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PubMed1997

The effects of cyclosporin A, dexamethasone and other immunomodulatory drugs on induced expression of IL-3, IL-4 and IL-8 mRNA in a human mast cell line.

We examined by RT-PCR the effect of a number of immunomodulatory compounds on cytokine gene expression at the level of mRNA in the HMC-1 human leukemic mast cell line. Resting cells expressed relatively low constitutive levels of mRNA for the cytokine genes IL-3, IL-4 and IL-8, and mRNA levels for each of these cytokines were significantly enhanced after 4-h stimulation with the calcium ionophore ionomycin. Treatment of the cells with the immunosuppressant CsA at 10(-5) M produced a significant inhibition of ionomycin-induced expression of IL-3, IL-4 and IL-8 mRNA, and at 10(-6) M produced a significant inhibition of induced expression of IL-3 and IL-8 but not IL-4. At both concentrations of CsA, expression of IL-3 was inhibited to a greater extent than that of the other two cytokines. Treatment of the cells with the corticosteroid DEX at 10(-5) M but not 10(-6) M significantly reduced the ionomycin-induced expression of IL-3 but not IL-4 or IL-8 mRNA. Progesterone and methotrexate were both inactive in modulation of induced cytokine expression in this cell line. In conclusion, this study shows that cytokine expression, particularly of IL-3, is inhibited in a human mast cell line by CsA and DEX. These findings may be relevant to the anti-allergic action of these drugs.

Adjuvants, Immunologic↗
PubMed2003

Calcium-regulated expression of activin A in RBL-2H3 mast cells.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Activins↗
PubMed2004

Estrogen regulation of nitric oxide synthesis in the porcine oocyte.

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.

Animals↗
PubMed1987

Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.

Amiloride↗
PubMed1989

Stimulation of phosphorylcholine turnover and diacylglycerol production in human polymorphonuclear leukocytes. Novel assay for phosphorylcholine.

Receptor-bypassing stimulants of human polymorphonuclear leukocytes (PMNLs), such as ionomycin or phorbol 12-myristate 13-acetate (PMA), generate an increase in diacylglycerol (DAG) which is independent of a phospholipase C specific for phosphatidylinositol 4,5,-bisphosphate (PIP2). Activation of a phospholipase C specific for phosphatidylcholine (PC) has been implicated as a source of DAG in other cells by measuring the release of radiolabelled phosphorylcholine. However, since PMNLs could not be labelled sufficiently with [3H]choline, we developed an h.p.l.c. assay to quantify mass levels of phosphorylcholine after enzymic conversion to [32P]CDP-choline with CTP-phosphorylcholine (choline phosphate) cytidylyltransferase (EC 2.7.7.15). This assay was linear to at least 20 nmol, and was sensitive to 10 pmol of phosphorylcholine. Baseline phosphorylcholine levels in unstimulated PMNLs were 2300 +/- 510 pmol/10(7) cells and were decreased by pretreatment with PMA (166 nM) or ionomycin (1 microM) for 10 min by 360 +/- 130 and 600 +/- 290 pmol/10(7) cells respectively (P less than 0.05). In contrast, baseline DAG levels were 147.6 +/- 11.7 pmol/10(7) cells in unstimulated PMNLs, and were increased by PMA or ionomycin by 1320 +/- 222 and 1891 +/- 264 pmol/10(7) cells respectively (P less than 0.05). Similarly, the chemoattractant fMet-Leu-Phe raised DAG levels by 731 +/- 111 pmol/10(7) cells and decreased phosphorylcholine levels by 180 +/- 60 pmol/10(7) cells. Activation of PMNLs by PMA, ionophore or fMet-Leu-Phe thus leads to the sustained production of DAG accompanied by the disappearance of phosphorylcholine. This suggests that these stimulants enhance PC turnover via a hydrolytic mechanism which is independent of phospholipase C, with activation of a PC-specific phospholipase D being a plausible mechanism.

Choline↗
PubMed1993

Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets.

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

Blood Platelets↗
PubMed1994

Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum.

