RESUMEN
BACKGROUND: Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). METHODS: PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. RESULTS: In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. CONCLUSION: PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.
Asunto(s)
Células Madre Embrionarias/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Fibroblastos/fisiología , Osteogénesis/fisiología , Ligamento Periodontal/citología , Fosfatasa Alcalina/análisis , Animales , Antracenos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/análisis , Medios de Cultivo Condicionados , Factor 4 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 7 de Crecimiento de Fibroblastos/análisis , Factores de Crecimiento de Fibroblastos/análisis , Flavonoides/farmacología , Encía/citología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteínas Quinasas Activadas por Mitógenos/análisis , Osteocalcina/análisis , Osteopontina/análisis , Piel/citologíaRESUMEN
BACKGROUND: PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are thought to be the central transducers of oncogenic signals in solid malignancies, and there has been a lot of enthusiasm for developing inhibitors of these pathways for cancer therapy. Some preclinical models have suggested that combining inhibitors of both parallel pathways may be more efficacious, but it remains unknown whether dual inhibition with high enough concentrations of the drugs to achieve meaningful target inhibition is tolerable in a clinical setting. Furthermore, the predictive factors for dual inhibition are unknown. METHODS: Non-small cell lung cancer (NSCLC) cell lines (n=12) with the most frequent oncogenic backgrounds (K-Ras mut n=3, EGFR mut n=3, ALK translocated n=3, and triple-negative n=3) were exposed to PI3K inhibitors (ZSTK474, PI-103) or MEK inhibitor (CI-1040) alone or in combination and analysed with an MTS growth/cytotoxicity assay and statistically by combination index analysis. The activity of the intracellular signaling pathways in response to the inhibitor treatments was analysed with a western blot using phospho-specific antibodies to AKT, ERK1/2, S6, and 4E-BPI. For the differential dosing schedule experiments, additional breast and colon cancer cell lines known to be sensitive to dual inhibition were included. RESULTS: Two of the 12 NSCLC cell lines tested, H3122 (ALK translocated) and H1437 (triple-negative), showed increased cytotoxicity upon dual MEK and PI3K inhibition. Furthermore, MDA-MB231 (breast) and HCT116 (colon), showed increased cytotoxicity upon dual inhibition, as in previous studies. Activation of parallel pathways in the dual inhibition-sensitive lines was also noted in response to single inhibitor treatment. Otherwise, no significant differences in downstream intracellular pathway activity (S6 and 4E-BPI) were noted between PI3K alone and dual inhibition other than the increased cytotoxicity of the latter. In the alternative dosing schedules two out of the four dual inhibition-sensitive cell lines showed similar cytotoxicity to continuous PI3K and short (15min) MEK inhibition treatment. CONCLUSIONS: Therapy with a dual PI3K and MEK inhibitor combination is more efficient than either inhibitor alone in some NSCLC cell lines. Responses to dual inhibition were not associated with any specific oncogenic genotype and no other predictive factors for dual inhibition were noted. The maximal effect of the dual PI3K and MEK inhibition can be achieved with alternative dosing schedules which are potentially more tolerable clinically.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Comunicación Celular/fisiología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Quimioterapia Combinada , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/fisiologíaRESUMEN
BACKGROUND: Typha angustata is a traditional Chinese medicine, commonly used for a variety of clinical disorders, including atherosclerosis and wound healing. In the present study, the protective effects of T. angustata pollen extract (TE) on the response of osteoblast to oxidative stress were evaluated. MATERIALS AND METHODS: Osteoblastic MC3T3-E1 cells were incubated with H(2)O(2) and/or TE, and markers of osteoblast function and oxidative damage were examined. RESULTS: TE treatment significantly (P < 0.05) reversed the cytotoxic effect of H(2)O(2) and this effect was blocked by ICI182780, suggesting that TE's effect might be partly involved in estrogen action. TE significantly (P < 0.05) increased collagen content, alkaline phosphatase activity, calcium deposition of osteoblasts in the presence of H(2)O(2) and these effects were blocked by rottlerin and PD98059, suggesting that the induction of differentiation by TE is associated with increased activation of protein kinase C and ERK. Moreover, H(2)O(2)-induced reduction of osteocalcin was significantly recovered in the presence of TE. Pretreatment with TE also decreased the increase in receptor activator of nuclear factor-kB ligand, malondialdehyde, and protein carbonyl induced by H(2)O(2). CONCLUSION: These results suggest that the pollen of T. angustata may be useful for the protection of H(2)O(2)-induced oxidative damage and dysfunction in osteoblasts.