RÉSUMÉ
PURPOSE: A radiofrequency identification (RFID) tag system was designed to streamline cryopreservation and thawing procedures. This study evaluated the usefulness of the RFID tag system for improving the efficiency of cryopreserving/thawing bovine ovarian tissue by the closed vitrification protocol. METHODS: Six participants carried out closed vitrification and thawing of bovine ovarian tissues procedures using either the conventional or the new RFID tag method, and the time required to perform each step of the respective methods was measured. After normality of data was confirmed by the Shapiro-Wilk test, the significance of differences was assessed by the unpaired t test. RESULTS: When closed vitrification was performed, the time required for each step showed a significant difference between the two methods (t(4) = 2.938, p = 0.042, d = 2.40), and the total cryopreservation time was 11 min shorter using the RFID tag system. When thawing was performed, the time required for each step also showed a significant difference between the two methods (t(4) = 2.797, p = 0.049, d = 2.28), and the total thawing time was 2 min shorter using the RFID tag system. CONCLUSION: The RFID tag system tested in this study seems to be suitable for managing biological samples stored in liquid nitrogen. Adoption of an RFID tag system by fertility centers may not only improve the efficiency of cryopreserving/thawing reproductive tissues but could also reduce human error.
Sujet(s)
Cryoconservation/méthodes , Ovaire/physiologie , Dispositif d'identification par radio-fréquence/méthodes , Animaux , Bovins , Cryoprotecteurs/pharmacologie , Femelle , Fécondité/effets des médicaments et des substances chimiques , Fécondité/physiologie , Ovocytes/effets des médicaments et des substances chimiques , Ovocytes/physiologie , Ovaire/effets des médicaments et des substances chimiques , Reproduction/effets des médicaments et des substances chimiques , Reproduction/physiologie , VitrificationRÉSUMÉ
Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.
Sujet(s)
Aquaporines/métabolisme , Protéines membranaires/métabolisme , Glande parotide/physiologie , Vésicules de sécrétion/métabolisme , Équilibre hydroélectrolytique/physiologie , Animaux , Aquaporine-5 , Mâle , Rats , Rat Sprague-DawleyRÉSUMÉ
Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such as Km values for l-arginine and EC(50) values for calmodulin and Ca(2+), were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca(2+) concentrations. These results suggest that the Ca(2+)- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP(2) regulate the NOS activity in the membrane site by regulating the effect of Ca(2+) in the rabbit submandibular gland.
Sujet(s)
Calcium/métabolisme , Calmoduline/métabolisme , Nitric oxide synthase/métabolisme , Phospholipides/métabolisme , Lapins/métabolisme , Glande submandibulaire/métabolisme , Animaux , Arginine/métabolisme , Membrane cellulaire/métabolisme , Cytosol/métabolisme , Immunotransfert , Acides phosphatidiques , Phosphatidylinositol diphosphate-4,5/métabolisme , Comptage de scintillations , Spectrométrie de fluorescence , TritiumRÉSUMÉ
Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6-7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N(omega)-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.
Sujet(s)
Animaux de laboratoire/métabolisme , Arginase/métabolisme , Arginine/analogues et dérivés , Glandes salivaires/enzymologie , Animaux , Arginase/antagonistes et inhibiteurs , Arginine/composition chimique , Arginine/métabolisme , Antienzymes/composition chimique , Antienzymes/métabolisme , Cochons d'Inde , Rein/enzymologie , Cinétique , Foie/enzymologie , Mâle , Souris , Ornithine/composition chimique , Ornithine/métabolisme , Physiologie comparée , Lapins , Rats , Rat Sprague-Dawley , Glandes salivaires/composition chimique , Spécificité d'espèce , Fractions subcellulaires/enzymologie , Glande submandibulaire/composition chimique , Glande submandibulaire/cytologie , Glande submandibulaire/enzymologieRÉSUMÉ
IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.
