Alkyne-Tagged Analogue of Jaspine†B: New Tool for Identifying Jaspine†B Mode of Action.

The first biologically relevant clickable probe related to the antitumor marine lipid jaspine B is reported. The concise synthetic route to both enantiomers relied on the supercritical fluid chromatography (SFC) enantiomeric resolution of racemic materials. The eutomeric dextrogyre derivative represents the first jaspine B analogue with enhanced cytotoxicity with IC50 down to 30 nm. These enantiomeric probes revealed a chiralitydependent cytoplasmic imaging of U2OS cancer cells by in situ click labeling.

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation.

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.

1-Deoxysphingolipids Encountered Exogenously and Made de Novo: Dangerous Mysteries inside an Enigma.

The traditional backbones of mammalian sphingolipids are 2-amino, 1,3-diols made by serine palmitoyltransferase (SPT). Many organisms additionally produce non-traditional, cytotoxic 1-deoxysphingoid bases and, surprisingly, mammalian SPT biosynthesizes some of them, too (e.g. 1-deoxysphinganine from L-alanine). These are rapidly N-acylated to 1-deoxy-"ceramides" with very uncommon biophysical properties. The functions of 1-deoxysphingolipids are not known, but they are certainly dangerous as contributors to sensory and autonomic neuropathies when elevated by inherited SPT mutations, and they are noticeable in diabetes, non-alcoholic steatohepatitis, serine deficiencies, and other diseases. As components of food as well as endogenously produced, these substances are mysteries within an enigma.

Substrate and stereocontrolled iodocycloetherification of highly functionalized enantiomerically pure allylic alcohols: application to synthesis of cytotoxic 2-epi jaspine B and its biological evaluation.

Stereoselectivities of electrophilic additions of molecular iodine to enantiomerically pure highly functionalized allylic alcohols with internal nucleophiles have been investigated. The intramolecular nucleophilic attack on the I2-π complex by an oxygen nucleophile to obtain tri- and tetrasubstituted THFs is highly regio-, stereoselective and substrate controlled. The application of this study has been shown by utilizing one of the THFs 4a as a key intermediate to complete the total synthesis of marine anti-cancer natural product 2-epi jaspine B.

Exogenous S1P Exposure Potentiates Ischemic Stroke Damage That Is Reduced Possibly by Inhibiting S1P Receptor Signaling.

Initial and recurrent stroke produces central nervous system (CNS) damage, involving neuroinflammation. Receptor-mediated S1P signaling can influence neuroinflammation and has been implicated in cerebral ischemia through effects on the immune system. However, S1P-mediated events also occur within the brain itself where its roles during stroke have been less well studied. Here we investigated the involvement of S1P signaling in initial and recurrent stroke by using a transient middle cerebral artery occlusion/reperfusion (M/R) model combined with analyses of S1P signaling. Gene expression for S1P receptors and involved enzymes was altered during M/R, supporting changes in S1P signaling. Direct S1P microinjection into the normal CNS induced neuroglial activation, implicating S1P-initiated neuroinflammatory responses that resembled CNS changes seen during initial M/R challenge. Moreover, S1P microinjection combined with M/R potentiated brain damage, approximating a model for recurrent stroke dependent on S1P and suggesting that reduction in S1P signaling could ameliorate stroke damage. Delivery of FTY720 that removes S1P signaling with chronic exposure reduced damage in both initial and S1P-potentiated M/R-challenged brain, while reducing stroke markers like TNF-α. These results implicate direct S1P CNS signaling in the etiology of initial and recurrent stroke that can be therapeutically accessed by S1P modulators acting within the brain.

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor.

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in "cation-free" solution and blocked by the nonspecific Cl(-) channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-ß-S. Notably, S1P(3) receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P(3)(-/-) mice, whereas in control S1P(1) floxed (S1P(1)(fl/fl)) mice and mice with a nociceptor-specific deletion of S1P(1)(-/-) receptor (SNS-S1P(1)(-/-)), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl(-) conductance that is largely mediated by S1P(3) receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.

Involvement of hydrogen peroxide in safingol-induced endonuclease G-mediated apoptosis of squamous cell carcinoma cells.

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator--endonuclease G (endo G)--and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H2O2) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H2O2 levels but not extracellular H2O2 levels, indicating the production of H2O2. The cell killing effect of safingol and H2O2 was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H2O2 and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H2O2 and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H2O2 and safingol. These results suggest that H2O2 is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.

