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Candida albicans can cause two major types of infections: superficial infection and systemic candidiasis. C. albicans infects diverse host niches, owing to a wide range of virulence factors and attributes, such as morphological transitions and phenotypic switching. C. albicans uses glycolysis, followed by alcoholic fermentation or mitochondrial respiration to rapidly generate ATP under aerobic conditions. In this study, we quantified the mRNA expression of several glycolysis-related enzymes associated with the initial phase of environmental changes using two strains: a type strain, NBRC 1385, and a strain from a patient with auto-brewery syndrome, LSEM 550. Additionally, we analyzed the regulation of a rate-limiting enzyme in glycolysis, phosphofructokinase 1 (PFK1). Our results showed that the mRNA expression of enzymes in the middle and last stages of glycolysis and alcoholic fermentation increased, and that of mitochondrial respiration enzymes decreased under short-term anaerobic conditions. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) administration showed similar results under anaerobic conditions. Moreover, PFK1 maintained its regulatory effect under different conditions; no significant change was observed in its mRNA expression. Our results suggest that C. albicans obtains energy via carbohydrate catabolism in the early phase of environmental change and survives in various parts of the host.
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Candida albicans , Candidiasis , Humanos , Anaerobiosis , Glucólisis , ARN Mensajero/metabolismoRESUMEN
Previous studies on dolphin electrocardiograms have shown that they are mainly composed of increased negative waves, similar to ungulates. The electrocardiogram waveform was determined by the distribution of the Purkinje fibers. Based on the waveform of the dolphin electrocardiogram, Hamlin predicted that the Purkinje fibers would be distributed within the ventricular muscle, as in ungulates. The purpose of this study was to confirm the histological distribution of Purkinje fibers in dolphins. In the present study, bottlenose dolphin hearts were observed both grossly and histologically, and the effects of Purkinje fiber distribution and cardiac morphology on electrocardiogram waveforms were examined. This study showed that the Purkinje fibers of dolphins run just below the endocardium, as in humans, dogs, and cats, whose electrocardiograms mainly show positive waves. When the cardiac morphology of dolphins was observed carefully, the right ventricle was found to be extremely dilated compared to that of terrestrial mammals. In human recreational divers, right ventricular dilatation is induced by diving. We hypothesized that the dolphin's heart is in a state similar to that of the right heart dilatation in terrestrial animals. The dolphin electrocardiogram waveform was considered to be due to right axis deviation. Based on the above, we concluded that the dolphin electrocardiogram waveform was due to its ability to live in water. We found that the dolphins are genetically related to ungulates, particularly the hippopotamus, but that their hearts have evolved differently.
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Delfín Mular , Animales , Humanos , Perros , Delfín Mular/fisiología , Tórax , Mamíferos , Electrocardiografía , Ventrículos CardíacosRESUMEN
Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2-) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2-, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2- generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.
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Neutrófilos , Factor de Activación Plaquetaria , Animales , Calcio , Perros , Neutrófilos/citología , Factor de Activación Plaquetaria/farmacología , Especies Reactivas de Oxígeno , Receptores Acoplados a Proteínas G , Transducción de Señal , Suramina/farmacologíaRESUMEN
OBJECTIVE: The physiological expression of cell division cycle 42 (cdc42) in major salivary glands, and paracellular transport of fluorescein isothiocyanate-dextran (FITC-dextran) in SMIE cells, which regulate cdc42 expression, was investigated to clarify the involvement of cdc42 in salivary production. DESIGN: The physiological expression of cdc42 in the rat submandibular gland, parotid gland, sublingual gland, and SMIE cells was detected using SDS-PAGE and western blotting. The paracellular transport of FITC-dextran in transwells was compared in transfected SMIE cells, exhibiting up- or downregulated cdc42 expression. RESULTS: Cdc42 was expressed in all major salivary glands and SMIE cells. SMIE cells transfected with the cdc42 plasmid had an increase efflux. In addition, SMIE cells transfected with the cdc42 siRNA showed decreased efflux. CONCLUSION: We suggest that cdc42 enhances paracellular transport in salivary glands without any morphological changes, including cell-cell adhesion.