Sujet(s)
Microdomaines membranaires/métabolisme , Microdomaines membranaires/physiologie , Phosphotyrosine/métabolisme , Récepteurs du fragment Fc des IgG/métabolisme , Récepteurs du fragment Fc des IgG/physiologie , Superoxydes/métabolisme , Ubiquitin-protein ligases , Cyclodextrines bêta , Cellules cultivées , Cyclodextrines/pharmacologie , Détergents/composition chimique , Antienzymes/pharmacologie , Cellules HL-60 , Humains , Phosphorylation/effets des médicaments et des substances chimiques , Transport des protéines , Protéines proto-oncogènes/métabolisme , Protéines proto-oncogènes c-cbl , Protéines proto-oncogènes pp60(c-src)/antagonistes et inhibiteurs , Pyrazoles/pharmacologie , Pyrimidines/pharmacologie , Transduction du signal , Trétinoïne/pharmacologie , src-Family kinases/métabolismeRÉSUMÉ
In rabbit parotid acinar cells, the muscarinic cholinergic agonist methacholine induced an increase in the intracellular Ca(2+) concentration and provoked nitric oxide (NO) generation. Ca(2+)-mobilizing reagents such as thapsigargin and the Ca(2+) ionophore A23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the removal of extracellular Ca(2+). Immunoblot analysis indicated that the antibody against the neuronal type of nitric oxide synthase (NOS) cross-reacted with NOS in the cytosol of rabbit parotid gland cells. Immunofluorescence testing showed that neuronal NOS is present in the cytosol of acinar cells but less in the ductal cells. NOS was purified approximately 8100-fold from the cytosolic fraction of rabbit parotid glands by chromatography on Sephacryl S-200, DEAE-Sephacel, and 29,59-ADP-Sepharose. The purified NOS was a NADPH- and tetrahydroxybiopterin-dependent enzyme and was activated by Ca(2+) within the physiological range in the presence of calmodulin. These results suggest that NO is generated by the activation of the neuronal type of NOS, which is regulated in rabbit parotid acinar cells by the increase in intracellular Ca(2+) levels induced by the activation of muscarinic receptors.
Sujet(s)
Calcium/métabolisme , Monoxyde d'azote/métabolisme , Glande parotide/cytologie , Glande parotide/métabolisme , Animaux , A-23187/pharmacologie , Calmoduline/pharmacologie , Relation dose-effet des médicaments , Antienzymes/pharmacologie , Immunotransfert , Ionophores/pharmacologie , Cinétique , Chlorure de méthacholine/pharmacologie , Microscopie de fluorescence , Agonistes muscariniques/pharmacologie , NADP/pharmacologie , Nitric oxide synthase/métabolisme , Liaison aux protéines , Lapins , Rats , Thapsigargine/pharmacologie , Facteurs tempsRÉSUMÉ
We have previously demonstrated that bradykinin potentiates prostaglandin E(2)release in human gingival fibroblasts pretreated with interleukin-1 beta (priming). In this study, we demonstrate a potentiating effect of bradykinin on cyclooxygenase-2 mRNA expression in the interleukin-1 beta-primed fibroblasts. Interleukin-1 beta (200 pg/ml) induced cyclooxygenase-2 mRNA expression, but not bradykinin (1 microM). However, bradykinin rapidly and markedly increased the cyclooxygenase-2 mRNA expression in the fibroblasts primed with interleukin-1 beta. In the primed fibroblasts, ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on the cyclooxygenase-2 mRNA expression. Dexamethasone and actinomycin D completely suppressed not only the interleukin-1 beta-induced cyclooxygenase-2 mRNA expression, but also the bradykinin-induced cyclooxygenase-2 mRNA expression in the interleukin-1 beta-primed fibroblasts, although cycloheximide did not inhibit the effects of interleukin-1 beta and bradykinin. These results suggest that bradykinin-induced prostaglandin E2 synthesis is regulated at the level of the transcription of cyclooxygenase-2 mRNA via Ca2+ mobilization in the interleukin-1 beta-primed human gingival fibroblasts.
Sujet(s)
Bradykinine/métabolisme , Calcium/métabolisme , Interleukine-1/métabolisme , Isoenzymes/génétique , Prostaglandin-endoperoxide synthases/génétique , ARN messager/métabolisme , Bradykinine/pharmacologie , Calcium/agonistes , Cellules cultivées , Cycloheximide/métabolisme , Cycloheximide/pharmacologie , Cyclooxygenase 2 , Dactinomycine/métabolisme , Dactinomycine/pharmacologie , Dexaméthasone/métabolisme , Dexaméthasone/pharmacologie , Fibroblastes/cytologie , Fibroblastes/métabolisme , Gencive/cytologie , Humains , Interleukine-1/pharmacologie , Ionomycine/métabolisme , Ionomycine/pharmacologie , Isoenzymes/effets des médicaments et des substances chimiques , Isoenzymes/métabolisme , Protéines membranaires , Prostaglandin-endoperoxide synthases/effets des médicaments et des substances chimiques , Prostaglandin-endoperoxide synthases/métabolisme , ARN messager/effets des médicaments et des substances chimiques , ARN messager/génétique , Thapsigargine/métabolisme , Thapsigargine/pharmacologieRÉSUMÉ
Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.
