Phorbol Myristyl Acetate Research Articles

Selection of Unique Molecules for Cancer Treatment by Distance-Based Method: Hypericin Effect on Respiratory Chain

The heterogeneous composition of tumors presents a significant obstacle to the selection of a single molecule as a potential universal inhibitor of tumor growth. Lipid signaling and cellular metabolism have become the main targets of anticancer treatment in recent years. The protein kinase C (PKC) regulators Gö6976, rottlerin, hypericin, and phorbol myristyl acetate have been identified as agents affecting cellular metabolism. Measurable parameters describing metabolism, endocytosis, and respiration were subjected to a distance-based computational procedure for higher dimensions to complement and extend the knowledge gained from experimental data. The mutual distances of the parameters of the substances applied to the cancer cells in the presence and absence of lipids were calculated within the Lp spaces. The distance-based methods and comparisons of the generalized distances suggested to us the exceptional role of hypericin in heterogeneous systems. Furthermore, our results are confirmed by Western blotting of the levels of respiratory chain proteins and enzymes active in oxidative stress defense in cancer cell monolayers and spheroids. PKCα and PKCδ have been studied for lipid-activated cell signaling. In this study, we attempt to apply the concept of parametric distance in cell signal transduction and activation where the above methods have not yet been used.

Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and NF90/NF110/ILF3.

Immediate early gene (IEG) transcription is rapidly activated by diverse stimuli. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 (encoded by ILF2) and its heterodimeric partner NF90/NF110 (encoded by ILF3) are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90/NF110 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90/NF110 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90/NF110 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90/NF110 chromatin association, while decreasing NF45 chromatin association at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90/NF110 or NF45, doxycycline-mediated knockdown of NF90/NF110 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of transcription, RNA and protein. Dynamic chromatin association of NF45 and NF90/NF110 at IEG promoters are observed upon stimulation, and NF45 and NF90/NF110 contribute to inducible transcription of IEGs. NF45 and NF90/NF110 operate as chromatin regulators of the immediate early response.

Changes in the Th1 : Th2 Cytokine Bias in Pregnancy and the Effects of the Anti-Inflammatory Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-ProstaglandinJ2

Pregnancy is a complex immunological state in which a bias towards T helper 2 (Th2) protects the fetus. Evidence suggests that proinflammatory cytokines increase the risk of poor neonatal outcome, independently of the direct effect of preterm labour. The anti-inflammatory prostaglandin 15-deoxy-Δ12,14-Prostaglandin J2 (15dPGJ2) inhibits nuclear factor Kappa B (NF-κB) in amniocytes and myocytes in vitro and is a ligand for the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor. Here we examine the Th1:Th2 cytokine bias in pregnancy and whether 15dPGJ2 could be used to inhibit the production of the proinflammatory cytokines through inhibition of NF-κB while simultaneously promoting Th2 interleukin 4 (IL-4) synthesis via CRTH2 in T helper cells. Peripheral blood mononuclear cells (PBMCs) from women at 28 weeks, term pre-labour, term labour as well as non-pregnant female controls were cultured with 15dPGJ2 or vehicle control and stimulated with phorbol myristyl acetate (PMA)/ionomycin. The percentage of CD4+ cells producing interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in response to PMA/ionomycin was significantly reduced in pregnancy. 15dPGJ2 reduced IFN-γ and TNF-α production in stimulated T helper cells, but did not alter IL-4 production in CRTH2+ve cells. 15dPGJ2 also reduced phospho-p65 in stimulated PBMCs. In summary, 15dPGJ2 suppresses the Th1 response of PBMCs during pregnancy and active labour whilst maintaining the Th2 response suggesting a therapeutic benefit in reducing neonatal morbidity in inflammation-induced PTL.

Aloe emodin inhibits colon cancer cell migration/angiogenesis by downregulating MMP-2/9, RhoB and VEGF via reduced DNA binding activity of NF-κB

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study we analyzed molecular mechanisms involved in the antimigratory and antiangiogenic activity of this hydroxy anthraquinone in colon cancer cell, WiDr. Our results show that a relatively non toxic concentration of AE suppressed the phorbol-12-myristyl-13-acetate (PMA) induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found AE downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. AE suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression. Taken together these data indicate that AE target multiple molecules responsible for cellular invasion, migration and angiogenesis. Inhibitory effect on angiogenic and metastatic regulatory processes make AE a sensible candidate as a specific blocker of tumor associated events.

