Radiofrequency identification tag system improves the efficiency of closed vitrification for cryopreservation and thawing of bovine ovarian tissues.

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.

Circadian rhythm of plasma aldosterone concentration in patients with primary aldosteronism.

Plasma aldosterone, cortisol, and renin activity were measured in nine recumbent patients with hyperaldosteronism, including seven with adenomas, one with idiopathic hyperplasia, and one with glucocorticoid suppressible hyperplasia. All had peak values of plasma aldosterone concentration from 3 a.m. to noon and lowest values at 6 p.m. or midnight. This rhythm was similar to the circadian pattern of plasma cortisol in the same patients. When these data were normalized to eliminate the wide variation in ranges of plasma aldosterone and cortisol between individuals, there was an excellent correlation (r = + 0.87, P < 0.005) between the two hormones. In contrast, plasma aldosterone concentrations did not correlate with plasma renin activity before or after normalization of data. Short term suppression of ACTH by administration of dexamethasone eliminated the circadian variation of plasma aldosterone in both patients with hyperplasia and in four of five patients with adenomas, while it markedly altered the rhythm in the fifth. Similar doses of dexamethasone were administered to four normal subjects and did not flatten the circadian rhythm of plasma aldosterone. These data suggest that patients with primary aldosteronism have a circadian rhythm of plasma aldosterone mediated by changes in ACTH.

Sphingosine increases inositol trisphosphate in rat parotid acinar cells by a mechanism that is independent of protein kinase C but dependent on extracellular calcium.

In rat parotid acinar cells prelabelled with [3H]-inositol, sphingosine stimulated the accumulation of [3H]-inositol polyphosphates. When the cells were exposed to sphingosine, [3H]-inositol trisphosphate (InsP3) was accumulated in a time- and dose-dependent manner. When the extracellular Ca2+ was chelated by 1 mM EGTA, the effect of sphingosine on InsP3 accumulation was completely inhibited. Ionophores, A23187 and ionomycin, had no significant effect on InsP3 accumulation. An inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), failed to stimulate InsP3 accumulation. In the homogenate of parotid acinar cells, InsP3 3-kinase and 5-phosphomonoesterase activities were not affected by sphingosine. These results suggest that sphingosine activates phosphoinositide turnover by a mechanism dependent upon extracellular Ca2+, but different from that of an ionophore, and independent of protein kinase C.

Ca2+/calmodulin-dependent protein phosphatase in bovine parotid gland: purification and characterization.

Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni2+ or Mn2+ and Ca2+ plus calmodulin. Ca2+/calmodulin-dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD - the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells.

Bradykinin regulates the histamine-induced Ca2+ mobilization via protein kinase C activation in human gingival fibroblasts.

We previously demonstrated that histamine and bradykinin evoke an increase in intracellular Ca2+ ([Ca2+]i) in human gingival fibroblasts by using a fluorescent Ca2+ indicator Fura-2. In this paper, we further demonstrate the regulation of the histamine-induced Ca2+ mobilization by bradykinin. In fibroblasts stimulated with bradykinin (1 microM), subsequent stimulation with histamine (100 microM) failed to mobilize Ca2+, whereas bradykinin induced an increase in [Ca2+]i in the cells pre-stimulated with histamine. The attenuation of the histamine response was dependent on the concentration of bradykinin for the first stimulation. Histamine also failed to induce the formation of inositol 1,4,5-trisphosphate in fibroblasts pretreated with bradykinin. In fibroblasts pretreated with bradykinin (1 microM) for 3 min and then washed with fresh medium, the effect of histamine on [Ca2+]i quickly returned to the control level. The activation of protein kinase C by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (PMA) elicited a marked decrease in histamine-induced Ca2+ mobilization. When the protein kinase C activity was inhibited with H7, a protein kinase C inhibitor, or was down-regulated by pretreatment with PMA for 20 h, the inhibitory effect of PMA on the histamine response was relieved. In the fibroblasts pretreated with H7 or PMA for 20 h, histamine evoked Ca2+ mobilization even after bradykinin stimulation. These results suggest that the histamine response is regulated by bradykinin receptor activation via the activation of protein kinase C in human gingival fibroblasts.

cGMP production is coupled to Ca(2+)-dependent nitric oxide generation in rabbit parotid acinar cells.

We investigated the mechanism of guanosine 3',5'-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.

Bradykinin induces a rapid cyclooxygenase-2 mRNA expression via Ca2+ mobilization in human gingival fibroblasts primed with interleukin-1 beta.

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.