C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells.

Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-D-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.

Ethylene-responsive AP2/ERF transcription factor MACD1 participates in phytotoxin-triggered programmed cell death.

To investigate plant programmed cell death (PCD), we developed the model system using phytotoxin AAL, which is produced by necrotrophic pathogen Alternaria alternata f. sp. lycopersici, and AAL-sensitive Nicotiana umbratica. We previously reported that ethylene (ET) signaling plays a pivotal role in AAL-triggered cell death (ACD). However, downstream signaling of ET to ACD remains unclear. Here, we show that the modulator of AAL cell death 1 (MACD1), which is an APETALA2/ET response factor (ERF) transcription factor, participates in ACD and acts downstream of ET signaling during ACD. MACD1 is a transcriptional activator and MACD1 overexpression plants showed earlier ACD induction than control plants, suggesting that MACD1 positively regulates factors affecting cell death. To investigate the role of MACD1 in PCD, we used Arabidopsis thaliana and a structural analog of AAL, fumonisin B1 (FB1). FB1-triggered cell death was compromised in ET signaling and erf102 mutants. The loh2 mutants showed sensitivity to AAL, and the loh2-1/erf102 double mutant compromised ACD, indicating that ERF102 also participates in ACD. To investigate the PCD-associated genes regulated by ERF102, we compared our microarray data using ERF102 overexpression plants with the database of upregulated genes by AAL treatment in loh2 mutants, and found genes under the control of ERF102 in ACD.

Toxic c17-sphinganine analogue mycotoxin, contaminating tunisian mussels, causes flaccid paralysis in rodents.

Severe toxicity was detected in mussels from Bizerte Lagoon (Northern Tunisia) using routine mouse bioassays for detecting diarrheic and paralytic toxins not associated to classical phytoplankton blooming. The atypical toxicity was characterized by rapid mouse death. The aim of the present work was to understand the basis of such toxicity. Bioassay-guided chromatographic separation and mass spectrometry were used to detect and characterize the fraction responsible for mussels' toxicity. Only a C17-sphinganine analog mycotoxin (C17-SAMT), with a molecular mass of 287.289 Da, was found in contaminated shellfish. The doses of C17-SAMT that were lethal to 50% of mice were 750 and 150 µg/kg following intraperitoneal and intracerebroventricular injections, respectively, and 900 µg/kg following oral administration. The macroscopic general aspect of cultures and the morphological characteristics of the strains isolated from mussels revealed that the toxicity episodes were associated to the presence of marine microfungi (Fusarium sp., Aspergillus sp. and Trichoderma sp.) in contaminated samples. The major in vivo effect of C17-SAMT on the mouse neuromuscular system was a dose- and time-dependent decrease of compound muscle action potential amplitude and an increased excitability threshold. In vitro, C17-SAMT caused a dose- and time-dependent block of directly- and indirectly-elicited isometric contraction of isolated mouse hemidiaphragms.

S1P1 receptor subtype inhibits demyelination and regulates chemokine release in cerebellar slice cultures.

Sphingosine-1-phosphate receptors (S1PRs) are drug targets for the compound FTY720, which is the first oral therapy developed for treatment of relapsing-remitting multiple sclerosis. S1PRs play a variety of functional roles in the differentiation, proliferation, survival and/or migration of neurons and glia. In this study, rat organotypic cerebellar slice cultures were used to assess whether S1PRs play a role in demyelination induced by lysolecithin (LPC). The data demonstrated that FTY720 and SEW2871 (a S1P1R-specific agonist) inhibited LPC-induced demyelination as assessed by myelin basic protein (MBP) immunofluorescence. Treatment with both drugs for 48 h also induced an increase in S1P1R expression in astrocytes. Moreover, FTY720 and SEW2871 inhibited the release of several chemokines in conditions of LPC-induced demyelination, including LIX (CXCL5), MIP-1alpha, and MIP-3alpha. Taken together, the data suggest that activation of S1P1Rs prevents LPC-induced demyelination via a mechanism involving a reduction of chemotactic chemokine release. The study supports the concept that FTY720 attenuates demyelination by not only preventing S1PR-mediated T cell migration into the CNS but also by limiting cytokine communication between cells of the immune system and the CNS.