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Glándula Sublingual , Glándula Submandibular , Proteína de Unión al GTP cdc42 , Animales , Western Blotting , Glándula Parótida , Ratas , Glándulas SalivalesRESUMEN
OBJECTIVE: Secretory immunoglobulin A (sIgA) is important for mucosal immunity due to the inhibition of pathogen infection. The submandibular gland is known to secrete more sIgA than the parotid and sublingual glands. In this study, we focused on the relationship between the secretion of accumulated intracellular sIgA and ß-adrenergic receptor stimulation, and clarified the autonomic regulatory mechanism of sIgA secretion in submandibular gland cells using dispersed gland cells. DESIGN: Sprague-Dawley rats (male, 6 weeks old, 200-250 g) were euthanized and their submandibular glands were removed. Dispersed submandibular gland cells placed in Krebs-Ringer-Bicarbonate solution were stimulated by autonomic nerve agonists. The concentration of secreted sIgA was measured using a rat IgA ELISA kit. The results were analysed using ANOVA and Tukey's test. RESULTS: Cells stimulated with the non-selective ß-adrenoreceptor agonist, isoprenaline, secreted significantly more sIgA compared with the unstimulated control. The ß2-adrenoreceptor agonist, fenoterol, caused significantly more sIgA secretion than the control, and more sIgA secretion than the ß1-adrenoreceptor agonist, xamoterol. sIgA secretion by isoprenaline stimulation was dose dependent. Inhibition of the ß receptor by propranolol completely blocked sIgA secretion following isoprenaline stimulation. CONCLUSION: Stimulation of ß receptors could result in more secretion of intracellularly accumulated sIgA compared with stimulation of other autonomic receptors in the autonomic modulation of mucosal immunity.
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Inmunoglobulina A Secretora , Glándula Submandibular , Agonistas Adrenérgicos beta/farmacología , Animales , Isoproterenol/farmacología , Masculino , Glándula Parótida , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta , SalivaRESUMEN
Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.
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Señalización del Calcio/fisiología , Saliva/metabolismo , Glándulas Salivales/metabolismo , Células Acinares/metabolismo , Animales , Calcio/metabolismo , Biología Computacional , Retículo Endoplásmico/metabolismo , Femenino , Canales Iónicos/metabolismo , Masculino , Ratones , Glándulas Salivales/patología , Glándula SubmandibularRESUMEN
INTRODUCTION: Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated. METHODS: The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca2+ concentrations after stimulation with KCl and l-glutamate were examined. RESULTS: The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca2+ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group. CONCLUSIONS: A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs.
RESUMEN
In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1-2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.
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Fructosa-Bifosfatasa/metabolismo , Riñón/enzimología , Hígado/enzimología , Fosfofructoquinasa-1/metabolismo , Animales , Perros , Femenino , Fructosa-Bifosfatasa/química , Gluconeogénesis , Glucólisis , Masculino , Fosfofructoquinasa-1/químicaRESUMEN
Pilocarpine is an M3 muscarinic agonist that is widely used for the treatment of xerostomia caused by various diseases and medical conditions. Pilocarpine induced the secretion of salivary fluid in perfused submandibular glands of rats. The secretion of salivary fluid observed after removal of pilocarpine was referred to as residual fluid secretion. The volume of fluid and time of the residual secretion depended on the dose of pilocarpine. Such a residual effect of pilocarpine was observed on fluid secretion via the paracellular pathway and oxygen consumption. When a muscarinic antagonist was added to the perfusate immediately after cessation of pilocarpine, residual secretion of salivary fluid did not occur. These observations indicate that the residual secretion of salivary fluid is a characteristics of the interaction of pilocarpine with muscarinic receptors.
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Perfusión , Pilocarpina/farmacología , Saliva/metabolismo , Glándula Submandibular/metabolismo , Animales , Masculino , Antagonistas Muscarínicos/farmacología , Consumo de Oxígeno , Piperidinas/farmacología , Ratas Wistar , Saliva/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , TetrodotoxinaRESUMEN
Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs.
