RESUMEN
Propolis is one of the most widely used products in traditional medicine. One of the most prominent types of Brazilian propolis is the red one, whose primary botanical source is Dalbergia ecastaphyllum (L.) Taub. Despite the potential of Brazilian red propolis for developing new products with pharmacological activity, few studies guarantee safety in its use. The objective of this study was the evaluation of the possible toxic effects of Brazilian red propolis and D. ecastaphyllum, as well as the cytotoxicity assessment of the main compounds of red propolis on tumoral cell lines. Hydroalcoholic extracts of the Brazilian red propolis (BRPE) and D. ecastaphyllum stems (DSE) and leaves (DLE) were prepared and chromatographed for isolation of the major compounds. RP-HPLC-DAD was used to quantify the major compounds in the obtained extracts. The XTT assay was used to evaluate the cytotoxic activity of the extracts in the human fibroblast cell line (GM07492A). The results revealed IC50 values of 102.7, 143.4, and 253.1 µg/mL for BRPE, DSE, and DLE, respectively. The extracts were also evaluated for their genotoxic potential in the micronucleus assay in Chinese hamster lung fibroblasts cells (V79), showing the absence of genotoxicity. The BRPE was investigated for its potential in vivo toxicity in the zebrafish model. Concentrations of 0.8-6.3 mg/L were safe for the animals, with a LC50 of 9.37 mg/L. Of the 11 compounds isolated from BRPE, medicarpin showed a selective cytotoxic effect against the HeLa cell line. These are the initial steps to determine the toxicological potential of Brazilian red propolis.
Asunto(s)
Dalbergia/química , Extractos Vegetales/farmacología , Própolis/farmacología , Animales , Brasil , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Própolis/química , Própolis/aislamiento & purificación , Pez CebraRESUMEN
Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de la Membrana/química , Conformación Proteica , Proteínas Quinasas/química , Transducción de Señal , Thermotoga maritima/enzimología , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Dimerización , Histidina Quinasa , Hidrólisis , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425-432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Deltanik-1 mutant, the Deltacos1/Deltacos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Deltanik-1 and Deltacos1/Deltacos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.
Asunto(s)
Candida albicans/fisiología , Genes Fúngicos/fisiología , Proteínas Quinasas/fisiología , Secuencia de Aminoácidos , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Histidina Quinasa , Datos de Secuencia Molecular , Mutación , Alineación de SecuenciaRESUMEN
Two-component signal transduction systems are most often found in prokaryotic organisms where they are responsible for mediating the cellular responses to many environmental stimuli. These systems are composed of an autophosphorylating histidine kinase and a response regulator. We have found evidence for the existence of two-component histidine kinases in the eukaryotic filamentous fungus Neurospora crassa based on screening with degenerate primers to conserved regions of these signaling proteins. Subsequent cloning and sequencing of one member of this newly discovered group, nik-1+, shows that the predicted protein sequence shares homology with both the kinase and response regulator modules of two-component signaling proteins. In addition, the N-terminal region of the protein has a novel repeating 90-amino acid motif. Deletion of the nik-1+ gene in N. crassa results in an organism that displays aberrant hyphal structure, which is enhanced under conditions of high osmostress. Increased osmotic pressure during growth on solid medium leads to restricted colonial growth, loss of aerial hyphae formation, and no subsequent conidiophore development. This finding may have implications for mechanisms of fungal colonization and pathogenicity.
Asunto(s)
Neurospora crassa/enzimología , Neurospora crassa/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN/genética , ADN de Hongos/genética , Proteínas Fúngicas/genética , Histidina Quinasa , Datos de Secuencia Molecular , Mutagénesis , Neurospora crassa/genética , Proteínas Quinasas/genética , Transducción de Señal , Equilibrio Hidroelectrolítico/genéticaRESUMEN
Autophosphorylating histidine kinase and response-regulator domains constitute the building blocks of two-component signaling systems. These systems use a unique phosphotransfer chemistry to regulate many aspects of bacterial physiology. Homologous systems are now being found in eukaryotes. Despite their common mechanism of phosphotransfer, the two-component systems display an extensive diversity in the arrangement of their domains, and flexibility in their roles in different signal transduction circuits.