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Osteoblastos/efectos de los fármacos , Oxidantes/toxicidad , Extractos Vegetales/farmacología , Polen , Typhaceae , Células 3T3 , Acetofenonas/farmacología , Fosfatasa Alcalina/efectos de los fármacos , Animales , Benzopiranos/farmacología , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Colágeno/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Flavonoides/farmacología , Fulvestrant , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/análisis , Ratones , Osteocalcina/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/antagonistas & inhibidores , Carbonilación Proteica/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Ligando RANK/efectos de los fármacosRESUMEN
Microvascular dysfunction occurs in insulin resistance and/or hyperinsulinaemia. Enhanced uptake of free fatty acids (FFA) and oxidised low-density lipoproteins (oxLDL) may lead to oxidative stress and microvascular dysfunction interacting with CD36, a PPARα/γ-regulated scavenger receptor and long-chain FFA transporter. We investigated CD36 expression and CD36-mediated oxLDL uptake before and after insulin treatment in human dermal microvascular endothelial cells (HMVECs), ± different types of fatty acids (FA), including palmitic, oleic, linoleic, arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. Insulin (10(-8) and 10(-7) M) time-dependently increased DiI-oxLDL uptake and CD36 surface expression (by 30 ± 13%, p<0.05 vs. untreated control after 24 hours incubation), as assessed by ELISA and flow cytometry, an effect that was potentiated by the PI3-kinase inhibitor wortmannin and reverted by the ERK1/2 inhibitor PD98059 and the PPARα/γ antagonist GW9662. A ≥ 24 hour exposure to 50 µM DHA or EPA, but not other FA, blunted both the constitutive (by 23 ± 3% and 29 ± 2%, respectively, p<0.05 for both) and insulin-induced CD36 expressions (by 45 ± 27 % and 12 ± 3 %, respectively, p<0.05 for both), along with insulin-induced uptake of DiI-oxLDL and the downregulation of phosphorylated endothelial nitric oxide synthase (P-eNOS). At gel shift assays, DHA reverted insulin-induced basal and oxLDL-stimulated transactivation of PPRE and DNA binding of PPARα/γ and NF-κB. In conclusion, omega-3 fatty acids blunt the increased CD36 expression and activity promoted by high concentrations of insulin. Such mechanisms may be the basis for the use of omega-3 fatty acids in diabetic microvasculopathy.
Asunto(s)
Antígenos CD36/metabolismo , Células Endoteliales/metabolismo , Ácidos Grasos Omega-3/metabolismo , Regulación de la Expresión Génica , Receptores Depuradores/metabolismo , Androstadienos/farmacología , Anilidas/farmacología , Antígenos CD36/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Dermis/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/farmacología , Resistencia a la Insulina/fisiología , Estrés Oxidativo , PPAR alfa/antagonistas & inhibidores , PPAR gamma/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores Depuradores/genética , WortmaninaRESUMEN
Perception of red light (400 µmol photon m²/s) by the shoot bottom turned off the greening process in wheat. To understand the signaling cascade leading to this photomorphogenic response, certain signaling components were probed in seedlings grown in different light regimes. Upon analysis the gene expression of heterotrimeric Gα and Gß were severely down-regulated in seedlings grown without vermiculite and having their shoot bottom exposed to red light (R/V-) and was similar to that of dark-grown seedlings. Supplementing the red-light-grown V- seedlings with blue light resulted in up-regulation of both Gα and Gß expression, suggesting that blue light is able to modulate G protein expression. Treatment of cytokinin analog benzyladenine to cytokinin-deficient red-light-grown R/V- seedlings resulted in up-regulation of gene expression of both Gα and Gß. To probe further, modulators of signal transduction pathway--AlF3 (G protein activator), LaCl3 (Ca(2+) channel blocker), NaF (nonspecific phosphatase inhibitor), or calmodulin (CaM) antagonists trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide (W-7)--were added along with Hoagland solution to the roots of 4-day-old etiolated seedlings, grown on germination paper and transferred to red light. AlF3, LaCl3, NaF failed to elicit any photomorphogenic response. However, CaM antagonists TFP and W-7 significantly reversed the red-light-induced suppression of photomorphogenesis. Phosphorylation of proteins assayed in the absence or presence of CaM antagonist TFP revealed respective up-regulation or down-regulation of phosphorylation of several plastidic proteins in R/V- seedlings. These suggest that signal transduction of red light perceived by the shoot bottom to suppress photomorphogenesis is mediated by CaM-dependent protein kinases.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Luz , Proteínas de Plantas/metabolismo , Transducción de Señal , Triticum/efectos de la radiación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Citocininas/antagonistas & inhibidores , Regulación de la Expresión Génica de las Plantas , Proteínas de Unión al GTP Heterotriméricas/genética , Fosforilación , Proteínas de Plantas/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/efectos de la radiación , Sulfonamidas/farmacología , Trifluoperazina/farmacología , Triticum/genética , Triticum/metabolismoRESUMEN
