Ceramide Activates the Stress-activated Protein Kinases

Tumor necrosis factor alpha (TNF alpha) activates the stress-activated protein kinases (SAPKs, also known as Jun nuclear kinases or JNKs) resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NF kappa B to the nucleus resulting in the stimulation of NF kappa B-dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNF alpha activates sphingomyelinases. We show that treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A2, C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C2-ceramide (5 microM) or TNF alpha (10 ng/ml). The prolonged stimulation of this kinase activity by C2-ceramide is similar to that previously reported for TNF alpha. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNF alpha treatment (1.5-fold) and are inhibited by C2-ceramide treatment. TNF alpha also potently stimulates NF-kappa B DNA binding activity and transcriptional activity, but these effects are not mimicked by addition of C2-ceramide or sphingomyelinase to intact cells. Furthermore, TNF alpha, sphingomyelinase, and C2-ceramide induce c-jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNF alpha's biochemical and biological effects

\u3ci\u3eArabidopsis\u3c/i\u3e Accelerated Cell Death 11, ACD11, Is a Ceramide-1-Phosphate Transfer Protein and Intermediary Regulator of Phytoceramide Levels

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1- phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, ‘‘sandwich’’ topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily

Nowa kronika wałbrzyska, t. 3

Wałbrzych i jego słynny „złoty pociąg”, od pierwszej wzmianki na jego temat, zajmuje obecnie szczególne miejsce w wielu informacjach publicystycznych i naukowych. Na nowo rozbudzona ciekawość historyczna naszym miastem i regionem zbiegła się z siedemdziesiątą rocznicą przejęcia miasta przez władze polskie. Każdy niemalże mieszkaniec Wałbrzycha odczuwa ten pozytywny wzrost zainteresowania. Przygraniczne położenie, wielonarodowe wpływy w rozwoju politycznym, gospodarczym i kulturalnym odcisnęły swoje piętno na dziejach miasta, widzianych przez nas jako ciągłość od czasów najdawniejszych po czasy współczesne. I właśnie ta różnorodność stała się nagle niezwykle atrakcyjnym elementem dla przybywających tu turystów zaintrygowanych tajemniczymi historiami. Kolejny, trzeci tom Nowej kroniki wałbrzyskiej stara się jak zwykle uwzględnić wyniki najnowszych prac naukowych lokalnych badaczy, jak i wykorzystać te ustalenia, które posiadają istotną wartość poznawczą. Jak pisze Andrzej Garlicki „Najtrudniejsze w historii jest zrozumienie. Czyli odpowiedź na pytanie, dlaczego wydarzenia potoczyły się tak, a nie inaczej”, dlatego też staramy się zapewnić Czytelnikom szerokie spectrum poruszanych problemów oraz różne wątki wałbrzyskiej historii, podzielone na cztery części.Elżbieta Kwiatkowska-Wyrwis

The challenge to verify ceramide's role of apoptosis induction in human cardiomyocytes - a pilot study

<p>Abstract</p> <p>Background</p> <p>Cardioplegia and reperfusion of the myocardium may be associated with cardiomyocyte apoptosis and subsequent myocardial injury. In order to establish a pharmacological strategy for the prevention of these events, this study aimed to verify the reliability of our human cardiac model and to evaluate the pro-apoptotic properties of the sphingolipid second messenger ceramide and the anti-apoptotic properties of the acid sphingomyelinase inhibitor amitryptiline during simulated cardioplegia and reperfusion ex vivo.</p> <p>Methods</p> <p>Cardiac biopsies were retrieved from the right auricle of patients undergoing elective CABG before induction of cardiopulmonary bypass. Biopsies were exposed to <it>ex vivo </it>conditions of varying periods of cp/rep (30/10, 60/20, 120/40 min). Groups: I (untreated control, n = 10), II (treated control cp/rep, n = 10), III (cp/rep + ceramide, n = 10), IV (cp/rep + amitryptiline, n = 10) and V (cp/rep + ceramide + amitryptiline, n = 10). For detection of apoptosis anti-activated-caspase-3 and PARP-1 cleavage immunostaining were employed.</p> <p>Results</p> <p>In group I the percentage of apoptotic cardiomyocytes was significantly (p < 0.05) low if compared to group II revealing a time-dependent increase. In group III ceramid increased and in group IV amitryptiline inhibited apoptosis significantly (p < 0.05). In contrast in group V, under the influence of ceramide and amitryptiline the induction of apoptosis was partially suppressed.</p> <p>Conclusion</p> <p>Ceramid induces and amitryptiline suppresses apoptosis significantly in our ex vivo setting. This finding warrants further studies aiming to evaluate potential beneficial effects of selective inhibition of apoptosis inducing mediators on the suppression of ischemia/reperfusion injury in clinical settings.</p