All- trans-retinoic acid and Erk1/2 signaling synergistically regulate the expression of CD300B in human monocytic cells

The regulation of the cell-surface receptors that constitute the gene cluster, CD300, also known as the Myeloid Activating/Inhibitory Receptor (MAIR) family, is poorly understood. In the present study, we tested the hypothesis that all- trans-RA (RA), a bioactive form of vitamin A long recognized for its role in regulation of immune cell activities, may be a potent regulator of the expression of human CD300B. In monocytic THP-1 cells, RA (20 nM) alone significantly increased CD300B mRNA within 2 h and up to 20-fold after 24 h; however, CD300B protein determined by flow cytometry and confocal microscopy showed little change. A search for coactivating molecules revealed that phorbol myristyl acetate (PMA), a mimetic of diacylglycerol, alone increased CD300B mRNA by less than 5-fold; however, the combination of at-RA and PMA increased CD300B mRNA nearly 60-fold. Moreover, CD300B protein was increased. CD300B molecules were mainly located on the plasma membrane and in the endosomal compartment, sharing a distribution/recycling pattern similar to transferrin receptor CD71. The induction of CD300B mRNA by PMA required signaling through the MEK/ERK branch of the MAP kinase pathway, as PD98059, a MEK1/2 inhibitor, abrogated this response, while SB203580, an inhibitor of the p38 pathway, had no effect. Our data suggest a model in which RA alone induces a CD300B mRNA response in which transcripts accumulate but remain untranslated and therefore “sterile,” whereas RA combined with signals from the ERK1/2 pathway results in both increased CD300B transcription and protein expression on the cell surface and in endocytic vesicles.

The Neutrophil Respiratory Burst and Bacterial Digestion in Crohn’s Disease

Neutrophils are a key part of the innate immune defence against microbes, using the respiratory burst (RB) to optimise killing and digestion. Previous studies of the neutrophil RB in Crohn's disease (CD) have yielded conflicting results. Superoxide production in response to phorbol-myristyl acetate (PMA) was measured in neutrophils from 100 patients with CD compared to 50 healthy controls (HCs) and 50 patients with ulcerative colitis (UC). A further 22 CD and 10 HCs were studied using f-Met-Leu-Phe (fMLP), and digestion of E. coli by neutrophils was also evaluated. The mean ± SEM PMA-stimulated RB (nmol O(2)/10(6) cells/min) was 10.86 ± 0.26 in HCs, 9.76 ± 0.23 in CD (P=0.02) and 10.04 ± 0.28 in UC (P=0.09 vs HC and 0.47 vs CD). No significant effect of age, gender or medication was observed. The RB in three patients with presumed CD was found to be in the range expected in patients with inherited neutrophil disorders. Stimulation with fMLP was calcium dependent and attenuated in patients on 5-ASA. Digestion of E. coli by neutrophils was not different in HC vs CD (21.6 vs 20.53%, P=0.60). The significant reduction in neutrophil RB in CD does not appear to result in defective bacterial digestion and is therefore unlikely play a major role in pathogenesis. Three patients in this cohort of patients with presumed idiopathic CD were found to have a profound defect of the neutrophil RB. A high index of suspicion for such patients is prudent, as their prognosis can be improved by altering or augmenting the conventional treatment regimens employed for CD.