Death-associated protein kinase (DAPK) is a serine/threonine kinase that participates in the modulation of apoptosis and tumor suppression. Our previous study revealed high levels of DAPK protein expression in differentiated endometrial adenocarcinoma cells. To clarify the role of DAPK in human endometrial adenocarcinomas, we down-regulated endogenous DAPK expression in HHUA cells, a well-differentiated endometrial adenocarcinoma cell line, using specific small-interfering RNAs (siRNAs). The suppression of endogenous DAPK expression triggered apoptosis in HHUA cells, as evidenced by an increase in the sub-G1 DNA content in flow cytometric analyses. The apoptosis induced by the DAPK siRNA transfections was caspase-dependent, as characterized by the activations of caspase-3, -8 and -9. RNase protection assays detected higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 transcripts in the DAPK siRNA-transfected HHUA cells than in the control siRNA-transfected cells. Consistent with these findings, enzyme-linked immunosorbent assays revealed that the DAPK siRNA transfections significantly increased the secretion of TRAIL protein from the cells. Treatment with recombinant human TRAIL protein dose-dependently suppressed the cell viability of HHUA cells. The present findings reveal that down-regulation of endogenous DAPK expression in HHUA cells induces caspase-dependent apoptosis, possibly through increased TRAIL, DR4 and DR5 signaling, thereby suggesting that DAPK expression is essential for HHUA cell survival. Consequently, endogenous DAPK mRNA may represent a potential candidate for molecularly targeted anticancer therapies.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Carcinoma Endometrioide/patología , Neoplasias Endometriales/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Carcinoma Endometrioide/tratamiento farmacológico , Carcinoma Endometrioide/genética , Proteínas Quinasas Asociadas a Muerte Celular , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Marcación de Gen , Humanos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Transfección , Células Tumorales CultivadasRESUMEN
Death-associated protein kinase (DAPK) is a serine/threonine protein kinase implicated in diverse programmed cell death pathways. DAPK is a promising target protein for the treatment of ischemic diseases. We identified novel potent and selective DAPK inhibitors efficiently by structure-based virtual screening, then further developed the hit compounds. In this paper, we describe the development of the hit compounds and the structure-activity relationship studies of the DAPK inhibitors in detail, including calculation of the solvated interaction energy (SIE), and verification of selectivity using a kinase panel.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Algoritmos , Biología Computacional , Simulación por Computador , Proteínas Quinasas Asociadas a Muerte Celular , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Death-associated protein kinases (DAPKs) function in the early stages of eukaryotic programmed cell death. DAPKs are now emerging as targets for drug discovery in novel therapeutic approaches for ischemic diseases in the brain, heart, kidney, and other organs. Using a structure-based virtual screening approach, we discovered potent and selective DAPKs inhibitors. 6 was found to be the most potent inhibitor with enzyme selectivity (IC(50) = 69 nM for DAPK1).
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Interfaz Usuario-Computador , Proteínas Reguladoras de la Apoptosis/química , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Cristalografía por Rayos X , Proteínas Quinasas Asociadas a Muerte Celular , Evaluación Preclínica de Medicamentos , Humanos , Cinética , Ligandos , Modelos Moleculares , Conformación ProteicaRESUMEN
Rutaecarpine is a quinazolinocarboline alkaloid isolated from a traditional Chinese medicinal fruit, Evodia rutaecarpa. In the present study, we investigated the effect of rutaecarpine on CYP1A1 expression mediated by [Ca(2+)] and the AhR pathway in mouse hepatoma Hepa-1c1c7 cells. Rutaecarpine also significantly increased CYP1A1 enzyme activity and mRNA and protein levels. Rutaecarpine markedly induced XRE and AhR binding activity. CH-223191, an AhR antagonist, blocked the rutaecarpine-induced CYP1A1 enzyme activity and mRNA and protein expression. In addition, rutaecarpine remarkably induced the phosphorylation of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK). W7 and BAPTA/AM, a CaM antagonist and an intracellular Ca(2+) chelator, respectively, blocked the rutaecarpine-induced CYP1A1 enzyme activity and mRNA and protein expression. These results indicate that rutaecarpine induces CYP1A1 expression through AhR- and calcium-dependent mechanisms.