In order to determine whether the endoplasmic reticulum (ER) luminal FK506-binding protein, FKBP13, shares properties of ER molecular chaperones, MDCK cells were treated with either tunicamycin or Ca2+ ionophores. By Northern-blot analysis, tunicamycin resulted in a 2-fold rise in FKBP13 mRNA, whereas ionophores (A23187 and ionomycin) caused a more impressive rise in FKBP13 mRNA (up to 5-fold with ionomycin). Actinomycin D chase experiments in ionomycin-treated cells revealed no change in the half-life of FKBP13 mRNA, indicating that the increase in FKBP13 mRNA observed was not due to greater message stability. Moreover, sequencing of the 5' flanking region of the gene for murine FKBP13 revealed significant similarity to similar regions in human BiP (immunoglobulin-binding protein) and the human glucose-regulated protein grp94, including a 37 bp sequence in FKBP13 with approximately 50% identity with the unfolded protein response element of the BiP gene. Together, these data suggest a role for FKBP13 in ER protein folding.

Animals↗
PubMed1997

Stimulation-induced calcium signalling and ion transport in rat pancreatic acini.

In the present study we have characterized receptor-mediated Ca2+ signalling patterns as well as Ca(2+)-mediated ion transport mechanisms in collagenase isolated rat pancreatic acini. Measurements of the initial Ca2+ response to maximal carbachol stimulation revealed a rapid increase in [Ca2+]i, which, in general, occurred synchronously throughout the cells. Less frequently, not all cells in the acinus responded to carbachol, but did respond to subsequent stimulation with bombesin, indicating that not all cells possess receptors for all the applied agonists. In view of the heterogeneity in the agonist-evoked Ca2+ responses, ionomycin was used to assess the role of Ca2+ in activating K+, Na+ and Cl- transport mechanisms, Ionomycin induced a rise in [Ca2+]i, thereby increasing Cl- permeability as well as stimulating K+ efflux, probably through non-specific cation channels. However, the resting K+ efflux was insensitive to blockers of non-specific cation channels, indicating the existence of a selective resting K+ conductance. Ionomycin also stimulated influx of Na+, which in part was mediated by non-specific cation channels. The changes in ion fluxes measured in the present study revealed that when [Ca2+]i is raised in rat pancreatic acini, they gain Na+ and Cl- and lose K+, with non-specific cation channels being essential for this process.

Animals↗
PubMed2002

In vitro development of porcine parthenogenetic and cloned embryos: comparison of oocyte-activating techniques, various culture systems and nuclear transfer methods.

The present study compares the development of porcine embryos following several oocyte activation techniques and culture systems using in-vitro-matured porcine oocytes. Two different nuclear transfer techniques (electrofusion and nuclear injection) were also tested using adult somatic cell (nucleus) and enucleated cytoplasm. In Experiment 1, oocytes activated by electric pulse, electric pulse + 6-dimethylaminopurine (6-DMAP), or ionomycin + 6-DMAP showed higher pronuclear formation rates (P<0.01) than those in the other groups (negative control and ionomycin treatment). Of these three groups, oocytes activated by electric pulse + 6-DMAP showed greater developmental rate to the blastocyst stage and higher cell numbers in blastocysts than those activated by electric pulse or ionomycin + 6-DMAP (P<0.05). In Experiment 2, activated oocytes were grouped and cultured as follows: (i) NCSU-PVA for 6 days (PVA-PVA); (ii) NCSU-BSA for 6 days (BSA-BSA); (iii) NCSU-FBS for 6 days (FBS-FBS); (iv) NCSU-PVA for 4 days followed by NCSU-BSA for 2 days (PVA-BSA); (v) NCSU-PVA for 4 days followed by NCSU-FBS for 2 days (PVA-FBS); and (vi) NCSU-BSA for 4 days followed by NCSU-FBS for 2 days (BSA-FBS). Cleavage rates in all experimental groups were not significantly different, but the embryos cultured in PVA-BSA or BSA-BSA showed higher blastocyst formation rates than those in other groups (P<0.05). In Experiment 3, the pseudo-pronuclear formation rate tended to be higher in the electrofusion group than in piezo-driven nuclear injection group, but the difference was not statistically significant (P=0.12). In addition, there was no significant difference between groups in cleavage, blastocyst formation, or the number of cells in blastocysts. The results indicate that porcine adult somatic cell nuclear transfer can be performed by the nuclear injection technique with a piezo-driven micromanipulator. In addition, activation by electrical pulse followed by 6-DMAP and in vitro culture in BSA-supplemented medium throughout the culture period was found to be the most efficient system for the production of porcine parthenogenetic embryos in vitro.