Sujet(s)
Antigènes de différenciation/métabolisme , Antienzymes/pharmacologie , Gangliosides/pharmacologie , N-Glycosyl hydrolases/antagonistes et inhibiteurs , NAD nucleosidase/métabolisme , ADP-ribosyl cyclase , Antigènes CD38 , Antigènes CD/métabolisme , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/enzymologie , Membrane cellulaire/métabolisme , Antienzymes/métabolisme , Espace extracellulaire/effets des médicaments et des substances chimiques , Espace extracellulaire/enzymologie , Espace extracellulaire/métabolisme , Cytométrie en flux , Gangliosides/métabolisme , Gangliosides/physiologie , Cellules HL-60 , Humains , Hydrolyse , Glycoprotéines membranaires , N-Glycosyl hydrolases/métabolisme , Oligosaccharides/métabolisme , Facteurs temps , Cellules cancéreuses en cultureRÉSUMÉ
Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein, found almost exclusively in mineralized connective tissues, that may function in the nucleation of hydroxyapatite crystals. We have found that expression of BSP in osteoblastic ROS 17/2.8 cells is stimulated by fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells. Stimulation of BSP mRNA with 10 ng/ml FGF2 was first evident at 3 h ( approximately 2.6-fold) and reached maximal levels at 6 h ( approximately 4-fold). From transient transfection assays, a FGF response element (FRE) was identified (nucleotides -92 to -85, "GGTGAGAA") as a target of transcriptional activation by FGF2. Ligation of two copies of the FRE 5' to an SV40 promoter was sufficient to confer FGF-responsive transcription. A sequence-specific protein-DNA complex, formed with a double-stranded oligonucleotide encompassing the FRE and nuclear extracts from ROS 17/2.8 cells, but not from fibroblasts, was increased following FGF2 stimulation. Several point mutations within the critical FRE sequence abrogated the formation of this complex and suppressed both basal and FGF2-mediated promoter activity. These studies, therefore, have identified a novel FRE in the proximal promoter of the BSP gene that mediates both constitutive and FGF2-induced BSP transcription.
Sujet(s)
Facteur de croissance fibroblastique de type 2/pharmacologie , Régions promotrices (génétique) , Éléments de réponse , Sialoglycoprotéines/génétique , Séquence nucléotidique , Cellules cultivées , Protéines de liaison à l'ADN/métabolisme , Régulation de l'expression des gènes , Sialoprotéine liant les intégrines , Mitogènes/pharmacologie , Données de séquences moléculaires , Protéines nucléaires/métabolisme , Liaison aux protéines , Inhibiteurs de protéines kinases , Stabilité de l'ARN , ARN messager/biosynthèse , Sialoglycoprotéines/biosynthèseRÉSUMÉ
Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. Parathyroid hormone (PTH), which regulates serum calcium through its actions on bone cells, increases the expression of BSP in the rat osteosarcoma cell line (ROS 17/2.8). At 10(-8) M PTH (human 1-34 PTH), stimulation of BSP mRNA was first evident at 3 h ( approximately 3.8-fold), reached maximal levels at 6 h ( approximately 4.7-fold), and declined slowly thereafter. The effects of PTH, which were abrogated by cycloheximide (28 microg/ml), did not alter the stability of the BSP mRNA. The increased transcription was mimicked by both forskolin (10(-6) M) and isoproterenol (10(-7) M), and was also increased by 3-isobutyl-1-methylxanthine (IBMX; 10(-5) M), while the transcriptional activity induced by PTH was inhibited by the protein kinase A inhibitor, H89 (5x10(-6) M). From transient transfection assays using various BSP promoter-luciferase constructs, a pituitary-specific transcription factor-1 (Pit-1) regulatory element (nts -111 to -105) was identified as the target of transcriptional activation by PTH. Thus, transcriptional activity of constructs including the Pit-1 was enhanced approximately 4.7-fold by 10(-8) M PTH while 5'-ligation of the Pit-1 element conferred PTH regulation in an SV40 promoter construct. Binding of a nuclear protein, recognized by anti-Pit-1 antibodies, to a radiolabelled Pit-1-BSP probe was decreased in nuclear extracts prepared from PTH, forskolin and isoproterenol-stimulated ROS 17/2.8 cells. Moreover, co-transfection of ROS cells with a double-stranded Pit-1 oligonucleotide also increased luciferase activity. Collectively, these results indicate that PTH acts through a protein kinase A pathway involving cAMP to stimulate BSP transcription by blocking the action of a Pit-1-related nuclear protein that suppresses BSP transcription by binding a cognate element in the BSP promoter. Thus, we have identified a novel Pit-1 suppressor element in the rat BSP gene promoter that is the target of PTH-stimulated transcription of the BSP gene.