Stimulation of human macrophages (THP-1) using Toll-like receptor-2 (TLR-2) agonist decorated nanocarriers

The purpose of this study was to prepare and characterize nanocarrier systems, which allow the application of pDNA vaccines and adjuvants to mucosal vaccination. Chitosan from a vegetal source (Agaricus bisporus) and of GMP quality was used to synthesize the derivative 6-O-carboxymethyl-N,N,N-trimethylchitosan (CM-TMC). Toll-like receptor-2 (TLR-2) agonist, Pam3Cys, was synthesized and coupled to CM-TMC through a polyethylene glycol (PEG) spacer. Successively, Pam3Cys decorated nanocarriers were prepared by complexation with plasmid DNA (pDNA) expressing green fluorescence protein (GFP), and characterized with respect to their physicochemical properties and protection of the included plasmid against DNase I enzymatic degradation. In vitro studies using phorbol 12-myristyl 13-acetate (PMA) stimulated macrophage-like THP-1 (mTHP-1) cells were focused on cytotoxicity of both polymers and particles, and their potential to stimulate IL-8 release via the TLR-2 pathway. Our results showed that the TLR-2 functionalized pDNA nanocarriers have the ability to complex and to protect pDNA against enzymatic degradation. pDNA nanocarriers were of around 400 nm in size, and displayed a positive zeta potential of 27.9 ± 1.6 mV. Chitosan, CM-TMC, and Pam3Cys-functionalized CM-TMC polymers displayed cytotoxicity on mTHP1 cells in a concentration-dependent manner, which decreased by 50-fold on complexation with pDNA. In addition, decorated pDNA nanocarriers induced IL-8 secretion by mTHP-1 macrophages, which was increased by 10-fold as compared to nondecorated carriers.

Activation of BKCa channels via cyclic AMP‐ and cyclic GMP‐dependent protein kinases by eugenosedin‐A in rat basilar artery myocytes

The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases. Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities. Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current. Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.

Use of Quantitative Flow Cytometry to MeasureEx VivoImmunostimulant Activity of Echinacea: The Case for Polysaccharides

When directly exposed to various echinacea fractions, human leukocytes ex vivo are strongly stimulated to proliferate and to produce immunostimulation and inflammatory cytokines. A comparison of fractions containing lipoidal small molecules and high-molecular-weight water-soluble polysaccharides indicates that the latter are substantially more potent as immunostimulants. Echinacea purpurea (L.) Moench, E. angustifolia DC, and E. pallida (Nutt.), Nutt. extracts, and each plant part contain significantly potent constituents. Flow cytometric techniques were utilized. This study was undertaken to determine whether flow cytometry could measure immunostimulant activity present in echinacea and, if so, which species produced more activity, which plant part was the most active, and whether the organic soluble or the aqueous extractables were more active. Ex vivo human clinical material was employed. Echinacea extracts were analyzed using flow cytometric techniques. The immunostimulation assays were measured in triplicate. Samples dissolved in dimethyl sulfoxide (DMSO) were added to 200 microL of heparinized blood mixed with 50 muL of phosphate buffer, vortexed, and incubated to allow adequate time for immune-cell stimulation. Fifty (50) microL of the stimulated blood samples were added to each of a reagent cocktail consisting of 20 microL of CD4FITC/CD69PE/CD3PerCP expressed on the helper/inducer T-lymphocyte subset; CD8FITC/CD69/PE/ CD3PerCP expressed on the human suppresser/cytotoxic T-lymphocytes and on a subset of natural killer lymphocytes; CD19FITC/CD69PE/CD45PerCP expressed on B-lymphocytes; or CD56FITC/CD69PE/CD45PerCP expressed on NK lymphocytes. Four hundred and fifty (450) microL of 1 X FACS lysing solution was added and incubated in the dark (rt, 30 minutes) and then subjected to flow cytometric analysis. All reported readings are the average of several determinations. Positive controls consisted of phorbol myristyl acetate (PMA) (50 ng/mL), phytohemagglutinin (10 microg/mL), CD2/CD2R (positive activation control)(5 microL/250 muL of reaction), and negative controls consisted of dimethyl sulfoxide (2% in RPMI-1640), RPMI-1640 medium, and cyclosporin A (10 microg/mL). The main immunostimulatory activity of echinacea resides in the water-soluble materials rather than the lipoidal small molecules. E. purpurea, E. Pallida, and E. angustifolia leaves, stems, flowering tops, and roots all produce substantial immunostimulatory activity. The use of flow cytometry demonstrates a link between the polysaccharides in echinacea and the biologic immunostimulatory effect that has therapeutic relevance, and strong evidence for this immunostimulant property is presented.