Animals↗
PubMed1996

Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity.

NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

Amino Acid Sequence↗
PubMed2005

Differences in eNOS activity because of subcellular localization are dictated by phosphorylation state rather than the local calcium environment.

Nitric oxide (NO) produced in the endothelium via the enzyme endothelial nitric-oxide synthase (eNOS) is an important vasoactive compound. Wild-type (WT) eNOS is localized to the plasma membrane and perinuclear/Golgi region by virtue of N-terminal myristoylation and palmitoylation. Acylation-deficient mutants (G2AeNOS) remain cytosolic and release less NO in response to Ca2+-elevating agonists; a disparity that we hypothesized was attributed to the greater distance between G2AeNOS and plasma membrane Ca2+ influx channels. The reduced activity of G2AeNOS versus WT was reversed upon disruption of cellular integrity with detergents or sonication. NO production from both constructs relied almost exclusively on the influx of extracellular Ca2+, and elevating intracellular Ca2+ to saturating levels with 10 microM ionomycin in the presence of 10 mM extracellular Ca2+ equalized NO production. To identify the contribution of calcium to the differences in activity between these enzymes, we created Ca2+/CaM-independent eNOS mutants by deleting the two putative autoinhibitory domains of eNOS. There was no difference in NO production between WT and G2A-targeted Ca2+-independent eNOS, suggesting that Ca2+ was the factor responsible. When eNOS constructs were fused in-frame to the bioluminescent probe aequorin, membrane-bound probes were exposed to higher [Ca2+] in unstimulated cells but upon ionomycin stimulation, the probes experienced equal amounts of Ca2+. The WT and G2A enzymes displayed significant differences in the phosphorylation state of Ser617, Ser635, and Ser1179, and mutating all three sites to alanine or restoring phosphorylation with the phosphatase inhibitor calyculin abolished the differences in activity. We therefore conclude that the disparity in NO production between WTeNOS and G2AeNOS is not caused by different localized [Ca2+] upon stimulation with ionomycin, but rather differences in phosphorylation state between the two constructs.

Animals↗
PubMed2006

Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation.

In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC(50) of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr(494) and Ser(498). Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr(494) and Ser(498) to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.

Amino Acid Substitution↗
PubMed2002

In vitro models of brain ischemia: the peptidergic drug cerebrolysin protects cultured chick cortical neurons from cell death.

Glutamate (1 mM), iodoacetate (0.01 mM) and ionomycin (0.25 micro M) are reported to induce several characteristics of ischemia and neuronal degeneration in vitro, e.g. glutamate and ionomycin lesion result in a disturbance of Ca(2+) homeostasis, iodoacetate impairment leads to an inhibition of energy metabolism, suppression of protein synthesis and generation of oxygen free radicals. In this study these three lesion models were used to investigate the effects of the nootropic drug Cerebrolysin (Cere) on the survival of cortical neurons in culture and on the occurrence of apoptosis. The viability of the cells was evaluated with the colorimetric MTT-reduction assay. Apoptosis was detected with Bisbenzimide (Hoechst:33258), a fluorescent DNA stain. Administration of Cere resulted in dose dependent neuroprotection independent from the kind of lesion. In the glutamate model the drug almost doubled neuronal viability compared to lesioned controls. After acute glutamate exposure Cere reduced the number of apoptotic cells significantly. In spite of the protective efficacy after cytotoxic hypoxia induced by iodoacetate, the drug significantly increased the number of apoptotic neurons, indicating a shift from necrosis to apoptosis. In contrast to previous studies investigating acute ionomycin lesions, the chronic Ca(2+)-overload used here did not increase the abundance of apoptosis compared to the unlesioned control. Summarizing the findings it can be suggested that Cere is able to stabilize Ca(2+) homeostasis, to protect protein synthesis and to counteract neuronal death in different in vitro medels of ischemia.

