntos
Poluentes do Solo/toxicidade , Testes de Toxicidade , Aliivibrio fischeri/efeitos dos fármacos , Animais , Bioensaio , Cádmio/análise , Cádmio/metabolismo , Cádmio/toxicidade , Dictyostelium/efeitos dos fármacos , Europa (Continente) , Germinação/efeitos dos fármacos , Chumbo/análise , Chumbo/metabolismo , Chumbo/toxicidade , Dose Letal Mediana , Lactuca/efeitos dos fármacos , Lactuca/crescimento & desenvolvimento , Metais/análise , Oligoquetos/efeitos dos fármacos , Oligoquetos/metabolismo , Petróleo/análise , Petróleo/toxicidade , Raphanus/efeitos dos fármacos , Medição de Risco , Solo/química , Poluentes do Solo/análise , Poluentes do Solo/metabolismo , Espanha , Zinco/análise , Zinco/metabolismo , Zinco/toxicidadeRESUMO
In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations.
Assuntos
Meios de Cultura/química , Dictyostelium/efeitos dos fármacos , Dictyostelium/crescimento & desenvolvimento , Estabilidade Enzimática/efeitos dos fármacos , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Dictyostelium/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenilalanina Hidroxilase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Toxicity profiles of two soils (a brownfield in Legazpi and an abandoned iron mine in Zugaztieta; Basque Country) contaminated with several metals (As, Zn, Pb and Cu in Legazpi; Zn, Pb, Cd and Cu in Zugaztieta) and petroleum hydrocarbons (in Legazpi) were determined using a multi-endpoint bioassay approach. Investigated soils exceeded screening values (SVs) of regulatory policies in force (Basque Country; Europe). Acute and chronic toxicity bioassays were conducted with a selected set of test species (Vibrio fischeri, Dictyostelium discoideum, Lactuca sativa, Raphanus sativus and Eisenia fetida) in combination with chemical analysis of soils and elutriates, as well as with bioaccumulation studies in earthworms. The sensitivity of the test species and the toxicity endpoints varied depending on the soil. It was concluded that whilst Zugaztieta soil showed very little or no toxicity, Legazpi soil was toxic according to almost all the toxicity tests (solid phase Microtox, D. discoideum inhibition of fruiting body formation and developmental cycle solid phase assays, lettuce seed germination and root elongation test, earthworm acute toxicity and reproduction tests, D. discoideum cell viability and replication elutriate assays). Thus, albeit both soils had similar SVs, their ecotoxicological risk, and therefore the need for intervening, was different for each soil as unveiled after toxicity profiling based on multiple endpoint bioassays. Such a toxicity profiling approach is suitable to be applied for scenario-targeted soil risk assessment in those cases where applicable national/regional soil legislation based on SVs demands further toxicity assessment.
Assuntos
Oligoquetos/efeitos dos fármacos , Petróleo/toxicidade , Poluentes do Solo/toxicidade , Aliivibrio fischeri , Animais , Bioensaio , Dictyostelium/efeitos dos fármacos , Dictyostelium/crescimento & desenvolvimento , Ecotoxicologia , Monitoramento Ambiental , Germinação/efeitos dos fármacos , Lactuca/efeitos dos fármacos , Lactuca/fisiologia , Oligoquetos/fisiologia , Petróleo/análise , Poluição por Petróleo/análise , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Raphanus/efeitos dos fármacos , Raphanus/fisiologia , Medição de Risco , Solo/química , Poluentes do Solo/análise , Testes de ToxicidadeRESUMO
Much of our knowledge of molecular cellular functions is based on studies with a few number of model organisms that were established during the last 50 years. The social amoeba Dictyostelium discoideum is one such model, and has been particularly useful for the study of cell motility, chemotaxis, phagocytosis, endocytic vesicle traffic, cell adhesion, pattern formation, caspase-independent cell death, and, more recently, autophagy and social evolution. As nonmammalian model of human diseases D. discoideum is a newcomer, yet it has proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections, for mitochondrial diseases, and for pharmacogenetic studies. The D. discoideum genome harbors several homologs of human genes responsible for a variety of diseases, -including Chediak-Higashi syndrome, lissencephaly, mucolipidosis, Huntington disease, IBMPFD, and Shwachman-Diamond syndrome. A few genes have already been studied, providing new insights on the mechanism of action of the encoded proteins and in some cases on the defect underlying the disease. The opportunities offered by the organism and its place among the nonmammalian models for human diseases will be discussed.