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Perros/metabolismo , Isoenzimas/metabolismo , Fosfofructoquinasa-1/metabolismo , Animales , Encéfalo/enzimología , Femenino , Hígado/enzimología , Masculino , Músculo Esquelético/enzimología , Fosfofructoquinasa-1/química , Distribución TisularRESUMEN
This study was undertaken to establish a method for measuring mRNA expression by using real-time RT-PCR in the diagnosis of canine meningiomas. When performing real-time RT-PCR, it is essential to include appropriate control tissues and to select appropriate housekeeping genes as an internal standard. Based on the results of our study, RPS18 constitutes a suitable internal standard for the comparison of mRNA expression between normal meninges and meningiomas. The results showed increased mRNA expression of VEGFA and EGFR; however, mRNA expression of KDR was reduced. Measuring mRNA expression by using real-time RT-PCR with appropriate control tissues and internal standards can provide useful information to understanding the pathogenesis of canine meningiomas, which corresponds with immunohistochemical findings.
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Neoplasias Meníngeas/veterinaria , Meningioma/veterinaria , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Animales , Perros , Femenino , Genes Relacionados con las Neoplasias , Masculino , Neoplasias Meníngeas/genética , Meningioma/genética , Clasificación del Tumor/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
OBJECTIVES: Salivary acinar and duct cells show different expression patterns of claudins, which may reflect their different functions. To study the role of claudins in saliva secretion, we examined alterations in the expression patterns of cell adhesion molecules in parotid glands of γ-irradiated rats and analyzed the influence of those changes on intercellular barrier function using primary cultures of parotid acinar cells. DESIGN: Rats were γ-irradiated with doses of 5, 15 or 20Gy, and expression levels of cell adhesion molecules were examined by immunoblotting analysis. Acinar cells were isolated from parotid glands and were cultured in the absence or presence of the Src kinase inhibitor PP1. Changes in protein and mRNA expression patterns were determined by immunoblotting and by RT-PCR analyses, respectively. Intercellular barrier function was examined by measuring transepithelial electrical resistance and the paracellular flux of FITC-dextran. RESULTS: In irradiated parotid glands, the expression of claudin-4 was enhanced at 15Gy or higher, levels that induce the hyposecretion of saliva, although that increase was transient. At 30days after irradiation, expression levels of cell adhesion molecules were decreased. In primary cultures, the expression of claudin-4 was also increased transiently but the expression of claudin-3 and E-cadherin was decreased. The barrier function of tight junctions was disrupted although the localization of occludin was maintained. The Src kinase inhibitor PP1 suppressed those changes in gene expression and retained the intercellular barrier function. CONCLUSIONS: These results suggest that the inhibition of Src signaling maintains the barrier functions of intercellular junctions in salivary glands, which can be lost due to tissue injury.
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Células Acinares/metabolismo , Claudina-3/metabolismo , Uniones Intercelulares/metabolismo , Glándula Parótida/metabolismo , Glándula Parótida/efectos de la radiación , Familia-src Quinasas/metabolismo , Animales , Cadherinas/metabolismo , Células Cultivadas , Immunoblotting , Masculino , Ocludina/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
The proinflammatory cytokine interleukin 1ß (IL-1ß) induces prostaglandin E2 (PGE2) production via upregulation of cyclooxygenase-2 (COX-2) expression in synovial fibroblasts. This effect of IL-1ß is involved in osteoarthritis. We investigated MAPK signaling pathways in IL-1ß-induced COX-2 expression in feline synovial fibroblasts. In the presence of MAPK inhibitors, IL-1ß-induced COX-2 expression and PGE2 release were both attenuated. IL-1ß induced the phosphorylation of p38, JNK, MEK, and ERK1/2. A JNK inhibitor prevented not only JNK phosphorylation but also MEK and ERK1/2 phosphorylation in IL-1ß-stimulated cells, but MEK and ERK1/2 inhibitors had no effect on JNK phosphorylation. A p38 inhibitor prevented p38 phosphorylation, but had no effect on MEK, ERK1/2, and JNK phosphorylation. MEK, ERK1/2, and JNK inhibitors had no effect on p38 phosphorylation. We also observed that in IL-1ß-treated cells, phosphorylated MEK, ERK1/2, and JNK were co-precipitated with anti-phospho-MEK, ERK1/2, and JNK antibodies. The silencing of JNK1 in siRNA-transfected fibroblasts prevented IL-1ß to induce phosphorylation of MEK and ERK1/2 and COX-2 mRNA expression. These observations suggest that JNK1 phosphorylation is necessary for the activation of the MEK/ERK1/2 pathway and the subsequent COX-2 expression for PGE2 release, and p38 independently contributes to the IL-1ß effect in synovial fibroblasts.