Neutrophil respiratory burst is decreased in scleroderma and normalized by near-infrared mediated hyperthermia

The production of reactive oxygen species (ROS) by fibroblasts has been suggested to contribute to scleroderma pathogenesis. Infrared-mediated hyperthermia has recently been shown to be of benefit in scleroderma. As the contribution of neutrophils and monocytes to ROS formation in scleroderma is unknown, we studied respiratory burst in these cell types. We also aimed to test the hypothesis that near-infrared (IRA) treatment may effect burst activity. We determined respiratory burst in patients with scleroderma (n = 22) and age- and sex-matched controls (n = 20) at baseline, and after high-level stimulation by phorbolmyristyl acetate (PMA) and low-level stimulation by non-opsonized zymosan. Respiratory burst was also assessed before and after a series of infrared-mediated hyperthermia treatments. Unexpectedly, we observed no increase but instead a slight but statistically significant reduction in baseline and zymosan-stimulated respiratory burst in scleroderma neutrophils (P < 0.001) and monocytes (P < 0.005). This decrease in burst activity was nonspecific, as it was also observed in patients with another active inflammatory disease, psoriasis. IRA treatment induced a cell-type-specific normalization of respiratory burst only in neutrophils, but not in monocytes. Intriguingly, neutrophil-specific normalization of ROS formation persisted for 6 weeks after the end of IRA treatment, in concordance with the previously reported clinical responses to this therapy. Neutrophils and monocytes do not exhibit cell-autonomous overproduction of ROS in scleroderma, thereby implicating fibroblasts as main source for clinically relevant ROS accumulation. Furthermore, repeated mild infrared-mediated hyperthermia exerts a lasting cell-type-specific effect on neutrophils.

Dual β-adrenergic modulation in the immune system: Stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between β-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which β-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that β-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.

RNA regulator NF90 is essential for T‐cell homeostasis and normal IL‐2 expression

T-cell expression of IL-2 is regulated at the levels of transcription, posttranscriptional mRNA stabilization and translation. NF90 is a double-stranded RNA-binding protein subunit of a native nuclear complex that binds to the nuclear factor of activated T-cells (NF-AT) target DNA sequence in the IL-2 promoter. NF90 binding to AU-rich elements (ARE) in the 3′ untranslated region (UTR) stabilizes IL-2 mRNA and facilitates nuclear export. Mice with targeted disruption of NF90 die after birth due to defective terminal differentiation of skeletal muscles and respiratory failure. The molecular mechanisms involve NF90 binding to the AREs in the 3′ UTRs of MyoD, myogenin and p21Cip1/Waf1 mRNAs, conferring stability and translational expression (Shi, Zhao, Qiu, Godfrey, Vogel, Rando, Hu and Kao, J Biol Chem 280:18981–18989, 2005). To characterize the contributions of NF90 to immune development and T-cell function, fetal liver cells from NF90 gene-targeted mice were transplanted into irradiated recombination activating gene (RAG)-2(−/−) and IL2R (−/−) adult mice that lack T-, B- and NK cells. Compared to NF90(+/+) and NF90(+/−) donors, NF90(−/−)-RAG reconstituted mice demonstrated decreased numbers of CD4+ and CD8+ T-cells in peripheral blood. NF90(−/−)-RAG thymic, splenic and purified splenic CD4+ T-cells demonstrated profound decreases in IL-2 secretion following primary stimulations with phorbol myristyl acetate + ionomycin, concanavalin A or anti-CD3 + CD28. Deficiency of NF90 is associated with T-cell lymphocytopenia in vivo and impaired IL-2 secretion in vitro in reconstituted T-cells. Supported by NIH R01AI39624.