Asunto(s)
Calcio/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Medicamentos Herbarios Chinos/farmacología , Alcaloides Indólicos/farmacología , Hígado/efectos de los fármacos , Quinazolinas/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Animales , Compuestos Azo/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular Tumoral , Quelantes/farmacología , Citocromo P-450 CYP1A1/genética , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inducción Enzimática , Hígado/enzimología , Ratones , Fosforilación , Regiones Promotoras Genéticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/metabolismo , Sulfonamidas/farmacologíaRESUMEN
OBJECTIVES: To define the role of protein kinase C delta (PKC delta) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model. METHODS: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC delta-specific inhibitor or (2) PKC delta-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured. RESULTS: The PKC delta inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC delta(-/-) and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor kappaB activation in both PKC delta(-/-) and wild type mice, although it was up to 50% less in PKC delta(-/-). CONCLUSIONS: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC delta-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.
Asunto(s)
Colecistoquinina/farmacología , Páncreas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteína Quinasa C-delta/metabolismo , Acetofenonas/farmacología , Amilasas/metabolismo , Animales , Benzopiranos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Immunoblotting , Indoles/farmacología , Masculino , Maleimidas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Páncreas/citología , Páncreas/metabolismo , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Ratas , Ratas Sprague-Dawley , Tripsinógeno/metabolismoRESUMEN
Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.
Asunto(s)
Antioxidantes/farmacología , Catequina/análogos & derivados , Monocitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Camellia sinensis/inmunología , Catequina/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Flavonoides/farmacología , Humanos , Inflamación , Inyecciones Intraperitoneales , Leucemia Basofílica Aguda/sangre , Leucemia Basofílica Aguda/tratamiento farmacológico , Leucemia Basofílica Aguda/inmunología , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , N-Formilmetionina Leucil-Fenilalanina/inmunología , Ratas , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/inmunología , Receptores de Formil Péptido/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Transfección , TransgenesRESUMEN
The objective of this study was to determine the ability of multiple-factor supplementation to augment derivation of mouse embryonic stem (mES) cells. Three factors, leukemia inhibitory factor (LIF), Parke-Davis 98059 (PD98059), and 6-bromoindirubin-3'-oxime (BIO), were added as supplements (individually or in a combination of all three) at two consecutive stages of culture; that is, from the start of blastocyst culture to the outgrowth stage, and from putting disaggregated outgrowth into culture medium to generation of primary mES colonies, respectively. The main outcome measure was the percentage of derivable mES cell lines, based on the number of blastocysts initially cultured. Three experiments demonstrated the following: (1) For the addition of individual single factor, only LIF yielded mES cell lines (6.2%), whereas a combination of all three factors resulted in the greatest number of mES cell lines (31.3%). (2) The advantages of a combination of multiple factors (LIF+PD98059+BIO) were manifested only when they were used during the first stage of the culture and not during the second stage (31.6% vs. 6.2%, respectively). (3) The quality of the inner cell mass (ICM) outgrowth obtained from first-stage culture was studied. After alkaline phosphatase and Oct-4 staining, which documented pluripotency of the embryonic stem cells, outgrowths cultured in multiple factors (LIF+PD98059+BIO) stained much stronger and in higher proportions than did those obtained after supplementation only with LIF (80% vs. 30%, respectively).