Journal Article↗
PubMed1986

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers.

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1-selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F-actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.

Actins↗
PubMed1990

Inhibition of Ly-6A antigen expression prevents T cell activation.

Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression. Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect of antisense oligonucleotides on stimulation of preactivated D10 cells. Preactivation of D10 cells with Con A + IL-1 renders them receptive to secondary stimulation by other lymphokines. In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with IL-2, IL-4 + IL-1, or PMA + ionomycin. We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.

Animals↗
PubMed2002

The effect of activation of Mammalian oocytes on remodeling of donor nuclei after nuclear transfer.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization is essential both for intracytoplasmic sperm injection and for nuclear transfer (NT). Therefore, with the goal of producing haploid activated oocytes, we evaluated whether butyrolactone I and bohemine, either alone or in combination with ionomycin, are able to activate young matured mammalian oocytes. Furthermore, the effect on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of cyclin-dependent kinases (CDKs) in metaphase II bovine oocytes induces parthenogenetic activation in a dose dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). The effect of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis, and development to blastocyst of embryos produced by somatic nuclear transfer were studied. Pronuclear formation was significantly higher when activation lasted 5 h compared to 3 h for both ethanol-cycloheximide and ionomycin-bohemine treatment. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12-h labeling 100% of embryos synthesized DNA in both groups. Analysis of two-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy, independent of the activation protocol.

Animals↗
PubMed2001

Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis.

BACKGROUND: Activated platelets have been recently reported to produce platelet microparticles and to enhance platelet-leukocyte interaction. The precise role of platelets in systemic inflammatory response syndrome (SIRS) has not been clarified. The objective of this study was to evaluate microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. METHODS: Twenty-six patients with severe SIRS (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 12 healthy volunteers were studied. The severe SIRS was caused by trauma in 12 patients and sepsis in 14. Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding, and platelet-polymorphonuclear leukocyte (PMNL) binding were measured by flow cytometry in the presence or absence of ionomycin, N-formyl-methionyl-leucyl-phenylalanine, or anti-CD62p monoclonal antibody. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood were also measured. RESULTS: Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding with or without ionomycin, and platelet-PMNL binding with ionomycin significantly increased in patients with severe SIRS in comparison with values in normal volunteers. The increased platelet-leukocyte binding in severe SIRS patients was markedly inhibited by P-selectin blockade and was not enhanced by N-formyl-methionyl-leucyl-phenylalanine. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood also increased in these patients. CONCLUSION: Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. Enhanced platelet-leukocyte interaction is dependent on P-selectin expression and may be involved in the systemic inflammatory response after severe inflammatory insult.

Adolescent↗
PubMed1989

Dendritic epidermal T cells: activation requirements and phenotypic characterization of proliferating cells.

Dendritic epidermal T cells (DETC) are CD45+, Thy-1+, CD5-, CD8-, CD4- murine lymphocytes that express surface-bound CD3 antigens associated with T cell receptor gamma/delta heterodimers. Using epidermal cells greatly enriched for DETC and depleted of Langerhans cells, we found that DETC have growth requirements quite different from those of accessory cell-depleted lymph node and splenic T cells. Although the latter cells strongly proliferate in response to phorbol myristate acetate (PMA) + ionomycin, DETC, when exposed to interleukin-1 (IL-1), interleukin-3 (IL-3), concanavalin A (ConA), PMA, and ionomycin used either alone or in combination, do not exhibit significant mitotic activity. Recombinant interleukin 2 (rIL-2), albeit ineffective by itself, leads to vigorous proliferation of DETC when used with either ConA or PMA + ionomycin + IL-1. In contrast, the combination of PMA and recombinant interleukin-4 (rIL-4), which triggers growth of lymph node T cells, does not induce proliferation of DETC. Although a portion of proliferating DETC expressed CD8 antigens, essentially none bore detectable amounts of surface-bound CD4 or CD5 antigens, or both. Continuing stimulation of primary DETC cultures with lectin/lymphokine-rich media results in the propagation of cells with the essential phenotypic features of resident DETC.

Animals↗