Characterization of two candidate genes, NCoA3 and IRF8, potentially involved in the control of HIV-1 latency

BackgroundThe persistence of latent HIV-1 reservoirs is the principal barrier preventing the eradication of HIV-1 infection in patients by current antiretroviral therapy. It is thus crucial to understand the molecular mechanisms involved in the establishment, maintenance and reactivation of HIV-1 latency. Since chromatin remodeling has been implicated in the transcriptional reactivation of the HIV-1 promoter, we assessed the role of the histone deacetylase inhibitor sodium butyrate (NaB) on two HIV-1 latently infected cell lines (U1 and ACH-2) gene expression.ResultsAnalysis of microarrays data led us to select two candidate genes: NCoA3 (Nuclear Receptor Coactivator 3), a nuclear receptor coactivator and IRF8 (Interferon Regulatory Factor 8), an interferon regulatory factor. NCoA3 gene expression is upregulated following NaB treatment of latently infected cells whereas IRF8 gene expression is strongly downregulated in the promonocytic cell line following NaB treatment. Their differential expressions were confirmed at the transcriptional and translational levels. Moreover, NCoA3 gene expression was also upregulated after treatment of U1 and ACH-2 cells with phorbol myristyl acetate (PMA) but not trichostatin A (TSA) and after treatment with NaB of two others HIV-1 latently infected cell lines (OM10.1 and J1.1). IRF8 gene is only expressed in U1 cells and was also downregulated after treatment with PMA or TSA. Functional analyses confirmed that NCoA3 synergizes with Tat to enhance HIV-1 promoter transcription and that IRF8 represses the IRF1-mediated activation through the HIV-1 promoter Interferon-stimulated response element (ISRE).ConclusionThese results led us to postulate that NCoA3 could be involved in the transcriptional reactivation of the HIV-1 promoter from latency and that IRF8 may contribute to the maintenance of the latent state in the promonocytic cell line. Implication of these factors in the maintenance or reactivation of the viral latency may provide potential new targets to control HIV-1 replication in latent viral reservoirs.

Generation of Tissue factor-rich microparticles in an ex vivo whole blood model

The aim of this study was to investigate the role of blood cells in the expression of tissue factor (TF) and P-selectin in platelets and microparticles from blood stimulated with lipopolysaccharide (LPS), with or without the further addition of phorbol myristyl acetate (PMA). TF activity was found to be associated with platelets after 2 h incubation of whole blood with LPS or LPS + PMA, while no TF activity was detected in microparticles from blood subjected to such stimulation. In blood stimulated for 6 and 24 h, addition of PMA to the samples led to a substantial increase in TF activity associated with microparticles, compared with stimulation with LPS alone. Addition of PMA to blood samples also led to a three-fold increase in the amount of P-selectin found in the isolated microparticle fraction, and a 50% reduction in P-selectin measured in platelets, compared with LPS alone used for stimulation. In a different experiment, TF-rich microparticles were shown to be absorbed very efficiently by neutrophils in a calcium-independent reaction. Our results imply that LPS stimulation of whole blood is associated with a direct transfer of TF from monocytes to platelets in the absence of free TF-rich microparticles, which probably is accounted for by the fusion of TF-rich microparticles with activated platelets exposing P-selectin. Further addition of PMA to samples generates both free TF-rich microparticles as well as enhanced transfer of TF from monocytes to platelets.

Specific cytoskeleton changes during apoptosis accompanying induced differentiation of HL-60 myeloid leukemia cells

Changes in actin filaments and microtubules were studied in the human myeloid leukemia HL-60 cell line during the process of apoptotic cell death accompanying induced differentiation. These cytoskeleton changes were assessed during a 6-day cultivation in the presence of 10(-6) M all-trans retinoic acid (ATRA), a specific inductor of both differentiation into granulocytes and apoptosis, or during a 18-day cultivation in the presence of 1.6 nM phorbol myristylacetate (PMA), which induces differentiation into macrophages. The processes were studied at the morphological level by fluorescence microscopy and, quantitatively, by flow cytometry. The results showed that the actin cytoskeleton underwent specific structural changes during the apoptotic process, but microtubules were not actively involved. In the initial stages of apoptosis, a fine meshwork of actin filaments turned into actin granules that, in the final stages, were transformed into a network of long actin fibres distributed throughout the cytoplasm. These actin structures were considered to play an active role in two main morphological events of apoptosis - formation of blebs and final cell disintegration into apoptotic bodies. In addition, high proportion of cells with apoptotic nuclei and completely destroyed actin structures were found in the differentiating ATRA-treated cell population. Flow cytometric measurement of cytoskeletal proteins content confirmed all these observed changes. Alterations and rearrangements of both cytoskeletal structures are common for the apoptotic cell death of HL-60 cells and they are independent on the course of differentiation.