Asunto(s)
Medios de Cultivo , Células Madre Embrionarias/citología , Animales , Blastocisto/citología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/administración & dosificación , Femenino , Fibroblastos , Flavonoides/administración & dosificación , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inmunohistoquímica , Indoles/administración & dosificación , Factor Inhibidor de Leucemia/administración & dosificación , Masculino , Ratones , Ratones Endogámicos ICR , Oximas/administración & dosificación , EmbarazoRESUMEN
Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. The aim of this study was to investigate the effect of pifithrin-alpha (PFTalpha; a specific p53 inhibitor) and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on apoptotic signaling transduction mechanism induced by Cin in human hepatoma PLC/PRF/5 (CD95-negative) cells. Using XTT assay, Cin exhibited a powerful cytotoxic effect and apoptotic induction in PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 microM Cin as characterized by morphological changes and the appearance of phosphatidylserine on the outer surface of the plasma membrane. Cin down-regulated the expression of Bcl-(XL), up-regulated mutant p53 and Bax proteins and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. This could be supported by the activation and phosphorylation of MAPKs, including JNK, ERK and p38 kinases. Pre-incubation with PFTalpha and specific MAPK inhibitors significantly diminished the effect of Cin-induced apoptosis. The activities of anti-apoptotic (Bcl-(XL)) and pro-apoptotic (Bax) proteins were remarkably affected by PFTalpha and PD98059 pre-treatment. PFTalpha effectively blocked PARP cleavage in cells treated with Cin, and also markedly prevented the phosphorylation of JNK, p38 and ERK proteins. These results suggest that p53 induction and MAPK signaling pathways are required for Cin-mediated apoptosis in PLC/PRF/5 cells.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Cinnamomum aromaticum , Fitoterapia , Extractos Vegetales/farmacología , Antracenos/farmacología , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/uso terapéutico , Benzotiazoles/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular Tumoral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Tolueno/análogos & derivados , Tolueno/farmacologíaRESUMEN
Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).
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Señalización del Calcio , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Oocitos/metabolismo , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Gránulos Citoplasmáticos/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis , Femenino , Fertilización In Vitro , Masculino , Ratones , Microinyecciones , Oocitos/efectos de los fármacos , Oocitos/enzimología , Partenogénesis , Péptidos/farmacología , ARN Complementario/metabolismo , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Superovulación , Zona Pelúcida/metabolismo , Cigoto/metabolismoRESUMEN
We recently reported that ANG II inhibits NBCe1 current and surface expression in Xenopus laevis oocytes (Perry C, Blaine J, Le H, and Grichtchenko II. Am J Physiol Renal Physiol 290: F417-F427, 2006). Here, we investigated mechanisms of ANG II-induced changes in NBCe1 surface expression. We showed that the PKC inhibitor GF109203X blocks and EGTA reduces surface cotransporter loss in ANG II-treated oocytes, suggesting roles for PKC and Ca(2+). Using the endosomal marker FM 4-64 and enhanced green fluorescent protein (EGFP)-tagged NBCe1, we showed that ANG II stimulates endocytosis of NBCe1. To eliminate the possibility that ANG II inhibits NBCe1 recycling, we demonstrated that the recycling inhibitor monensin decreases surface expression, accumulates NBCe1-EGFP in endosomes, and inhibits NBCe1 current. Monensin and ANG II applied together produce greater inhibition of NBCe1 current than either did alone. This additive effect of monensin and ANG II suggests that ANG II stimulates internalization of NBCe1. We used the calmodulin (CaM) antagonist W13, which controls recycling by blocking the exit of the endocytosed cargo from early endosomes, to determine the role of CaM in NBCe1 trafficking. We demonstrated that W13 decreases surface expression of NBCe1, accumulates NBCe1-EGFP in endosomal-like formations, and inhibits NBCe1 current. W13 and ANG II applied together produce greater inhibition of NBCe1 current than either does alone, while W13 and monensin applied together do not. The additive effect of ANG II and W13 and lack of additive effect of monensin and W13 suggest that CaM is not involved in ANG II stimulation of internalization but controls recycling of endocytosed NBCe1. The CaM-activated enzyme CaM kinase II (CaMKII) applied with ANG II also gives an additive inhibitory effect, suggesting a role for CaMKII in NBCe1 recycling.
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Angiotensina II/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Calmodulina/fisiología , Simportadores de Sodio-Bicarbonato/fisiología , Proteínas de Xenopus/fisiología , Animales , Bencilaminas/farmacología , Biotina/fisiología , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Calmodulina/antagonistas & inhibidores , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Colorantes Fluorescentes , Humanos , Ionóforos/farmacología , Microscopía Confocal , Monensina/farmacología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteína Quinasa C/metabolismo , Sulfonamidas/farmacología , Xenopus laevisRESUMEN
INTRODUCTION: The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). CONCLUSION: The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Ramos Subendocárdicos/fisiopatología , Sulfonamidas/farmacología , Torsades de Pointes/prevención & control , Animales , Técnicas Electrofisiológicas Cardíacas , Femenino , Fluoroquinolonas/toxicidad , Técnicas In Vitro , Ramos Subendocárdicos/efectos de los fármacos , Conejos , Torsades de Pointes/inducido químicamente , Torsades de Pointes/fisiopatologíaRESUMEN
The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca(2+)/calmodulin-dependent kinase II (W7) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes.