Sujet(s)
Protéines de liaison à l'ADN/génétique , Hormone parathyroïdienne/pharmacologie , Régions promotrices (génétique) , Sialoglycoprotéines/génétique , Facteurs de transcription/génétique , Motifs d'acides aminés , Animaux , Séquence nucléotidique , Lignée cellulaire , ADN/génétique , ADN/métabolisme , Amorces ADN/génétique , Sialoprotéine liant les intégrines , Ostéoblastes/effets des médicaments et des substances chimiques , Ostéoblastes/métabolisme , Stabilité de l'ARN , ARN messager/génétique , ARN messager/métabolisme , Rats , Facteur de transcription Pit-1 , Transcription génétique/effets des médicaments et des substances chimiques , TransfectionRÉSUMÉ
The immediate-early cyclooxygenase-2 (COX-2) gene encodes an inducible prostaglandin synthase enzyme which is implicated in inflammatory and proliferative diseases. COX-2 is highly induced during cell activation by various factors, including mitogens, hormones and cytokines. Since pro-inflammatory cytokine IL-1beta has been shown to induce prostaglandin E2 (PGE2) release in human gingival fibroblasts (HGF), here we analyzed the effect of IL-1beta on the expression of COX-2 and the activation of NFkappaB in HGF. Northern hybridization analysis revealed that IL-1beta (200 pg/ml) increased the expression of COX-2 mRNA in HGF. The effect of IL-1beta was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1beta-induced PGE2 release was blocked by the tyrosine kinase inhibitor and increased by the tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human COX-2 promoter (nt -1432 approximately +59) ligated to a luciferase reporter gene indicated that IL-1beta stimulated the transcriptional activity approximately 1.5-fold. Gel mobility shift assays with a radiolabelled COX-2-NFkappaB oligonucleotide (nts-223 to-214) revealed an increase in the binding of nuclear proteins from IL-1beta-stimulated HGF. This increase of DNA-protein complex formation induced by IL-1beta was blocked by herbimycin A and another tyrosine kinase inhibitor, genistein. These results suggest that NFkappaB is an important transcription factor for IL-1beta-induced COX-2 gene expression, and is involved in inducing COX-2 gene transcription through tyrosine phosphorylation in HGF.