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Ciclooxigenasa 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/fisiología , Interleucina-1beta/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Transducción de Señal , Animales , Gatos , Células Cultivadas , Dinoprostona/metabolismoRESUMEN
Acute kidney injury (AKI) is characterized by a sudden loss of renal function. Early recognition of AKI, especially in critically ill patients, is essential for adequate therapy. Currently, neutrophil gelatinase-associated lipocalin (NGAL) is considered to be an effective biomarker of AKI; however, the regulation of its expression and function in renal tubular cells remains unclear. In this study, we investigated the regulation of the expression and function of NGAL in IL-1ß-treated Madin-Darby canine kidney (MDCK) cells as a model of renal tubular cells. IL-1ß induced a disturbance in the localization of E-cadherin and zonaoccludin-1 (ZO-1). The transepithelial electrical resistance (TER) also decreased 5 days after IL-1ß treatment. IL-1ß induced NGAL mRNA expression and protein secretion in a time- and dose-dependent manner, which occurred faster than the decrease in TER. In the presence of ERK1/2 and p38 inhibitors, IL-1ß-induced NGAL mRNA expression and protein secretion were significantly attenuated. In the presence of recombinant NGAL, IL-1ß-induced disturbance in the localization of E-cadherin and ZO-1 was attenuated, and the decrease in TER was partially maintained. These results suggest that NGAL can be used as a biomarker for AKI and that it functions as a protector from AKI.
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Interleucina-1beta/metabolismo , Túbulos Renales/citología , Túbulos Renales/metabolismo , Lipocalina 2/metabolismo , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Animales , Cadherinas/metabolismo , Perros , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Túbulos Renales/efectos de los fármacos , Lipocalina 2/genética , Células de Riñón Canino Madin Darby , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The Japanese wild population of the medaka fish (Oryzias latipes species complex) comprises two genetically distinct groups, the Northern and the Southern Populations, with boundary populations having a unique genotype. It is thought that the boundary populations have been formed through introgressive hybridization between the two groups, because they are fixed with the Northern alleles at two allozymic loci, with the Southern alleles at two other loci, and have a unique allele at one locus. In this study, we examined the genetic population structure of the boundary populations using genome-wide single nucleotide polymorphism (SNP) data. Most SNPs of the Toyooka population, a typical boundary population, were shared with the Northern Population, some were shared with the Southern Population, and the remaining SNPs were unique to this population, suggesting that the boundary populations originated and diverged from the Northern Population. Further analyses of different populations using SNPs at eight genomic loci indicated that the boundary populations at different locations share similar genomic constitutions, and can be genetically distinguished from typical Northern Populations by unique SNPs. In addition, the boundary populations in the Maruyama River Basin had Northern mitochondrial DNA (mtDNA), while others, from the Fukuda and Kishida River Basins and from the Kumihama Bay area, had Southern mtDNA. These findings suggested that the boundary populations originated from the Northern Population, and then their genomes diverged as a result of geographical isolation, followed by mtDNA introgression from the Southern Population that occurred independently in some populations.
