Concentration Of PMA Research Articles

Fluorescence enhancement of near infrared cell membrane probe by β-cyclodextrin supramolecular interaction

In this paper, oxazolepyridine and coumarin units were combined to prepare cell membrane fluorescent probe (PM-A) through aldol condensation reaction. PM-A had long wavelength emission (665–739 nm), high molar extinction coefficient (around 3.14 × 104 M−1cm−1) and large Stokes shift (150–219 nm). The photo-stability test showed that PM-A had a high relative residual absorption (93%) after 6 h of continuous illumination. In addition, the supramolecular interaction between PM-A and β-cyclodextrin was evaluated and applied to cell imaging. Probe PM-A had no fluorescence in water, but had obvious fluorescence signal in β-cyclodextrin solution. With the addition of β-cyclodextrin (0–800 eq), the fluorescence quantum yield of PM-A increased from 0% to 45%. Laser confocal experiments showed that when only PM-A was added, the probe concentration needed to be 15 μM to image the cell membrane obviously. In sharp contrast, with the help of β-cyclodextrin solution, the concentration of probe PM-A only needed 0.5 μM to quick and high specificity image cell membranes. Moreover, PM-A had high photo-bleaching resistance and long retention time after adding β-cyclodextrin. These excellent properties of PM-A were of great significance to the study of cell membrane.

Corrigendum for Oda et al., volume 291, 2006, p. C104–C113

CorrigendumCorrigendum for Oda et al., volume 291, 2006, p. C104–C113Published Online:19 Apr 2021https://doi.org/10.1152/ajpcell.00614.2005_COROriginal articleMoreSectionsPDF (336 KB)Download PDFDownload PDFPlus ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Oda T, Hirota K, Nishi K, Takabuchi S, Oda S, Yamada H, Arai T, Fukuda K, Kita T, Adachi T, Semenza GL, Nohara R. Activation of hypoxia-inducible factor 1 during macrophage differentiation. Am J Physiol Cell Physiol 291: C104–C113, 2006. First published February 15, 2006; doi:10.1152/ajpcell.00614.2005.—In the above article, blots in Figs. 2 and 7 were published incorrectly. Below are the corrected figures and revised Fig. 7 legend. The authors assert that the corrections do not affect the conclusions of the figures or the study.Figure 2.Dose- and time-dependent effect of differentiation-inducing agents on HIF-1 protein expression under 20% O2 conditions. THP-1 cells were exposed to the indicated concentration of PMA for 72 h (A), 100 nM PMA for the indicated time (B), or 1 μM ATRA for the indicated time (C), and harvested for immunoblot assay using anti-HIF-1α (top), HIF-1β (middle), or β-actin (bottom) antibody. D: Jurkat T cells were exposed to 100 nM PMA for the indicated time and harvested for immunoblot assay using anti-HIF-1α (top), HIF-1β (middle), or β-actin (bottom) antibody.Download figureDownload PowerPointFigure 7.MAPK, AKT, the translational regulators p70S6K, S6 ribosomal protein, 4E-BP1, and eIF-4E phosphorylation are induced by PMA treatment. A, B, and D: whole cell lysates were prepared and subjected to immunoblot assays using antibodies specific for phosphorylated (Thr-202/Tyr-204), total p42ERK2/p44ERK1 MAPK, phosphorylated (Ser-473) or total AKT. THP-1 cells were exposed to 100 nM PMA for the indicated time under 20% O2 conditions. Three time points in the original figure were spliced out to prepare 7B to combine bands corresponding to time points 48 h and 72 h next to each other, which is indicated in the figure as well. C: human peripheral blood monocytes (mono) were allowed to differentiate to macrophages (macro). In D, whole cell lysates were prepared and subjected to immunoblot assays using antibodies specific for phosphorylated (Thr-389) p70S6K (PS6K), phosphorylated (Ser-235/Ser-236) S6 ribosomal protein (S6R), phosphorylated (Ser-65) 4E-BP1 (P4E-BP1), and phosphorylated (Ser-209) eIF-4E (PeIF-4E).Download figureDownload PowerPointThis article has no references to display. Previous Back to Top FiguresReferencesRelatedInformationRelated articlesActivation of hypoxia-inducible factor 1 during macrophage differentiation 01 Jul 2006American Journal of Physiology-Cell Physiology More from this issue > Volume 320Issue 4April 2021Pages C666-C667 Crossmark Copyright & PermissionsCopyright © 2021 the American Physiological Society.https://doi.org/10.1152/ajpcell.00614.2005_CORPubMed33871285History Published online 19 April 2021 Published in print 1 April 2021 PDF download Metrics Downloaded 256 times