Sujet(s)
Régulation de l'expression des gènes codant pour des enzymes/physiologie , Gencive/enzymologie , Interleukine-1/pharmacologie , Facteur de transcription NF-kappa B/métabolisme , Prostaglandin-endoperoxide synthases/génétique , Animaux , Lignée cellulaire , Cellules cultivées , Cyclooxygenase 2 , Dinoprostone/métabolisme , Fibroblastes/cytologie , Fibroblastes/enzymologie , Régulation de l'expression des gènes codant pour des enzymes/effets des médicaments et des substances chimiques , Gencive/cytologie , Humains , Isoenzymes/génétique , Protéines membranaires , Protein Tyrosine Phosphatases/antagonistes et inhibiteurs , ARN messager/génétique , Rats , Protéines recombinantes/biosynthèse , Transcription génétique/effets des médicaments et des substances chimiques , Transfection , Vanadates/pharmacologieRÉSUMÉ
Fructose-1,6-bisphosphatase is one of the regulatory enzymes of gluconeogenesis in kidney cortex. The effect of ribose 1,5-bisphosphate on fructose-1,6-bisphosphatase purified from rat kidney cortex was studied. Rat kidney cortex, fructose-1,6-bisphosphatase exhibited hyperbolic kinetics with regard to its substrate, but the activity was inhibited by ribose 1,5-bisphosphate at nanomolar concentrations. The inhibitory effect of ribose 1,5-bisphosphate on the fructose-1,6-bisphosphatase was enhanced in the presence of AMP, one of the inhibitors of fructose-1,6-bisphosphatase. Fructose-2,6-bisphosphate, which is an inhibitor of fructose-1,6-bisphosphatase, inhibited rat kidney cortex fructose-1,6-bisphosphatase activities at a low concentration of fructose-1,6-bisphosphate but a high concentration of fructose-1,6-bisphosphate relieved fructose-1,6-bisphosphatase from fructose-2,6-bisphosphate-dependent inhibition. On the contrary, fructose-1,6-bisphosphate was not effective for the recovery of fructose-1,6-bisphosphatase from ribose 1,5-bisphosphate-dependent inhibition. These results suggest that ribose 1,5-bisphosphate is a potent inhibitor and is involved in the regulation of fructose-1,6-bisphosphatase in rat kidney cortex.
Sujet(s)
Fructose-1,6-diphosphatase/antagonistes et inhibiteurs , Cortex rénal/enzymologie , Pentose phosphates/pharmacologie , Animaux , Antienzymes/métabolisme , Antienzymes/pharmacologie , Fructose-1,6-diphosphatase/métabolisme , Pentose phosphates/métabolisme , Rats , Spécificité du substratRÉSUMÉ
Interleukin-1beta, a proinflammatory cytokine, causes a slow increase in prostaglandin E(2) release. On the other hand, bradykinin, a chemical mediator for inflammation, induces a rapid prostaglandin E(2) release. Simultaneous stimulation with interleukin-1beta (200 pg/ml) and bradykinin (1 microM) evoked a moderately synergistic increase in prostaglandin E(2) release in human gingival fibroblasts. However, in the human gingival fibroblasts pretreated with interleukin-1beta, bradykinin drastically enhanced prostaglandin E(2) release. NS-398, a specific inhibitor of cyclooxygenase-2, inhibited not only interleukin-1beta-induced prostaglandin E(2) release but also bradykinin-induced prostaglandin E(2) release in the human gingival fibroblasts pretreated with interleukin-1beta. Transcriptional and translational inhibitors such as actinomycin D, cycloheximide, and dexamethasone also suppressed the interleukin-1beta-induced prostaglandin E(2) release and the bradykinin-induced prostaglandin E(2) release in interleukin-1beta-pretreated human gingival fibroblasts. In the fibroblasts pretreated with interleukin-1beta, Ca(2+)-mobilizing reagents such as ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on prostaglandin E(2) release. These results suggest that interleukin-1beta- and bradykinin-induced prostaglandin E(2) release is dependent on cyclooxygenase-2 and the potentiated effect of bradykinin in the human gingival fibroblasts primed with interleukin-1beta is caused by Ca(2+) mobilization.
Sujet(s)
Bradykinine/pharmacologie , Calcium/métabolisme , Dinoprostone/métabolisme , Fibroblastes/effets des médicaments et des substances chimiques , Interleukine-1/pharmacologie , Cellules cultivées , Cycloheximide/pharmacologie , Cyclooxygenase 2 , Inhibiteurs de la cyclooxygénase 2 , Inhibiteurs des cyclooxygénases/pharmacologie , Dactinomycine/pharmacologie , Relation dose-effet des médicaments , Synergie des médicaments , Fibroblastes/cytologie , Fibroblastes/métabolisme , Gencive/cytologie , Humains , Ionomycine/pharmacologie , Ionophores/pharmacologie , Isoenzymes/effets des médicaments et des substances chimiques , Protéines membranaires , Nitrobenzènes/pharmacologie , Inhibiteurs de la synthèse d'acide nucléique/pharmacologie , Prostaglandin-endoperoxide synthases/effets des médicaments et des substances chimiques , Inhibiteurs de la synthèse protéique/pharmacologie , Sulfonamides/pharmacologie , Facteurs tempsRÉSUMÉ
Nitric oxide (NO) acts as an inter- and intracellular signalling molecule of various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from L-arginine. Here the characteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For full activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca(2+) and calmodulin were needed as cofactors, because the activity was clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca(2+), or in the absence of calmodulin and presence of trifluoperazine, a calmodulin antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca(2+), and activated by them in a dose-dependent manner; EC(50) for calmodulin and Ca(2+) were 10 and 340 nM, respectively. The K(m) for L-arginine was 1.57 microM. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cytosolic fraction cross-reacted with a rabbit polyclonal antibody against human brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concentrations of calmodulin and Ca(2+).