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Distribución Animal/fisiología , Oryzias/genética , Animales , Secuencia de Bases , ADN Mitocondrial/genética , Variación Genética , Genoma , Japón , Datos de Secuencia Molecular , FilogeniaRESUMEN
Feline calicivirus (FCV) is an important veterinary pathogen that causes acute upper respiratory tract diseases and, occasionally, highly contagious febrile hemorrhagic syndrome in cats. Many viruses have adopted mechanisms for evading IFN-α/ß signaling, particularly by directly or indirectly suppressing activation of IRF-3. In this study, we investigated whether nonstructural proteins of FCV possess these mechanisms. When p39, a nonstructural protein of FCV, was transiently expressed in 293T cells, it suppressed IFN-ß and ISG15 mRNA production induced by dsRNA. Expression of p39 also suppressed phosphorylation and dimerization of IRF-3 induced by dsRNA. These results suggest that p39 suppresses type 1 IFN production by preventing IRF-3 activation. This may become an important factor in understanding the pathogenesis and virulence of FCV.
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Infecciones por Caliciviridae/veterinaria , Calicivirus Felino/inmunología , Enfermedades de los Gatos , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Matriz Viral/metabolismo , Animales , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Enfermedades de los Gatos/inmunología , Enfermedades de los Gatos/virología , Gatos , Factor 3 Regulador del Interferón/genética , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Proteínas de la Matriz Viral/genéticaRESUMEN
Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.
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Amilasas/metabolismo , Colecistoquinina/farmacología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Páncreas/metabolismo , Proteína Quinasa C/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colecistoquinina/antagonistas & inhibidores , Citosol/efectos de los fármacos , Citosol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Páncreas/efectos de los fármacos , Páncreas/enzimología , Fosforilación , Proteína Quinasa C-delta/efectos de los fármacos , Proteína Quinasa C-delta/metabolismo , Proteínas Qa-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Translocación GenéticaRESUMEN
INTRODUCTION: Calcium carbonate biominerals participate in diverse physiological functions. Despite intensive studies, little is known about how mineralization is initiated in organisms. RESULTS: We analyzed the medaka spontaneous mutant, ha, defective in otolith (calcareous ear stone) formation. ha lacks a trigger for otolith mineralization, and the causative gene was found to encode polyketide synthase (pks), a multifunctional enzyme mainly found in bacteria, fungi, and plant. Subsequent experiments demonstrate that the products of medaka PKS, most likely polyketides or their derivatives, act as nucleation facilitators in otolith mineralization. The generality of this novel PKS function is supported by the essential role of echinoderm PKS in calcareous skeleton formation together with the presence of PKSs in a much wider range of animals from coral to vertebrates. CONCLUSION: The present study first links PKS to biomineralization and provides a genetic cue for biogeochemistry of carbon and calcium cycles.
RESUMEN
Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Neuronas/citología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Células Madre Mesenquimatosas/citología , Proteínas Asociadas a Microtúbulos/genética , Neuronas/efectos de los fármacos , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
The proinflammatory cytokine interleukin-1ß (IL-1ß) induced cyclooxygenases-2 (COX-2) mRNA expression and lipid mediator prostaglandin E2 release and in a time- and dose-dependent manner in canine dermal fibroblasts. The MEK inhibitor U0126 and the ERK inhibitor FR180204 clearly inhibited IL-1ß-induced prostaglandin E2 release and COX-2 mRNA expression. IL-1ß enhanced ERK1/2 phosphorylation, which was attenuated by inhibitors of MEK and ERK. The NF-κB inhibitor BAY 11-7082 also suppressed IL-1ß-induced prostaglandin E2 release and COX-2 mRNA expression. Treatment of fibroblasts with IL-1ß led to the phosphorylation of p65 and degradation of IκBα occurred, indicating that IL-1ß treatment activated NF-κB. MEK and ERK1/2 inhibitors had no effect on the phosphorylation of p65 subunit induced by IL-1ß, whereas the NF-κB inhibitor completely blocked IL-1ß-induced phosphorylation of ERK1/2. We also observed that IκBα-knockdown enhanced the phosphorylation of p65 and ERK1/2. These findings suggest that stimulation of MEK/ERK signaling pathway by NF-κB activation regulates IL-1ß-induced COX-2 expression and subsequent prostaglandin E2 release in canine dermal fibroblasts.