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.This work was supported by Fundacao para a Ciencia e Tecnologia through projects PTDC/BIA-BCM/69448/2006 and PEst-C/BIA/UI4050/2011, and fellowships to A. P. (SFRH/BPD/65003) and F. A. (SFRH/BD/80934/2011), as well as by FEDER through POFC - COMPETE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Neuronal Conduction of Excitation without Action Potentials Based on Ceramide Production

International audienceBACKGROUND: Action potentials are the classic mechanism by which neurons convey a state of excitation throughout their length, leading, after synaptic transmission, to the activation of other neurons and consequently to network functioning. Using an in vitro integrated model, we found previously that peripheral networks in the autonomic nervous system can organise an unconventional regulatory reflex of the digestive tract motility without action potentials. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we used combined neuropharmacological and biochemical approaches to elucidate some steps of the mechanism that conveys excitation along the nerves fibres without action potentials. This mechanism requires the production of ceramide in membrane lipid rafts, which triggers in the cytoplasm an increase in intracellular calcium concentration, followed by activation of a neuronal nitric oxide synthase leading to local production of nitric oxide, and then to guanosine cyclic monophosphate. This sequence of second messengers is activated in cascade from rafts to rafts to ensure conduction of the excitation along the nerve fibres. CONCLUSIONS/SIGNIFICANCE: Our results indicate that second messengers are involved in neuronal conduction of excitation without action potentials. This mechanism represents the first evidence-to our knowledge-that excitation is carried along nerves independently of electrical signals. This unexpected ceramide-based conduction of excitation without action potentials along the autonomic nerve fibres opens up new prospects in our understanding of neuronal functioning

Nuclear Interactions Of Super High Energy Cosmic-rays Observed In Mountain Emulsion Chambers