Establishment of PMA Real-Time PCR Method to Detect Viable Cells of Listeria monocytogenes and Salmonella spp. in Milk and Dairy Products

The infection with Salmonella spp., Listeria monocytogenes are the most contamination events in the milk and dairy products. There are many disadvantages of conventional culture-based methods, which are still recognized as the “gold standard” for identifying pathogenic bacteria. Thus, the aim of current study was to develop reliable and rapid method for the detection of bacteria causing foodborne. For the establishment of PMA Real-time PCR method for co-detection of Salmonella spp., Listeria monocytogenes , bacteria strains, including Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 19115; ATCC 19111), Salmonella enterica (ATCC 19115), Staphylococcus aureus (ATCC 25923), Vibrio parahaemolyticus (ATCC 17802), Shigella flexneri (ATCC 12022), Bacillus aureus (ATCC 11778) were enrolled into the establishment of current protocol. The concentration of PMA, primer concentration, probe concentration, and primer annealing temperature, specificity and sensitivity were evaluated. As the results, we successfully established the protocol of PMA Real-time PCR for co-detection of Salmonella spp. and Listeria monocytogenes on milk product. The concentration of PMA was determined as 50 I¼M. The sensitivity of established protocol were 101 CFU/ml for detection of Salmonella spp., and 102 CFU/ml for detection of Listeria monocytogenes . The current PMA Real-time PCR protocol was applied to detect the contamination of twenty local milk samples. No sample was co-contaminated with Salmonella spp. and Listeria monocytogenes . These results were similar to its performed by conventional culture-based methods. In summary, the current established PMA (50 I¼M) Real-time PCR could be applied for the co-detection of Salmonella spp. and Listeria monocytogenes on milk and dairy food.

Ethanol Inhibition of a T‐Type Ca2+ Channel Through Activity of Protein Kinase C

T-type calcium channels (T-channels) are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol (EtOH) effects and the underlying mechanism of action in human embryonic kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans rats. Whole-cell voltage-clamp recordings were performed at 32 to 34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10-minute application of EtOH or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate [PMA]). We recorded T-type Ca²⁺ currents (T-currents) from 3 channel isoforms (CaV3.1, CaV3.2, and CaV3.3) before and during administration of EtOH. We found that only 1 isoform, CaV3.2, was significantly affected by EtOH. EtOH reduced current density as well as producing a hyperpolarizing shift in steady-state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T-currents from DRG neurons that are known to be enriched in CaV3.2. A myristoylated PKC peptide inhibitor (MPI) blocked the major EtOH effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of EtOH, while higher concentration PMA (1 μM) did not, suggesting that the EtOH effects operate through activation of PKC and were mimicked by lower concentration of PMA. EtOH primarily affects the CaV3.2 isoform of T-type Ca²⁺ channels acting through PKC, highlighting a novel target and mechanism for EtOH effects on excitable membranes.

Effect of fluvastatin on apoptosis in human CD4 + T cells

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4 + T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4 + T cells but not in CD4 + T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4 + T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4 + T cells treated with fluvastatin (10 μM). In strongly activated CD4 + T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4 + T cells. Treatment with fluvastatin (10 μM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4 + T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4 + T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.

Analysis by Fluorescence Resonance Energy Transfer of the Interaction between Ligands and Protein Kinase Cδ in the Intact Cell

The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mumol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [3H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mumol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission. Polymyxin B (7 and 21 mumol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mumol/l. Polymyxin B (21 mumol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mumol/l), 8-bromo-cyclic AMP (90 mumol/l), tetraethylammonium chloride (300 mumol/l), and idazoxan (0.1 mumol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 mumol/l) by itself inhibited the S-I outflow of radioactivity. In the presence of a concentration of PMA (0.1 mumol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mumol/l) and 8-bromo-cyclic AMP (90 mumol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

UCN-01 (7-Hydroxystauorsporine) Blocks PMA-Induced Maturation and Reciprocally Promotes Apoptosis in Human Myelomonocytic Leukemia Cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21CIP1 induction, p27KIP1 cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21CIP1 mRNA antisense construct, suggesting that p21CIP1 expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program. Key Words:PMA, UCN-01, Differentiation, Apoptosis

A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.