Asunto(s)
Ácido Aspártico/metabolismo , Histidina/metabolismo , Animales , Fosforilación , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiologíaRESUMEN
During the past several years, it has become apparent that prokaryotic organisms process much of their sensory information through families of similar proteins that make up two-component signal transducing systems. Biochemical and genetic analyses have clarified our understanding of how these proteins mediate the signaling circuitry necessary to allow an appropriate response. Recent advances in this field suggest that 'primitive' signaling systems may involve molecular strategies and mechanisms that are conserved in more complex organisms.
Asunto(s)
Proteínas Bacterianas/fisiología , Células Eucariotas/enzimología , Proteínas Fúngicas/fisiología , Células Procariotas/enzimología , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional , Transducción de Señal/fisiología , Quimiotaxis , Histidina Quinasa , Modelos Biológicos , Fosforilación , Conformación Proteica , Especificidad por SustratoRESUMEN
We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis/fisiología , Proteínas de Escherichia coli , Escherichia coli/fisiología , Receptores de Superficie Celular , Transducción de Señal , Técnicas Biosensibles , Células Quimiorreceptoras , Enzimas Inmovilizadas , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina Quinasa , Cinética , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Mutación , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
The Tar receptor is a transmembrane protein that regulates bacterial chemotaxis in response to changes in the level of aspartic acid in the medium. The extracellular portion of the protein can bind aspartate, and the cytoplasmic portion modulates CheA kinase activity. The receptor can either activate or inhibit the kinase. The cytoplasmic portion of the receptor can be modified by carboxymethylation of specific glutamic acid residues. To test the effects of differential methylation on receptor function, we prepared membranes from cells that have specifically modified forms of the receptor and tested the relative ability of each of these forms to activate or inhibit CheA kinase. Completely demethylated receptor was a potent inhibitor and poor activator of the kinase, while the fully modified receptor was an excellent activator but an inefficient inhibitor. Partially modified receptor could act both as an effective inhibitor and as an activator. Reversible modification provides a mechanism that allows the cell to accumulate a population of receptor molecules capable of generating a wide range of signaling intensities.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular , Secuencia de Aminoácidos , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Proteínas Bacterianas/genética , Sitios de Unión , Células Quimiorreceptoras , Factores Quimiotácticos/metabolismo , Quimiotaxis/efectos de los fármacos , Escherichia coli/genética , Histidina Quinasa , Cinética , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
The genes frhA (1217 bp), frhB (845 bp), and frhG (710 bp) encoding the three known subunits, alpha, beta, and gamma, of the 8-hydroxy-5-deazaflavin (F420) reducing hydrogenase (FRH) from the thermophilic methanogen Methanobacterium thermoautotrophicum delta H have been cloned, sequenced, and shown to be tightly linked, indicative of a single transcriptional unit. The DNA sequence contains a fourth open reading frame, designated frhD (476 bp), encoding a polypeptide (delta) that does not copurify with the active enzyme. Expression of the frh gene cluster in Escherichia coli shows that four polypeptides are synthesized. When analyzed by SDS-PAGE, the proteins migrate with mobilities consistent with their calculated molecular weights. In order to understand the mechanism of H2 oxidation by this enzyme, localization of redox cofactors (Ni, Fe/S, FAD) to specific subunits and information on their structure is needed. This has been hindered due to the refractory nature of the enzyme to denaturation methods needed in order to obtain individual subunits with cofactors intact. In this paper we discuss the possible localization of the redox cofactors as implicated from the DNA-derived protein sequences of the subunits. The amino acid sequences of the subunits of the FRH are compared with those of other Ni-containing hydrogenases, including the methyl viologen reducing hydrogenase (MVH) of M. thermoautotrophicum delta H.