Here we present a summary of joint discussions on the results of three mountain experiments with large-scale emulsion chambers, at Pamir, Mt. Fuji and Chacaltaya. Observations cover gamma quanta, hadrons and their clusters (called "families"). The following topics are covered, concerning the characteristics of nuclear interactions the energy region 1014-1016 eV: (i) rapid dissipation seen in atmospheric diffusion of high-energy cosmic-rays; (ii) multiplicity and Pt increase in produced pi-mesons in the fragmentation region; (iii) existence of large-Pt jets, (iv) extremely hadron-rich family of the Centauro type; (v) exotic phenomena in the extremely high energy region beyond 1016 eV. © 1981.1911125(1977) Acta Univ. Lodz ser. II, (60)(1973) 13th Int. Cosmic-ray Conf., 3, p. 2228(1975) 14th Int. Cosmic-Ray Conf., 7, p. 2365(1979) AIP Conf. Proc. no. 49, p. 334(1979) 16th Int. Cosmic-ray Conf., 6, p. 344(1979) 16th Int. Cosmic-ray Conf., 7, p. 6816th Int. Cosmic-ray Conf. (1979) 16th Int. Cosmic-ray Conf., 7, p. 284(1979) 16th Int. Cosmic-ray Conf., 7, p. 294(1979) 16th Int. Cosmic-ray Conf., 13, p. 87(1979) 16th Int. Cosmic-ray Conf., 13, p. 92(1979) 16th Int. Cosmic-ray Conf., 13, p. 98(1979) AIP Conf. Proc. no. 49, p. 94(1979) AIP Conf. Proc. no. 49, p. 145(1979) AIP Conf. Proc. no. 49, p. 317(1979) 16th Int. Cosmic-ray Conf., 6, p. 350(1979) 16th Int. Cosmic-ray Conf., 6, p. 356(1979) 16th Int. Cosmic-ray Conf., 6, p. 362Nikolsky, Proc. 9th Int. High-energy Symp. (1978) CSSR, 21. , ToborMiyake, (1978) Proc. 19th Int. Conf. on High-energy physics, p. 433Vernov, (1977) Physica, 3, p. 1601Khristiansen, (1978) JETP Lett., 28, p. 124(1973) 13th Int. Cosmic-ray Conf., 3, p. 2219Izv. Acad. Nauk USSR, ser Phys. (1974) Izv. Acad. Nauk USSR, ser Phys., 38, p. 918(1975) 14th Int. Cosmic-ray Conf., 7, p. 2365(1979) 16th Int. Cosmic-ray Conf., 7, p. 68Dunaevsky, Urysson, Emelyanov, Shorin, Tashimov, (1975) FIAN preprint no. 150Dunaevsky, Urysson, Emelyanov, Shorin, Tashinov, (1979) Acta Univ. Lodz ser. II, (60), p. 199Ivanenko, Kanevskya, Roganova, (1978) JETP Lett., 40, p. 704Ivanenko, Kanevsky, Roganova, (1979) 16th Int. Cosmic-ray Conf., 7, p. 101Ivanenko, Kanevsky, Roganova, (1979) 16th Int. Cosmic-ray Conf., 7, p. 198Wrotniak, (1977) Acta Univ. Lodz ser. II, (60), p. 165Krys, Tomaszevski, Wrotniak, (1979) 16th Int. Cosmic-ray Conf., 7, p. 182Krys, Tomaszevski, Wrotniak, (1979) 16th Int. Cosmic-ray Conf., 7, p. 186Fomin, Kempa, Khristiansen, Levina, Piotrowska, Wdowczyk, (1977) 15th Int. Cosmic-ray Conf., 7, p. 248Fomin, Kempa, Khristiansen, Levina, Piotrowska, Wdowczyk, (1979) 16th Int. Cosmic-ray Conf., 13, p. 82Azimov, Mullazhanov, Yuldashbayev, (1979) 16th Int. Cosmic-ray Conf., 7, p. 262Azimov, Mullazhanov, Yuldashbayev, (1977) Acta Univ. Lodz ser. II, (60), p. 275Kasahara, Torri, Yuda, (1979) 16th Int. Cosmic-ray Conf., 13, p. 70Kasahara, Torii, Yuda, (1979) 16th Int. Cosmic-ray Conf., 13, p. 79Shibata, (1979) 16th Int. Cosmic-ray Conf., 7, p. 176H. Semba, T. Shibata and T. Tabuki, Suppl. Prog. Theor. Phys., to be publishedZhdanov, Roinishvilli, Smorodin, Tomaszevski, (1975) FIAN preprint no. 163Lattes, Fujimoto, Hasegawa, Hadronic interactions of high energy cosmic-ray observed by emulsion chambers (1980) Physics Reports, 65, p. 152Ellsworth, Gaisser, Yodh, (1981) Phys. Rev., 23 D, p. 764Baradzei, Smorodin, (1974) FIAN preprint nos. 103, 104Baradzei, Smorodin, (1977) Acta Univ. Lodz ser. II, (60), p. 51Zhdanov, (1980) FIAN preprint no. 140H. Semba, T. Shibata and T. Tabuki, Suppl. Prog. Theor. Phys., to be publishedShibata, (1980) Phys. Rev., 22 D, p. 100Slavatinsky, (1980) Proc. 7th European Symp. on Cosmic rays, , Leningrad, to be published(1979) AIP Conference Proc. no. 49, p. 145Azimov, Abduzhamilov, Chudakov, (1963) JETP (Sov. Phys.), 45, p. 40713th Int. Cosmic-ray Conf. (1973) 13th Int. Cosmic-ray Conf., 5, p. 326Acharya, Rao, Sivaprasad, Rao, (1979) 16th Int. Cosmic-ray Conf., 6, p. 289Ellsworth, Goodman, Yodh, Gaisser, Stanev, (1981) Phys. Rev., 23 D, p. 771Bariburina, Guseva, Denisova, (1980) Acta Univ. Lodz, 1, p. 9415th Int. Cosmic-ray Conf. (1977) 15th Int. Cosmic-ray Conf., 7, p. 184(1979) AIP Conf. Proc. no. 49, p. 33

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination

Irradiation-Induced Up-Regulation of HLA-E on Macrovascular Endothelial Cells Confers Protection against Killing by Activated Natural Killer Cells

BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury