RESUMEN
High speed atomic force microscopy can provide the possibility of many new scientific observations and applications ranging from nano-manufacturing to the study of biological processes. However, the limited imaging speed has been an imperative drawback of the atomic force microscopes. One of the main reasons behind this limitation is the excitation of the AFM dynamics at high scan speeds, severely undermining the reliability of the acquired images. In this research, we propose a piezo based, feedforward controlled, counter actuation mechanism to compensate for the excited out-of-plane scanner dynamics. For this purpose, the AFM controller output is properly filtered via a linear compensator and then applied to a counter actuating piezo. An effective algorithm for estimating the compensator parameters is developed. The information required for compensator design is extracted from the cantilever deflection signal, hence eliminating the need for any additional sensors. The proposed approach is implemented and experimentally evaluated on the dynamic response of a custom made AFM. It is further assessed by comparing the imaging performance of the AFM with and without the application of the proposed technique and in comparison with the conventional counterbalancing methodology. The experimental results substantiate the effectiveness of the method in significantly improving the imaging performance of AFM at high scan speeds.
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Microscopía de Fuerza Atómica/instrumentación , Algoritmos , Diseño de Equipo , Factores de TiempoRESUMEN
Improving the imaging speed of atomic force microscopy (AFM) requires accurate nanopositioning at high speeds. However, high speed operation excites resonances in the AFM's mechanical scanner that can distort the image, and therefore typical users of commercial AFMs elect to operate microscopes at speeds below which scanner resonances are observed. Although traditional robust feedforward controllers and input shaping have proven effective at minimizing the influence of scanner distortions, the lack of direct measurement and use of model-based controllers have required disassembling the microscope to access lateral scanner motion with external sensors in order to perform a full system identification experiment, which places excessive demands on routine microscope operators. Further, since the lightly damped instrument dynamics often change from experiment to experiment, model-based controllers designed from offline system identification experiments must trade off high speed performance for robustness to modeling errors. This work represents a new way to automatically characterize the lateral scanner dynamics without addition of lateral sensors, and shape the commanded input signals in such a way that disturbing dynamics are not excited. Scanner coupling between the lateral and out-of-plane directions is exploited and used to build a minimal model of the scanner that is also sufficient to describe the nature of the distorting resonances. This model informs the design of an online input shaper used to suppress spectral components of the high speed command signals. The method presented is distinct from alternative approaches in that neither an information-complete system identification experiment nor microscope modification are required. Because the system identification is performed online immediately before imaging, no tradeoff of performance is required. This approach has enabled an increase in the scan rates of unmodified commercial AFMs from 1-4 lines s(-1) to over 40 lines s(-1).
RESUMEN
We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to micro ARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the micro ARCS format. IC(50) values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of micro ARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Inhibidores Enzimáticos/química , Fluorescencia , Histona Desacetilasas/metabolismo , Humanos , Células K562 , Miniaturización/métodosRESUMEN
A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.
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Potenciales de la Membrana , Canales de Potasio/efectos de los fármacos , Animales , Células Cultivadas , Colorantes , Cobayas , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Técnicas de Placa-ClampRESUMEN
Gel permeation methods have been commonly used to screen combinatorial libraries synthesized on a solid support. We report here three screens of combinatorial libraries using gel permeation assays. These include a simple enzymatic assay to identify inhibitors of the influenza enzyme neuraminidase, and two more complex assays designed to screen for inhibitors of the interleukin-8 (IL-8)-IL-8 receptor and the urokinase-urokinase receptor interactions, respectively. The IL-8 ligand-receptor assay makes use of IL-8 receptor-expressing cells attached to a membrane, thus enabling washing steps as part of the assay. The urokinase ligand-receptor assay employs an enzyme-linked immunosorbent assay-type format, previously thought to be amenable only to well-based assays. The results of these three screens are reported here, including the discovery of a novel series of acyclic inhibitors of neuraminidase. The development of complex assays in a gel permeation format allows for the routine screening of combinatorially as well as noncombinatorially made compound collections against virtually any kind of target, and is being widely used in our high throughput screening operations.
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Cromatografía en Gel/métodos , Técnicas Químicas Combinatorias , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Interleucina-8/metabolismo , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/enzimología , Unión Proteica , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells. After screening more than 60,000 compounds, A-105972 was identified and selected for further testing. A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells. The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type. The potency of A-105972 is similar in cells expressing wild-type or mutant p53. A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle. Using [3H]A-105972, we found that A-105972 bound to purified tubulin. Unlabeled A-105972 competed with [3H]A-105972 binding with an IC50 value of 3.6 microL. Colchicine partially inhibited [3H]A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect. Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies. DNA ladder studies also indicated that A-105972 induced apoptosis. A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation. A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388. These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells. A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.
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Antineoplásicos/farmacología , Oxadiazoles/farmacología , Animales , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Leucemia P388/tratamiento farmacológico , Leucemia P388/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Oxadiazoles/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Identification of individual cyclic AMP binding proteins in tumor cytosolic extracts is possibly owing to the ability of (32)P-labeled 8-azido cyclic AMP to act as an effective analog of cAMP, to bind specifically to its protein effector sites, and on photo activation to incorporate covalently to these sites by means of a highly reactive nitrene derivative. Resolution of labeled proteins can be achieved by SDS-PAGE and visualization by autoradiography (1).
RESUMEN
To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.
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Ensayo de Amplificación de Señal de ADN Ramificado/métodos , Evaluación Preclínica de Medicamentos/métodos , Interleucina-8/genética , ARN Mensajero/análisis , Juego de Reactivos para Diagnóstico , Recuento de Células , Línea Celular , Sondas de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Inmunoensayo , Interleucina-1/farmacología , Interleucina-8/análisis , Interleucina-8/biosíntesis , Mediciones Luminiscentes , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Increases in category access (CA) and items recalled per category (IPC) are associated with increases in relational and item-specific processing, respectively. However, it has also been shown that CA increases as recall level increases and that CA scores following relational processing are actually below CA scores for randomly recalled items. These results prompted M. D. Murphy (1979) to suggest that, after adjusting for recall-level differences, relational processing decreases CA scores. Results of Experiment 1, along with a reanalysis of previously published data, showed that relational processing produces lower CA scores than purely item-specific processing (or random recall), but an increase in relational processing produces an increase in CA scores even when the CA and IPC scores are adjusted for recall-level differences. These results suggest a curvilinear relationship between relational processing and CA scores.
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Recuerdo Mental , Vocabulario , HumanosRESUMEN
The repeated-testing paradigm is used to study both retroactive interference and hypermnesia (the improvement in memory across repeated tests). Considerable theoretical progress has been made by separately analyzing the 2 components of hypermnesia: the recovery of previously unrecalled items on later tests (item gains) and the forgetting of previously recalled items on later tests (item losses). Item gains increase with increases in item-specific processing, whereas item losses decrease with increases in relational processing. The authors suggest that separate analysis of item gains and losses in retroactive interference research may also prove fruitful. Three experiments showed that an interpolated list affects item gains but not losses, whereas processing similarity between the target and interpolated lists affects losses but not gains. These results are interpreted within the relational-item-specific processing framework.
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Amnesia/diagnóstico , Recuerdo Mental , Amnesia/complicaciones , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/diagnóstico , Humanos , Pruebas Neuropsicológicas , Distribución Aleatoria , Índice de Severidad de la Enfermedad , Factores de Tiempo , VocabularioRESUMEN
The intracellular mechanisms that regulate the function of human neutrophils are not well understood. Receptor-initiated signaling events result in the production of several second messengers (e.g., Ca2+, diacylglycerol, phosphatidic acid, and arachidonic acid) with the potential to activate members of the protein kinase C (PKC) family of signaling enzymes. The mixture of second messenger signaling molecules produced usually varies, depending on the particular receptor engaged. Previous work suggests that PKC has complex regulatory effects on neutrophil function. This may be due to the presence of multiple isoforms of the enzyme family, responding differentially to the second messengers produced. In studies to identify the PKC isoforms present in human neutrophils, we discovered the presence of the PKC isoform delta in these cells. Like other previously identified isoforms (alpha, beta I, beta II, and zeta), delta is a cytosolic enzyme in unstimulated neutrophils and partially translocates to membrane-containing fractions in cells stimulated by either the PKC activator PMA or the chemoattractant FMLP. Partial purification of cytosolic PKC gave two peaks of activity. The beta isoforms predominated in peak I, while the delta isoform predominated in peak II. The identification of delta indicates that neutrophils contain at least one member of the Ca(2+)-independent, diacylglycerol-dependent subfamily of PKC isoforms. Thus, this isoform may participate in Ca(2+)-independent, but diacylglycerol-dependent signaling events in these cells.
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Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Neutrófilos/enzimología , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/metabolismo , Transporte Biológico , Calcio/fisiología , Diglicéridos/fisiología , Humanos , Isoenzimas/sangre , Proteína Quinasa C/sangre , Proteína Quinasa C-delta , Transducción de Señal/fisiologíaRESUMEN
To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.
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Regulación Enzimológica de la Expresión Génica , Isoenzimas/metabolismo , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Bases , Sangre , Colagenasas/genética , Humanos , Datos de Secuencia Molecular , Activación TranscripcionalRESUMEN
BACKGROUND: 8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is undergoing phase I clinical trials as an anticancer drug. However, there is debate as to whether it is a prodrug for its 8-Cl-adenosine metabolite. DESIGN: Pharmacokinetics, metabolism and tumour disposition studies have been performed in 7 breast cancer patients receiving continuous infusion (28 day) 8-Cl-cAMP (0.54 or 1.08 mg/kg/day) and tumour biopsies were obtained before and on the last day of infusion. Parallel studies were performed in nude mice bearing the HT29 human colon cancer xenograft after continuous infusion (7 day) of active drug doses (50 or 100 mg/kg/day). RESULTS: Steady state plasma levels (Css) of 8-Cl-cAMP in patients ranged from 0.15-0.72 microM but 8-Cl-adenosine was not detected in plasma. In contrast, 8-Cl-cAMP was not detectable in 3 tumour biopsies but 8-Cl-adenosine was present in 2 samples at high concentrations (1.33 and 2.02 microM). In mice, Css of 8-Cl-cAMP ranged from 3.2-4.6 microM and 8-Cl adenosine was present in plasma only at the higher dose (100 mg/kg/day, peak concentration of 2.3 microM). In the HT29 xenograft, 8-Cl-cAMP levels were considerably lower than in plasma (0.37-1.22 microM) while 8-Cl-adenosine was present at 5.3-21.0 microM and 8-Cl-AMP was found at 11.3-35.7 microM. CONCLUSIONS: The fate of 8-Cl-cAMP in human tumours is characterised by extensive metabolism to products which are not generally observed in plasma. These data raise the possibility that 8-Cl-cAMP is a prodrug for a product of its metabolism in human tumours.
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8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/farmacocinética , Neoplasias de la Mama/metabolismo , Neoplasias del Colon/metabolismo , Profármacos/farmacocinética , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacocinética , Animales , Antineoplásicos/metabolismo , Neoplasias del Colon/patología , Humanos , Infusiones Intravenosas , Ratones , Ratones Desnudos , Profármacos/metabolismo , Distribución Tisular , Trasplante HeterólogoRESUMEN
Relatively little is known about the substrate specificity of individual protein kinase C (PKC) isozymes, particularly with respect to physiologically relevant substrates. One class of prominent cellular substrates for PKC is represented by the myristoylated alanine-rich C kinase substrate, or MARCKS, protein. In the present study, we have used a baculovirus expression system to coexpress human MARCKS with eight different isozymes of PKC, to determine which isozymes are capable of phosphorylating MARCKS in intact cells. In Sf9 cells, coexpression of MARCKS with individual PKC isozymes led to the following increases in MARCKS phosphorylation: alpha, 3.6-fold; beta iota, 4.6-fold; beta mu, 2.7-fold; gamma, 4.8-fold; delta, 3.0-fold; epsilon, 4.3-fold; and eta, 4.9-fold. In most cases, stimulation of cells with a phorbol ester led to a slight increase (20-30%) in MARCKS phosphorylation. PKC zeta did not phosphorylate MARCKS to any appreciable extent above control. In addition, in vitro kinetic analysis of PKC zeta showed that it has a 1000-fold lower affinity for a synthetic peptide comprising the MARCKS phosphorylation site domain compared to mixed conventional PKC isozymes from rat brain. These data indicate that MARCKS is a substrate in intact cells for at least seven isozymes of PKC: alpha; beta iota; beta mu; gamma; delta; epsilon; and eta. The isozyme PKC zeta does not appear to phosphorylate MARCKS in vivo or with significant affinity in vitro. Thus, PKC zeta, which is not activated by phorbol esters or diacylglycerol, also appears to behave differently with respect to this class of important cellular PKC substrates.