Assuntos
Dictyostelium/citologia , Animais , Doenças da Medula Óssea/genética , Quimiotaxia , Dictyostelium/efeitos dos fármacos , Dictyostelium/fisiologia , Avaliação Pré-Clínica de Medicamentos , Insuficiência Pancreática Exócrina/genética , Interações Hospedeiro-Patógeno , Humanos , Doença de Huntington/genética , Lipomatose/genética , Doenças Mitocondriais/genética , Modelos Biológicos , Fagocitose , Proteínas de Protozoários/fisiologia , Síndrome de Shwachman-DiamondRESUMO
The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.
Assuntos
Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Vesículas Secretórias/metabolismo , Acil Coenzima A/metabolismo , Centrifugação , Detergentes/farmacologia , Dictyostelium/citologia , Dictyostelium/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vesículas Secretórias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Medicinal drugs do not always have clearly understood mechanisms of action, especially as regards psychiatric treatment. Identification of genes involved in drug resistance is an important step toward elucidating the genetic basis of disease and the molecular mechanism of drug action. However, this approach is impractical in higher animals, as ablation and screening of every gene in an animal is not currently possible. Dictyostelium has proven a good model system for molecular pharmacological research as a result of its genetic tractability, ease of gene knockout, and creation of isogenic lines. In this system, we have identified genes that confer resistance to bipolar disorder drugs. This work has implicated inositol (1,4,5) trisphosphate (InsP3) signaling as a common mechanism of action for these drugs in patients.
Assuntos
Antimaníacos/farmacologia , Transtorno Bipolar , Dictyostelium , Resistência a Medicamentos/genética , Inositol 1,4,5-Trifosfato/análise , Farmacogenética , Animais , Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/genética , Elementos de DNA Transponíveis , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Deleção de Genes , Marcação de Genes , Humanos , Mapeamento por RestriçãoRESUMO
Chemotaxing cells, such as Dictyostelium and mammalian neutrophils, sense shallow chemoattractant gradients and respond with highly polarized changes in cell morphology and motility. Uniform chemoattractant stimulation induces the transient translocations of several downstream signaling components, including phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In contrast, static spatial chemoattractant gradients elicit the persistent, amplified localization of these molecules. We have proposed a model in which the response to chemoattractant is regulated by a balance of a local excitation and a global inhibition, both of which are controlled by receptor occupancy. This model can account for both the transient and spatial responses to chemoattractants, but alone does not amplify the external gradient. In this article, we develop a model in which parallel local excitation, global inhibition mechanisms control the membrane binding of PI3K and PTEN. Together, the action of these enzymes induces an amplified PI(3,4,5)P3 response that agrees quantitatively with experimentally obtained plekstrin homology-green fluorescent protein distributions in latrunculin-treated cells. We compare the model's performance with that of several mutants in which one or both of the enzymes are disrupted. The model accounts for the observed response to multiple, simultaneous chemoattractant cues and can recreate the cellular response to combinations of temporal and spatial stimuli. Finally, we use the model to predict the response of a cell where only a fraction is stimulated by a saturating dose of chemoattractant.
Assuntos
Quimiotaxia/fisiologia , Dictyostelium/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Quimiotaxia/efeitos dos fármacos , Simulação por Computador , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos , PTEN Fosfo-HidrolaseRESUMO
Dictyostelium discoideum is a single-cell, eukaryotic microorganism that can undergo multicellular development in order to produce dormant spores. We investigated the capacity of D. discoideum to be used as a rapid screening system for potential developmental toxicity of compounds under development as pharmaceuticals. We used a set of four transgenic D. discoideum strains that expressed a reporter gene under the control of promoters that are active at certain time periods and in distinct cell types during D. discoideum development. We found that teratogens such as valproic acid, tretinoin, or thalidomide interfered to various extents with D. discoideum development, and had different effects on prestalk and prespore cell-specific reporter gene expression. Phenytoin was inactive in this assay, which may point to limitations in metabolization of the compound in Dictyostelium required to exert developmental toxicity. D. discoideum cell culture is cheap and easy to handle compared to mammalian cell cultures or animal teratogenicity models. Although the Dictyostelium-based assay described in this report may not securely predict the teratogenic potential of these drugs in humans, this organism may be qualified for rapid large-scale screenings of synthetic compounds under development as new pharmaceuticals for their potential to interfere with developmental processes and thus help to reduce the amount of teratogenicity tests in animal models.
Assuntos
Dictyostelium/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Reprodução/efeitos dos fármacos , Teratogênicos/toxicidade , Animais , Meios de Cultura , Dictyostelium/química , Dictyostelium/fisiologia , Dietilestilbestrol/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Modelos Biológicos , Fenitoína/toxicidade , Talidomida/toxicidade , Tretinoína/toxicidadeRESUMO
The physiological effects of a drug depend not only on its molecular structure but also on the time-pattern of its administration. One of the main reasons for the importance of temporal patterns in drug action is biological rhythms--particularly those of circadian period. These rhythms affect most physiological functions as well as drug metabolism, clearance, and dynamic processes that may alter drug availability and target cell responsiveness with reference to biological time. We present an overview of the importance of time-patterned signals in physiology focused on the insights provided by a modeling approach. We first discuss examples of pulsatile intercellular communication by hormones such as gonadotropin-releasing hormone, and by cyclic adenosine monophosphate (cAMP) signals in Dictyostelium amoebae. Models based on reversible receptor desensitization account in both cases for the existence of optimal patterns of pulsatile signaling. Turning to circadian rhythms, we examine how models can be used to account for the response of 24h patterns to external stimuli such as light pulses or gene expression, and to predict how to restore the physiological characteristics of altered rhythms. Time-patterned treatments of cancer involve two distinct lines of research. The first, currently evaluated in clinical trials, relies on circadian chronomodulation of anticancer drugs, while the second, mostly based on theoretical studies, involves a resonance phenomenon with the cell-cycle length. We discuss the implications of modeling studies to improve the temporal patterning of drug administration.
Assuntos
Cronoterapia , Modelos Biológicos , Preparações Farmacêuticas/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Comunicação Celular , Ritmo Circadiano/fisiologia , Cianobactérias/fisiologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/fisiologia , Hormônios/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Transdução de SinaisRESUMO
Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by pisatin is probably not via an alteration of membrane sterols. The inducible pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation.
Assuntos
Antifúngicos/farmacologia , Benzopiranos/farmacologia , Dictyostelium/efeitos dos fármacos , Nistatina/farmacologia , Extratos Vegetais/farmacologia , Pterocarpanos , Animais , Cromatografia Gasosa , Dictyostelium/genética , Resistência a Medicamentos , Genes de Protozoários , Espectrometria de Massas , Mutagênese , Sesquiterpenos , Esteróis/biossíntese , Terpenos , FitoalexinasRESUMO
Organisms use different mechanisms to detect and repair different types of DNA damage, and different species vary in their sensitivity to DNA damaging agents. The cellular slime mold Dictyostelium discoideum has long been recognized for its unusual resistance to UV and ionizing radiation. We have recently cloned three nucleotide excision repair (NER) genes from Dictyostelium , the rep B, D and E genes (the homologs of the human xeroderma pigmentosum group B, D and E genes, respectively). Each of these genes has a unique pattern of expression during the multicellular development of this organism. We have now examined the response of these genes to DNA damage. The rep B and D DNA helicase genes are rapidly and transiently induced in a dose dependent manner following exposure to both UV-light and the widely used chemotherapeutic agent cisplatin. Interestingly, the rep E mRNA level is repressed by UV but not by cisplatin, implying unique signal transduction pathways for recognizing and repairing different types of damage. Cells from all stages of growth and development display the same pattern of NER gene expression following exposure to UV-light. These results suggest that the response to UV is independent of DNA replication, and that all the factors necessary for rapid transcription of these NER genes are either stable throughout development, or are continuously synthesized. It is significant that the up-regulation of the rep B and D genes in response to UV and chemical damage has not been observed to occur in cells from other species. We suggest that this rapid expression of NER genes is at least in part responsible for the unusual resistance of Dictyostelium to DNA damage.