Ca(2+)-regulated nitric oxide generation in rabbit parotid acinar cells.

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.

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver.

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.

Parathyroid hormone regulation of bone sialoprotein (BSP) gene transcription is mediated through a pituitary-specific transcription factor-1 (Pit-1) motif in the rat BSP gene promoter.

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.

Sphingosine stimulates calcium mobilization in rat parotid acinar cells.

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.

Endogenous inhibitor of the ADP-ribosylation of (a) G-protein(s) as catalyzed by pertussis toxin is present in rat liver.

The inhibitor activity of the ADP-ribosylation of (a) G-protein(s) as catalyzed by pertussis toxin was found in the membrane extract of rat liver. The inhibitor activity was found in the fractions of DEAE-Sephacel column chromatography at 50-120 mM NaCl. The inhibitor activity is not due to the degradation of NAD nor to the reverse reaction of pertussis toxin (removal of incorporated ADP-ribose). The present result suggests the presence of an endogenous inhibitor of the ADP-ribosylation reaction of (a) G-protein(s).

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.

Histamine H1 receptor-induced Ca2+ mobilization and prostaglandin E2 release in human gingival fibroblasts. Possible role of receptor-operated Ca2+ influx.

Stimulation of human gingival fibroblasts with histamine elicited an increase in the intracellular concentration of free calcium ([Ca2+]i) and the formation of inositol 1,4,5-trisphosphate (InsP3) in a concentration- and time-dependent manner. The histamine-induced increase in [Ca2+]i was attenuated completely by chlorpheniramine, an H1 antagonist, but not by cimetidine, an H2 antagonist. The histamine-induced Ca2+ response consisted of an initial transient peak response and a subsequent sustained increase. The transient phase can be largely attributed to Ca2+ release from intracellular InsP3-sensitive stores since the increased [Ca2+]i effect of histamine completely disappeared after depletion of intracellular Ca2+ stores with thapsigargin in the absence of extracellular Ca2+. The sustained phase was due to Ca2+ influx which was attenuated in the absence of extracellular Ca2+. The Ca2+ influx required the continuous binding of histamine to the receptor, since chlorpheniramine attenuated the increase in [Ca2+]i observed when extracellular Ca2+ was re-applied to the cells after stimulation with histamine in the absence of extracellular Ca2+. Pretreatment with the Ca2+ channel blocker SK&F96365 inhibited the Ca2+ influx component, suggesting that histamine stimulates Ca2+ influx through an H1 receptor-operated Ca2+ channel. Histamine also evoked a concentration- and time-dependent release of prostaglandin E2 (PGE2). The histamine-evoked PGE2 release was reduced markedly by exclusion of extracellular Ca2+ or pretreatment with SK&F96365 or an H1 antagonist. These results indicate that histamine stimulates both the intracellular Ca2+ release from InsP3-sensitive stores and the H1 receptor-operated Ca2+ influx from extracellular sites. The increased [Ca2+]i due to the Ca2+ influx causes PGE2 release in human gingival fibroblasts.

Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside.

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.

Bradykinin potentiates prostaglandin E(2) release in the human gingival fibroblasts pretreated with interleukin-1beta via Ca(2+) mobilization.

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.

Studies on Ca2+-stimulated adenosine triphosphatase in rat submandibular glands: its distribution and properties.

Intracellular distribution and some characteristics of a Ca2+-stimulated adenosine triphosphatase (ATPase) in rat submandibular glands were investigated. This enzyme was activated by calcium alone, and magnesium was not necessary for its activation. Mg2+-stimulated ATPase also was investigated in the same enzyme preparations.

Developmental changes of calcium-stimulated adenosine triphosphatase in rat submandibular gland.

Developmental changes in Ca2-+ ATPase activity were determined in submandibular glands of the rat from the fetus to the 300-day-old rat. Ca2+-ATPase in the homogenate fraction showed a rapid increase from the fetus to the 5-day-old rat, and from the 20- to 30-day-old rat. In the microsomal fraction, enzyme activity increased from the fetus to the 10-day-old rat, and after that is remained at almost the same level. Oscillatory phenomena of Ca2+-ATPase were observed in zymogen and mitochondrial fractions.

Properties and distribution of inorganic pyrophosphatase in rabbit dental pulp.

Some properties and the intracellular distriubtion of inorganic pyrophosphatase in rabbit dental pulp were determined. This enzyme was sensitive to Mg2+, and not inhibited by imidazol and CN- which are inhibitors of alkaline phosphatase. Inorganic pyrophosphatase was found predominantly in the supernatant fraction.