Browsing by Subject "Cytochemistry"
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- PublicationOpen AccessAn ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 1998) Jiang, X.; Kobayashi, T.; Nahirney, P. C.; García del Saz, E.; Seguchi, H.Alkaline phosphatase (ALPase) activity was examined by cerium-based uitracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 nglml PMA or 10-7 M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.
- PublicationOpen AccessChanges in extranucleolar transcription during actinomycin D-induced apoptosis(Murcia : F. Hernández, 2005) Fraschini, A.; Bottone, M.G.; Scovassi, A.I.; Denegri, M.; Risueño, M.C.; Testillano, P.S.; Martin, T.E.; Biggiogera, M.; Pellicciari, C.Actinomycin D (AMD) inhibits DNAdependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 µg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromouridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD.
- PublicationOpen AccessCytochemical and biochemical evidences for a complex tridimensional structure of the hamster zona pellucida(Murcia : F. Hernández, 2009) Saavedra Leos, María Dolores; Gutiérrez Gallego, Ricardo; Fayrer Hosken, Richard; Ballesta Germán, José; Avilés Sánchez, Manuel; Castells Mora, María Teresa; Izquierdo Rico, María José; Jíménez Movilla, María; Martínez Alonso, Emma; Biología CelularZona pellucida (ZP) is an extracellular matrix that surrounds eggs and pre-implantation embryos and is required for in vivo fertility. A key event in successful fertilization is sperm binding to the surface of the ZP. It has been previously described that the hamster sperm binds mainly the outer region of the ZP which corresponds to the porous region in contact with the cumulus cells. Using ultrastructural cytochemistry approaches with an antibody developed against porcine ZP, this study shows that the pig ZP shares epitopes with some rodent species like hamster, rat and mouse. In the hamster, these epitopes are located mainly in the outer region of the ZP of preovulatory and ovulated oocytes. By means of biochemical approaches it was demonstrated that 1) the antibody is specific for the native hamster ZP3, 2) four different bands with a molecular weight of 67, 60, 48 and 38 kDa after Nlinked deglycosylation suggesting that the hamster ZP is formed by four proteins, and 3) the different composition observed in the outer region of the hamster ZP could be due to a specific supramolecular structure that makes some epitopes accessible for the antibodies. In summary, this study provides evidence that the different composition observed in the different regions of the ZP is mediated by a different organization of the components of the ZP produced during the oocyte maturation. This different organization could be responsible for the different sperm binding affinity observed for sperm to the outer region versus the inner region of the ZP.
- PublicationOpen AccessCytochemical localization of Na+/K+ -ATPase activity in cochlear strial marginal cells after various catecholamine administrations(Murcia : F. Hernández, 2001) Kanoh, N.; Dai, C.F.; Mohri, D.; Hori, S.Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a ceriumbased cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Stria1 K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+- NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.
- PublicationOpen AccessEndometrial epithelial cell organoids as tools for studying the CD39 family of enzymes and for validating enzyme inhibitors(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Rodríguez Martínez, Aitor; Torrejón Escribano, Benjamín; Eritja, Núria; Dorca Arévalo, Jonatan; Gabaldón, Clara; Sévigny, Jean; Matias Guiu, Xavier; Martín Satué, MireiaExtracellular adenosine triphosphate (ATP) conducts a complex dynamic system of broadly represented cell signaling. Ectonucleotidases are the enzymes with nucleotide hydrolytic ability that regulate ATP levels in physiological and pathological conditions, thus playing a key role in the so-called purinergic signaling. Altered ectonucleotidase expression has been reported in cancer, and the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family of enzymes, with its best-known form NTPDase1 (CD39), is targeted in cancer immunotherapy. The tandem of enzymes CD39-CD73 is responsible for the generation of immuno-suppressive adenosine in the tumor microenvironment, and inhibition strategies are of great interest. Organoids have emerged as very convenient models for the study of tumors since they are three-dimensional cultures that retain many of the features of tissue. The present study aims to contribute to improving the methodology and the molecular tools needed for the study of ecto-nucleotidases in healthy and disease conditions. The study, performed in an endometrial cancer cell model, could be extended to other types of tumors and pathologies in which the purinergic system is involved. We generated organoids from endometrial cancer cells overexpressing NTPDase2 (CD39L1) and NTPDase3 (CD39L3) as fusion proteins with EGFP, and we performed functional assays by adapting in situ cytochemistry protocols. This allowed us to simultaneously detect enzyme activity and protein expression and to demonstrate that organoids can be used to test ectonucleotidase inhibitors—a result that can be used to develop new cancer treatment options
- PublicationOpen AccessIn situ detection of cyclic AMP-phosphodiesterase activity in the heart of Lewis and Sprague-Dawley rats: the effect of restraint stress or amphetamine application(Murcia : F. Hernández, 2004) Okruhlicová, L.; Klenerová, V.; Hynie, S.; Sída, P.Cyclic AMP plays an important role in heart functions under normal as well as pathological conditions. Since phosphodiesterase (PDE), responsible for the hydrolysis of cAMP, is equally important as synthesizing adenylyl cyclase, we decided to determine its activity by cytochemical procedure after exposure of rats to restraint stress or an acute dose of amphetamine. Sprague-Dawley (S-D) and Lewis (LE) rats, the latter known to have a deficient hypothalamo-pituitary-adrenal axis activity, were used in order to disclose the possible significance of rat strain on PDE activity. Animals were divided into 3 groups: controls, rats treated with an acute dose of amphetamine (8 mg/kg, i.p., for 60 min) and rats under restraint stress for 60 min. Control hearts of both strains revealed PDE activity on sarcolemma of cardiomyocytes and plasmalemma of endothelial cells of microvessels. In LE rats we observed an additional enzyme reaction in junctional sarcoplasmic reticulum. In addition, cardiomyocytes of LE rats revealed a higher PDE activity when compared to S-D rats. Restraint stress decreased PDE activity in cardiomyocytes of LE rats while amphetamine markedly inhibited enzyme activity in cardiomyocytes of S-D rats. Endothelial PDE was more resistant to stress. Our results indicate differences in PDE localization and variations in sensitivity of myocardial cAMP-PDE of LE and S-D rat strains to restraint stress and amphetamine application
- PublicationOpen AccessNovel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes(Murcia : F. Hernández, 1999) Kobayashi, T.; Seguchi, H.We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.