Assuntos
Cisplatino/farmacologia , Reparo do DNA/genética , Dictyostelium/genética , Regulação Fúngica da Expressão Gênica , Raios Ultravioleta , Animais , Dano ao DNA , Dictyostelium/efeitos dos fármacos , Dictyostelium/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Mutação , Proteínas ras/genéticaRESUMO
Results from a number of laboratories suggest that intracellular Ca2+ is involved in the regulation of Dictyostelium discoideum growth and development. To learn more about the regulation and function of intracellular Ca2+ in this organism, we have cloned and sequenced cDNAs that encode a putative P-type Ca2+ ATPase designated patA. The deduced protein product of this gene (PAT1) has a calculated molecular mass of 120,718 daltons. It exhibits about 46% amino acid identity with Ca2+ ATPases of the plasma membrane Ca2+ ATPase family and lower identity with sarco(endo)plasmic reticulum Ca2+ ATPase family members and monovalent cation pumps. However, PAT1 lacks the highly conserved calmodulin-binding domain present in the C-terminal region of most plasma membrane Ca2+ ATPase-type enzymes. When Dictyostelium amoebae are adapted to grow in the presence of 80 mM CaCl2, both the patA message and protein product are up-regulated substantially. These cells also exhibit an increase in the rate and magnitude of intracellular P-type Ca2+ uptake activity. Immunofluorescence analysis indicates that PAT1 colocalizes with bound calmodulin to intracellular membranes, probably components of the contractile vacuole complex. The presence of PAT1 on the contractile vacuole suggests that in Dictyostelium this organelle might function in Ca2+ homeostasis as well as in water regulation.
Assuntos
ATPases Transportadoras de Cálcio/biossíntese , Cálcio/farmacologia , Dictyostelium/enzimologia , Regulação Enzimológica da Expressão Gênica , Aclimatação , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/química , Calmodulina/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar , Dictyostelium/efeitos dos fármacos , Dictyostelium/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Genes Fúngicos , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.
Assuntos
Equorina/genética , Apoproteínas/genética , Cálcio/metabolismo , AMP Cíclico/farmacologia , DNA Complementar/genética , Dictyostelium/metabolismo , Cifozoários/genética , Animais , Calcimicina/farmacologia , Cloreto de Cálcio/farmacologia , Diferenciação Celular , Citosol/metabolismo , Dictyostelium/efeitos dos fármacos , Luminescência , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Two pyrophosphate analogues, dichloromethane diphosphonate (Cl2MDP), and 1-hydroxyethane-1,1-diphosphonate (EHDP), at concentrations of 0.5-1 mM, efficiently inhibited the growth of amoebae of the slime mould Dictyostelium discoideum. Cell viability decreased markedly upon incubation with the diphosphonates. The mechanism of toxicity was investigated by in vivo 31P NMR spectroscopy and the formation of analogues of ATP [adenosine 5'-(beta, gamma-dichloromethane triphosphate) and adenosine 5'-(beta, gamma-1-hydroxyethane triphosphate)] was demonstrated. These two compounds were identified from their 31P NMR spectra in perchloric acid extracts prepared from amoebae poisoned with Cl2MDP or EHDP and may have been synthesized by reversible pyrophosphate exchange catalysed by cytosolic aminoacyl-tRNA synthetases.
Assuntos
Ácido Clodrônico/toxicidade , Dictyostelium/efeitos dos fármacos , Ácido Etidrônico/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Clodrônico/química , Dictyostelium/citologia , Dictyostelium/metabolismo , Ácido Etidrônico/química , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Nucleotídeos/biossíntese , Fósforo , FosforilaçãoRESUMO
Pretreatment of Dictyostelium discoideum amoebae with a sublethal concentration of the pea phytoalexin pisatin was shown to induce nondegradative resistance to subsequent challenges with inhibitory concentrations. An alteration of membrane sterol composition either with the azasterol A25822B or by mutations in nysC that confer resistance to the polyene antibiotic nystatin suppressed the induction of pisatin resistance. Wild-type cells grown on pisatin medium acquired resistance to nystatin; however, after transfer to nystatin medium, they lost their pisatin resistance phenotype but remained nystatin resistant. To account for this asymmetry in the induction and maintenance of cross-resistance after growth on pisatin and nystatin media, we propose a model in which the two resistance phenotypes are governed by distinct mechanisms. This model presumes that growth on pisatin induces membrane alterations that predispose cells to acquire nystatin resistance but that the pisatin-induced membrane alterations are not maintained in the absence of pisatin.
Assuntos
Benzopiranos/farmacologia , Dictyostelium/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pterocarpanos , Animais , Colestadienóis/farmacologia , Dictyostelium/genética , Dictyostelium/metabolismo , Resistência a Medicamentos/genética , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Mutação , Nistatina/farmacologia , Sesquiterpenos , Esteróis/metabolismo , Terpenos , FitoalexinasRESUMO
Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.