Polycyclic aromatic hydrocarbons enhance the production of phorbol 12-myristate 13-acetate-induced superoxide ions in human monocytes

Monocytes, separated from peripheral blood, preincubated with a mixture of polycyclic aromatic hydrocarbons (PAHs) show an enhanced production of superoxide ions (O 2 −) when the cells are stimulated with phorbol 12-myristate 13-acetate (PMA, direct activator of protein kinase C). When opsonized-zymosan is used as a stimulus (receptor-dependent stimulus), no enhanced production of O 2 − is observed. Superoxide production increases dose dependently up to a PAH concentration of 5 μg/ml. Although the effect was rather small (125–145% of the control value), it was significant and reproducible. Similar enhancing activity was also observed in the production of hydrogen peroxide (H 2O 2) excluding an inhibitory effect of PAHs on the enzyme superoxide dismutase (SOD). Since the effect is related to the concentration of PMA and in the absence of stimulus, the O 2 − is undetectable in both the control and in the PAHs-treated cells, it is concluded that the over production of O 2 − is due to an increased activity of the NADPH oxidase.

Regulatory Effects of Soluble Growth Factors on Choriocapillaris Endothelial Growth and Survival

The effects of soluble growth factors on regulating the survival and growth of choriocapillaris endothelial (CCE) cells were investigated in vitro. CCE cells were cultured in a serum-free medium in the presence or absence of various soluble growth factors. Cell growth and cell viability were assessed by counting viable cells. Results showed that acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) significantly stimulated CCE growth in a dose-dependent manner. Platelet-derived growth factor BB (PDGF, 0.4–10 ng/ml), insulin-like growth factor (IGF, 0.4–10 ng/ml) or insulin (0.4–10 µg/ml) alone did not affect the growth of CCE cells. In the presence of insulin (10 µg/ml), however, PDGF stimulated CCE growth in a dose-dependent manner. By contrast, transforming growth factor β1 (TGF-β1) induced CCE death in the absence of other growth factors and inhibited the CCE growth induced by aFGF, bFGF or VEGF. When CCE cells had been cultured in media containing aFGF, bFGF, VEGF or the combination of PDGF and insulin for 48 h, withdrawal of aFGF, bFGF or VEGF, but not PDGF, from the media resulted in CCE death. The CCE death induced by either an introduction of TGF-β1 or withdrawal of aFGF, bFGF or VEGF was defined as apoptosis based on morphologic characteristics (condensation and fragmentation of nuclei, shrinkage of cells in size) and DNA fragmentation in multiples of approximately 180 base pairs. Phorbol 12-myristate 13-acetate (PMA, 2 or 5 nM) protected CCE cells against apoptosis induced by the introduction of TGF-β1 and withdrawal of aFGF, bFGF or VEGF, while H7 (50 µM), but not HA1004 (50 µM), abrogated the protective effect of PMA on CCE apoptosis. However, cycloheximide (0.1 µM), a protein synthesis inhibitor, was only effective to protect CCE cells from apoptosis induced by aFGF, bFGF or VEGF withdrawal, but not that induced by the introduction of TGF-β1. Moreover, cycloheximide and a low concentration of PMA (2 nM) showed an additive effect on protecting CCE cells from apoptosis as the result of a growth factor withdrawal. These data may suggest that some growth factors, such as aFGF, bFGF, VEGF and TGF-β1, play a more critical role than the others, such as PDGF and IGF, in the regulation of CCE growth and survival. CCE apoptosis as a result of withdrawal of a survival factor or of receiving a death factor such as TGF-β1 may involve different mechanisms, which merits further study.

Cytokines and cross-linking of sIgM augment PMA-induced activation of human leukaemic CD5+ B cells.

Purified leukaemic CD5+ B cells obtained from patients with B cell chronic lymphocytic leukaemia (B-CLL) undergo activation and differentiation following in vitro stimulation with optimal concentrations of the phorbol ester PMA. This paper examines the ability of exogenous cytokines, anti-Ig antibodies, or combinations of these, to enhance or replace the activation signal provided by PMA to different populations of leukaemic B cells. Proliferation induced by PMA was enhanced 2-20-fold when the cells were co-cultured with either anti-Ig, IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha. Moreover, the combination of anti-Ig, PMA and any one of the above cytokines further enhanced proliferation. Anti-Ig and exogenous cytokines were also capable of inducing proliferation in leukaemic B cells cultured with a non-mitogenic concentration of PMA. When taken together with the finding that IL-2, IL-4, IL-13, IFN-gamma and TNF-alpha prevent in vitro apoptosis of leukaemic CD5+ B cells, the results presented here suggest that these cytokines, in conjunction with signals delivered via sIg, may play a role in the proliferation of leukaemic B cells in vivo and, consequently, the pathogenesis of B-CLL.

Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester.

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down-regulation of these PKC isozymes PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.

Persistently enhanced sensitivity of pancreatic islets from ob/ob mice to PKC-stimulated insulin secretion.

Islets from 2-wk-old ob/ob and lean littermate mice were cultured for 4-12 days and then perifused or statically incubated to identify early-onset differences in their regulation of insulin secretion. Islets from these young ob/ob and lean mice increased insulin secretion similarly in response to glucose (10 or 20 mM), whereas responsiveness to glucose plus acetylcholine (10 microM) was greater in islets from ob/ob mice than lean mice. This phenotype-specific effect of acetylcholine was mimicked by phorbol 12-myristate 13-acetate (PMA, 100 nM), a protein kinase C (PKC) agonist, whereas prior downregulation of PKC abolished this phenotype-specific effect of acetylcholine. A high concentration of PMA (1 microM) equally and substantially increased insulin secretion from islets of ob/ob and lean mice, suggesting an enhanced regulatory sensitivity rather than altered responsiveness of the PKC system in islets of ob/ob mice. Addition of BAY K 8644, a Ca2+ channel agonist, to the perifusate enhanced acetylcholine-induced insulin secretion from islets of lean mice to attain the high rates observed in islets from ob/ob mice exposed to acetylcholine alone. We propose that acetylcholine-induced PKC regulation of insulin secretion is altered in islets from ob/ob mice, that this alteration may directly or indirectly involve Ca2+ channels, and that it persists even when islets are cultured for up to 12 days.

Identification of the Proteolytic Enzyme Which Cleaves Human p75 TNF Receptorin Vitro

The extracellular domains of the human 55 and 75 kD TNF receptors (p55 and p75 TNF-R) are proteolytically cleaved to produce 30 and 40 kD soluble fragments, respectively. In this study, the enzymatic activity involved in the cleavage of human p75 TNF-R, named TNF-R releasing enzyme (TRRE), was identified in the culture supernatant of PMA-stimulated THP-1 cells using an activity assay system established by our group. When THP-1 cells were stimulated with PMA, TRRE was released rapidly into the supernatant, reaching maximal activity within 3 hours. The release of TRRE into the culture supernatant depended on the concentration of PMA and FCS. TRRE activity was partially inhibited by chelating agents, suggesting that TRRE may be a metalloprotease-like enzyme. This is the first successful attempt to establish a stable TRRE source with a reliable assay system.

Dynamic-Mechanical Study of the Dynamics of Polymer Blends near the Glass Transition

Poly(methyl acrylate) (PMA) and its blends with poly(4-hydroxystyrene) and a poly(4-hydroxy-co-4-methoxystyrene) have been studied by dynamic-mechanical spectroscopy at temperatures close to the calorimetric T g and frequencies between 0.3 and 30 Hz. The results for the imaginary part of the elastic modulus lead to broad master curves, which width increases with the content of the concentration of the second polymer. Even though the results can be described reasonably well using a Kohlrausch-Williams-Watts relaxation function, rather low values of the stretching parameter β are necessary in order to obtain good agreement with the experimental data. The Havriliak-Negami relaxation function leads to slightly worse agreement with experiment. The fragility parameter of the blends decreases with the concentration of PMA. The qualitative trend of the results may be interpreted in terms of the existence of concentration fluctuations in the blends

Interleukin-4 Inhibits Both Paracrine and Autocrine Tumor Necrosis Factor-α-Induced Proliferation of B Chronic Lymphocytic Leukemia Cells

AbstractThe proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)- alpha and PMA. The concentration of PMA was found to be critical and showed a sharp optimum. In most cases maximal proliferation was obtained with as little as 0.1 ng/mL PMA. In all cases tested, TNF- alpha-induced proliferation could be inhibited completely by the addition of low doses of interleukin-4 (IL-4). Maximal inhibition was already found with 400 pg/mL IL-4. Inhibition by IL-4 was not caused by a downmodulation of TNF receptors. Apart from TNF-alpha, IL-2 was also in synergy with PMA able to induce proliferation in B-CLL cells of some patients. This IL-2-induced proliferation could be inhibited both by IL- 4 and by neutralizing anti-TNF-alpha antibodies. This shows that TNF- alpha also can act as an autocrine growth factor. These data indicate that TNF-alpha is an important growth factor for neoplastic B-CLL cells and that IL-4 provides a tool to interfere with this TNF-alpha response.

Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells

The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)- alpha and PMA. The concentration of PMA was found to be critical and showed a sharp optimum. In most cases maximal proliferation was obtained with as little as 0.1 ng/mL PMA. In all cases tested, TNF- alpha-induced proliferation could be inhibited completely by the addition of low doses of interleukin-4 (IL-4). Maximal inhibition was already found with 400 pg/mL IL-4. Inhibition by IL-4 was not caused by a downmodulation of TNF receptors. Apart from TNF-alpha, IL-2 was also in synergy with PMA able to induce proliferation in B-CLL cells of some patients. This IL-2-induced proliferation could be inhibited both by IL- 4 and by neutralizing anti-TNF-alpha antibodies. This shows that TNF- alpha also can act as an autocrine growth factor. These data indicate that TNF-alpha is an important growth factor for neoplastic B-CLL cells and that IL-4 provides a tool to interfere with this TNF-alpha response.

Interleukin-1 beta induction of TNF-alpha gene expression: involvement of protein kinase C.

In the human astroglioma cell line CH235-MG, interleukin-1 beta (IL-1 beta) induces transcriptional activation of the tumor necrosis factor-alpha (TNF-alpha) gene, resulting in expression of TNF-alpha mRNA and biologically active TNF-alpha protein. This study was undertaken to elucidate intracellular signaling pathways involved in IL-1 beta induction of the TNF-alpha gene. We demonstrated that the protein kinase C (PKC) activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) in concert with Ca++ ionophore A23187 induced expression of TNF-alpha mRNA and protein, whereas an inactive PMA analogue (alpha PMA) had no effect. Various cyclic nucleotide activators such as 8-Bromo cAMP, cholera toxin, and forskolin had no effect on TNF-alpha production. Two PKC inhibitors, H7 and staurosporine (SS), abrogated IL-1 beta induced TNF-alpha expression in a dose-dependent fashion. Treatment of CH235-MG cells with a high concentration of PMA (1 microM) for an extended period of time (48 h) caused a greater than 90% reduction in total PKC activity. Further strengthening a role for PKC in this cytokine response is the fact that IL-1 beta was no longer able to induce TNF-alpha expression in these PKC depleted cells. Last, IL-1 beta treatment produced an increase of total PKC activity in CH235-MG cells. Taken together, these data demonstrate that IL-1 beta induces TNF-alpha gene expression in CH235-MG cells in a PKC-dependent manner.

Activation of the CD3/T cell receptor (TcR) complex or of protein kinase C potentiate adenylyl cyclase stimulation in a tumoral T cell line: involvement of two distinct intracellular pathways.

A monoclonal antibody (OKT3) directed against the T cell receptor (TcR)/CD3 molecular complex, as well as a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) were added to a culture of tumoral Jurkat T cells, in order to precise the sequence of intracellular signals leading to T cell activation. The experiments were performed in the presence or in absence of various stimulators of adenylate cyclase (AC) such as forskolin (FK), cholera toxin (CT) or prostaglandin E2 (PGE2). OKT3 increased inositol phosphate (IP) production; in parallel, it induced a slight accumulation of cAMP. The effect was markedly potentiated in presence of FK or CT, and to a lesser extent in the presence of PGE2. FK stimulated adenylate cyclase of Jurkat cell membranes, but the effect was not potentiated by OKT3, suggesting that potentiation of cAMP accumulation requires intact cells and is not mediated by direct receptor coupling. On the other hand, elevated cAMP accumulation induced a negative feedback on IP production. The effect of OKT3 on cAMP was mimicked by A23187, a Ca2+ ionophore, and abolished in the absence of extracellular Ca2+. PMA had the same effect as OKT3 on basal or FK- and CT-induced accumulation of cAMP. In contrast, it inhibited the PGE2 effect on the cyclic nucleotide. After desensitization of PKC by pretreatment with a high concentration of PMA, the phorbol ester was no longer effective. Under those conditions, facilitation by OKT3 of FK-induced accumulation of cAMP was preserved, whereas potentiation by the monoclonal antibody of the PGE2 stimulation of AC was even enhanced. The data indicate that cAMP accumulation indirectly elicited by phospholipase C activation is, at least partly, mediated by IP-dependent Ca2+ mobilization, while PKC is preferentially effective as an inhibitor of PGE2 stimulation.