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Citocromo P-450 CYP2B1/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Hígado/enzimología , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Animales , Western Blotting/métodos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/genética , Inhibidores Enzimáticos del Citocromo P-450 , ADN Complementario/biosíntesis , Dioxanos/farmacología , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética/métodos , Activadores de Enzimas/farmacología , Inducción Enzimática/efectos de los fármacos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hígado/efectos de los fármacos , Masculino , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/aislamiento & purificación , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sulfonamidas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesisRESUMEN
We have found that honokiol [4-allyl-2-(3-allyl-4-hydroxy-phenyl)-phenol] can promote neurite outgrowth and mobilize intracellular Ca2+ store in primary cultured rat cortical neurons. In this study, we examined the effects of honokiol on extracellular signal-regulated kinases (ERK1/2) and Akt, and their possible relationship to neurite outgrowth and Ca2+ mobilization. Honokiol-induced neurite outgrowth in the cultured rat cortical neurons was significantly reduced by PD98059, a mitogen-activated protein kinase kinase (MAPKK, MAPK/ERK kinase MEK, direct upstream of ERK1/2) inhibitor, but not by LY294002, a phosphoinositide 3-kinase (PI3K, upstream of Akt) inhibitor. Honokiol also significantly enhanced the phosphorylation of ERK1/2 in a concentration-dependent manner, whereas the effect of honokiol on Akt phosphorylation was characterized by transient enhancement in 10 min and lasting inhibition after 30 min. The phosphorylation of ERK1/2 enhanced by honokiol was inhibited by PD98059 as well as by KN93, a Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor. Moreover, the products of the phosphoinositide specific phospholipase C (PLC)-derived inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) were measured after honokiol treatment. Together with our previous findings, these results suggest that the signal transduction from PLC, IP3, Ca2+, and CaMK II to ERK1/2 is involved in honokiol-induced neurite outgrowth.
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Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuritas/efectos de los fármacos , Animales , Bencilaminas/farmacología , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Corteza Cerebral , Cromonas/farmacología , Diglicéridos/biosíntesis , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Feto , Flavonoides/farmacología , Inositol 1,4,5-Trifosfato/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Morfolinas/farmacología , Neuritas/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacologíaRESUMEN
At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Egtácico/análogos & derivados , Transportador 2 de Aminoácidos Excitadores/metabolismo , Simportadores/metabolismo , Ácido Aspártico/metabolismo , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Quelantes/farmacología , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN Complementario/biosíntesis , ADN Complementario/genética , Ácido Egtácico/farmacología , Transportador 1 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores , Espacio Extracelular/enzimología , Espacio Extracelular/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Humanos , Plásmidos/genética , Proteína Quinasa C/antagonistas & inhibidores , TransfecciónRESUMEN
The objective of this study was to investigate the role of calmodulin-dependent protein kinase II (CaMKII) during fertilization in the pig. Since it has been reported that CaMKII is involved in the capacitation and acrosome reaction of spermatozoa, we tested whether supplementation with the CaMKII inhibitor, KN-93, in the fertilization medium affected sperm penetration. The results showed that the addition of KN-93 in the fertilization medium significantly reduced the rate of sperm penetration into oocytes. However, pre-treatment with KN-93 before in vitro fertilization (i.v.f.) did not significantly affect sperm penetration, but it did affect pronuclear formation in a dose-dependent manner. In the oocytes pre-treated with KN-93 before i.v.f. and then co-cultured with spermatozoa without the drug, the decrease in p34cdc2 kinase and the cyclin B1 level were significantly suppressed as compared with those in penetrated oocytes without treatment with KN-93. However, the decrease in MAP kinase activity was not affected by KN-93. Additional treatment with KN-93 after Ca2+ ionophore treatment also inhibited the reduction in p34cdc2 kinase activity and the cyclin B1 level, but not MAP kinase activity. Treatment with KN-92, an inactive KN-93 analogue, did not significantly affect sperm penetration and pronuclear formation. In conclusion, the activation of CaMKII by artificial stimuli or sperm stimulated the disruption of cyclin B1 and the inactivation of p34cdc2 kinase, but did not affect MAP kinase inactivation during oocyte activation in pigs.