Protein kinase C inhibits the transplasma membrane influx of Ca2+ triggered by 4-aminopyridine in Jurkat T lymphocytes.

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).

Expression and tyrosine phosphorylation of Cbl regulates macrophage chemokinetic and chemotactic movement.

Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway.

Comparison of the antioxidant properties of wound dressing materials–carboxymethylcellulose, hyaluronan benzyl ester and hyaluronan, towards polymorphonuclear leukocyte-derived reactive oxygen species

In chronic wounds, factors are released which perpetuate inflammatory processes, including polymorphonuclear leukocyte (PMN)-derived reactive oxygen species (ROS), such as superoxide radical (O 2 − ) and hydroxyl radical ( OH) species. The glycosaminoglycan, hyaluronan, has established antioxidant properties towards ROS, although the antioxidant potential of wound dressing biomaterials, such as 75% benzyl esterified hyaluronan (BEHA) and carboxymethylcellulose (CMCH), are less characterised. This study compared the antioxidant properties of high and low molecular weight hyaluronan (HMWT HA and LMWT HA), BEHA and CMCH towards ROS, generated by stimulated PMN in vitro. The antioxidant capacities of each biomaterial were assessed by their inhibition of O 2 − -induced cytochrome C reduction, generated via PMN stimulation by phorbol myristyl acetate (PMA); and their inhibition of OH-induced 2-deoxy- d-ribose degradation, generated by PMA stimulated PMN in the presence of a ferric chloride-EDTA chelate. All biomaterials, except LMWT HA, possessed dose-dependent antioxidant properties against O 2 − , BEHA having greatest antioxidant potential, followed by HMWT HA and CMCH. HMWT HA exhibited the highest dose-dependent antioxidant properties towards OH, followed by BEHA and CMCH. LMWT HA demonstrated no antioxidant properties towards OH. These antioxidant activities, particularly towards O 2 − , may be beneficial in removing the initial source of ROS necessary for the secondary formation of OH, implicated as a causal factor for the extensive metabolic alterations observed in chronic wounds.

Differential Internalization of the Prostaglandin F2α Receptor Isoforms: Role of Protein Kinase C and Clathrin

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F(2alpha) (PGF(2alpha)). Alternative mRNA splicing gives rise to two isoforms, FP(A) and FP(B), which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP(A) and FP(B) receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF(2alpha) and were examined by immunofluorescence microscopy. In the absence of PGF(2alpha), FP(A)-expressing cells were labeled predominantly on the cell surface; however, FP(B)-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP(B) isoform. After treatment with PGF(2alpha), FP(A)-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP(A)-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF(2alpha)- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP(B) isoform, suggesting that the mechanisms of receptor internalization differ between the FP(A) and FP(B) isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF(2alpha)-induced internalization of the FP(A) isoform but did not block constitutive internalization of the FP(B) isoform. In conclusion, the FP(A) receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP(B) isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.

HDL-holoparticle uptake by alveolar type II cells: effect of vitamin E status.

Alveolar type II cells accumulate vitamin E preferentially from high-density lipoproteins (HDL) and express at least three receptors that are specific for HDL. The expression of these receptors increases in response to vitamin E deficiency. Beside receptors for specific lipid transfer from HDL, cubilin and megalin, several other receptors that mediate HDL-particle uptake were found in the lung. We hypothesize that alveolar type II cells also exhibit the HDL-particle uptake and that this process can be regulated by the vitamin E status. By confocal laser microscopy and flow cytometry we showed that type II cells accumulate protein-labeled HDL-particle. Vitamin E depletion in rats increased HDL-particle uptake in alveolar type II cells and the expression of megalin. The expression of cubilin did not change. Refeeding with vitamin E reversed HDL-particle uptake and megalin expression. Long-time incubation of type II cells with phorbol myristyl acetate (PMA) reduced HDL-holoparticle uptake and megalin expression. We assume that alveolar type II cells exhibit HDL-holoparticle uptake mediated by megalin and cubilin. Megalin represents the regulated element of the megalin/cubilin receptor-cooperation and can be modulated by protein kinase C.