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Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/metabolismo , Proteínas de la Membrana , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Animales , Baculoviridae/genética , Secuencia de Bases , Western Blotting , Células Cultivadas , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Pruebas de Precipitina , Spodoptera , TransfecciónRESUMEN
The item-order distinction has been useful in explaining memory dissociations in short-term retention tasks. It generally has been assumed that serial order information is beneficial to long-term retention as well, although the distinction has received little empirical attention. Recently, it was shown that generating items at input, rather than simply reading them, hinders processing of serial order information. This reduction in order processing has been implicated in the lack of generation effects in between-list designs. Experiment 1, using typical generation effect procedures, showed that generation inhibited order reconstruction performance. Experiment 2 showed that order reconstruction was hindered even when categorically related lists were used. Experiment 3 demonstrated that generation inhibited order reconstruction in an incidental learning procedure. The results suggest that order processing is relatively automatic and that generation constantly inhibits it. The results support the view that the item-order distinction may be a powerful explanatory tool in long-term memory research.
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Atención , Retención en Psicología , Aprendizaje Seriado , Adulto , Femenino , Humanos , Masculino , Memoria a Corto Plazo , Motivación , Valores de ReferenciaRESUMEN
The regulatory subunits of protein kinase A, or cyclic AMP-binding proteins, were measured in a series of 107 human ovarian tumors (89 malignant, 7 borderline, and 11 benign tumors) and related to tumor clinicopathological features and patient survival. Total cyclic AMP-binding protein levels were not significantly different between malignant tumors and either borderline or benign tumors. However, serous tumors showed significantly higher levels of total cyclic AMP-binding proteins than other malignant tumors (P = 0.007). Poorly differentiated tumors also possessed significantly higher levels of binding proteins as compared with well/moderately differentiated tumors (P < 0.01). Retrospective analysis of follow-up data also revealed a significant trend for patients with high tumor cyclic AMP-binding proteins to have poorer survival (P = 0.03). Individual binding proteins were identified by photoaffinity labeling, and the RI (Mr 48,000) protein was expressed as a percentage of total cyclic AMP-binding proteins detected. The percentage of the RI protein was not significantly different among malignant, borderline, or benign pathologies and was not associated with tumor stage, differentiation, or debulk status. The percentage of RI was significantly increased in serous tumors compared to other common epithelial malignancies (P = 0.01). In malignant tumors there was a significant positive correlation between the percentage of the RI protein and total cyclic AMP-binding proteins (P = 0.01). These data indicate that high tumor levels of cyclic AMP-binding proteins are associated with serous histology, poor differentiation, and poor patient survival.
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Proteína Receptora de AMP Cíclico/análisis , Proteínas de Neoplasias/análisis , Neoplasias Ováricas/química , Proteínas Portadoras , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Humanos , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Tasa de SupervivenciaRESUMEN
The bizarre imagery effect, better memory for bizarre stimuli than for common stimuli, is now an established finding. However, the mnemonic benefits of bizarre imagery are subject to several constraints (e.g., the use of mixed lists and free-recall tests). A further constraint on the bizarreness effect is demonstrated here. In each of two experiments, subjects were given either incidental or intentional study instructions and were asked to rate the vividness of the images they formed from the bizarre and common sentences. Contrary to conclusions based on available evidence, the bizarreness effect in free recall was manifested only with the incidental learning instructions. This additional constraint on the effect is consistent with the item-order account of bizarreness.
RESUMEN
Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.
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Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Antineoplásicos/farmacología , Baculoviridae , Secuencia de Bases , Sitios de Unión , Brioestatinas , División Celular/efectos de los fármacos , Línea Celular , Cartilla de ADN , Humanos , Lactonas/farmacología , Lamina Tipo B , Laminas , Leucemia Eritroblástica Aguda , Macrólidos , Mitógenos/farmacología , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
In a variety of intact cells, phorbol esters are known to activate phospholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was 2-3-fold more effective than ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol deplete of protein kinase C by chromatography on threnoine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane plasma membrane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta 1 > alpha > gamma; the beta 2, delta, epsilon, eta, and sigma isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.