Sujet(s)
Nitric oxide synthase/métabolisme , Glande parotide/enzymologie , Animaux , Chromatographie sur agarose/méthodes , Cytosol/composition chimique , Cytosol/enzymologie , Immunotransfert/méthodes , Mâle , Nitric oxide synthase/analyse , Nitric oxide synthase/isolement et purification , Glande parotide/composition chimique , Rats , Rat Sprague-Dawley , Spectrophotométrie/méthodesRÉSUMÉ
6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.
Sujet(s)
Fructose diphosphate/métabolisme , Fructose phosphate/métabolisme , Foie/métabolisme , Animaux , Fructose-1,6-diphosphatase/composition chimique , Foie/composition chimique , Mâle , Pentose phosphates/composition chimique , Phosphofructokinase-1/composition chimique , Rats , Rat Sprague-DawleyRÉSUMÉ
The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.
Sujet(s)
Antigènes CD , Antigènes de différenciation/composition chimique , Antigènes de différenciation/métabolisme , Gangliosides/composition chimique , Gangliosides/métabolisme , NAD nucleosidase/composition chimique , NAD nucleosidase/métabolisme , ADP-ribosyl cyclase , Antigènes CD38 , Antigènes de différenciation/génétique , Protéines de transport/composition chimique , Protéines de transport/génétique , Protéines de transport/métabolisme , Cristallisation , Protéines de liaison au maltose , NAD nucleosidase/génétique , Conformation des protéines , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Diffraction des rayons XRÉSUMÉ
Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.
Sujet(s)
Cyclic AMP-Dependent Protein Kinases/métabolisme , Protéines membranaires/métabolisme , Glande parotide/métabolisme , Protéines du transport vésiculaire , Animaux , Calcium/métabolisme , Membrane cellulaire/métabolisme , AMP cyclique/métabolisme , Activation enzymatique , Glande parotide/cytologie , Tests aux précipitines , Liaison aux protéines , Protéines Qa-SNARE , Protéines R-SNARE , Rats , Protéines SNARERÉSUMÉ
In the absence of external Ca2+, 100 microM histamine evoked a transient increase in intracellular Ca2+ ([Ca2+]i), and subsequent addition of Ca2+ to the medium resulted in a sustained increase in [Ca2+]i in fura-2-loaded human gingival fibroblasts. These Ca2+ mobilizations are attributed to Ca2+ release from intracellular stores and Ca2+ entry, respectively. When the histamine H1 antagonist chlorpheniramine was added after the histamine-induced transient increase in [Ca2+]i, the Ca2+ entry induced by the addition of Ca2+ was inhibited. In the fibroblasts pretreated with cyclooxygenase inhibitors, indomethacin (1 microM) or aspirin (100 microM), histamine-induced Ca2+ entry was significantly inhibited, but not the transient [Ca2+]i increase. These results suggest that the histamine-induced Ca2+ entry requires the continuous binding of histamine to the H1 receptors and is regulated by prostaglandins, which are probably produced due to the H1 receptor activation.
Sujet(s)
Calcium/métabolisme , Gencive/métabolisme , Prostaglandines/physiologie , Récepteur histaminergique H1/physiologie , Acide acétylsalicylique/pharmacologie , Calcium/pharmacologie , Canaux calciques/physiologie , Calcium-Transporting ATPases/antagonistes et inhibiteurs , Cellules cultivées , Chlorphénamine/pharmacologie , Inhibiteurs des cyclooxygénases/pharmacologie , Relation dose-effet des médicaments , Fibroblastes/métabolisme , Fura-2 , Gencive/cytologie , Histamine/pharmacologie , Antihistaminiques/pharmacologie , Antihistaminiques des récepteurs H1/pharmacologie , Humains , Indométacine/pharmacologie , Thapsigargine/pharmacologieRÉSUMÉ
Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.