Phosphorylation of Ser45 and Ser59 of αB-crystallin and p38/extracellular regulated kinase activity determine αB-crystallin-mediated protection of rat brain astrocytes from C2-ceramide- and staurosporine-induced cell death.

We previously demonstrated that αB-crystallin and protease-activated receptor (PAR) are involved in protection of astrocytes against C2-ceramide- and staurosporine-induced cell death [Li et al. (2009) J. Neurochem.110, 1433-1444]. Here, we further investigated the mechanism of cytoprotection by αB-crystallin. Our current data revealed that after down-regulation of αB-crystallin by siRNA, cell death caused by C2-ceramide and staurosporine is increased. Furthermore, we investigated the mechanism of cytoprotection of astrocytes by intracellular αB-crystallin. Application of specific inhibitors of p38 and extracellular regulated kinase (ERK) abrogates the protection of astrocytes by over-expression of αB-crystallin. Thus, p38 and ERK contribute to protective processes by αB-crystallin. To reveal the molecular mechanism of αB-crystallin-mediated cytoprotection, we mimicked phosphorylation or unphosphorylation of αB-crystallin. In these experiments, we found that the phosphorylation of αB-crystallin at Ser45 and Ser59 is required for protection. Ser19 phosphorylation of αB-crystallin does not contribute to protection. Moreover, we detected that PAR-2 activation increases the phosphorylation level of αB-crystallin at Ser59, but does not affect the expression level of αB-crystallin. Thus, endogenous αB-crystallin has protective capacity employing a mechanism, which involves regulation of the phosphorylation status of αB-crystallin and p38 and ERK activity. Moreover, we report that PAR-2 activation evokes the phosphorylation of αB-crystallin to increase astrocytes survival.

Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects.

BACKGROUND: Sphingosylphosphorylcholine (SPC) acts as a potent lipid mediator and signaling molecule in various cell types. In the present study, we investigated the effects of SPC on melanogenesis and SPC-modulated signaling pathways related to melanin synthesis. METHODS: Melanin production was measured in Mel-Ab cells. A luciferase assay was used to detect transcriptional activity of the MITF promoter. Western blot analysis was performed to examine SPC-induced signaling pathways. RESULTS: SPC produced significant hypopigmentation effects in a dose-dependent manner. It was found that SPC induced not only activation of Akt but also stimulation of mTOR, a downstream mediator of the Akt signaling pathway. Moreover, SPC decreased the levels of LC3 II, which is known to be regulated by mTOR. Treatment with the mTOR inhibitor rapamycin eliminated decreases in melanin and LC3 II levels by SPC. Furthermore, we found that the Akt inhibitor LY294002 restored SPC-mediated downregulation of LC3 II and inhibited the activation of mTOR by SPC. CONCLUSIONS: Our data suggest that the mTOR signaling pathway is involved in SPC-modulated melanin synthesis.

The Sphingosine-1 Phosphate receptor agonist FTY720 dose dependently affected endothelial integrity in vitro and aggravated ventilator-induced lung injury in mice.

Lung barrier protection by Sphingosine-1 Phosphate (S1P) has been demonstrated experimentally, but recent evidence suggests barrier disruptive properties of high systemic S1P concentrations. The S1P analog FTY720 recently gained an FDA approval for treatment of multiple sclerosis. In case of FTY720 treated patients experiencing multiple organ dysfunction syndrome the drug may accumulate due to liver failure, and the patients may receive ventilator therapy. Whereas low doses of FTY720 enhanced endothelial barrier function, data on effects of increased FTY720 concentrations are lacking. We measured transcellular electrical resistance (TER) of human umbilical vein endothelial cell (HUVEC) monolayers, performed morphologic analysis and measured apoptosis by TUNEL staining and procaspase-3 degradation in HUVECs stimulated with FTY720 (0.01-100 µM). Healthy C57BL/6 mice and mice ventilated with 17 ml/kg tidal volume and 100% oxygen for 2 h were treated with 0.1 or 2 mg/kg FTY720 or solvent, and lung permeability, oxygenation and leukocyte counts in BAL and blood were quantified. Further, electron microscopic analysis of lung tissue was performed. We observed barrier protective effects of FTY720 on HUVEC cell layers at concentrations up to 1 µM while higher concentrations induced irreversible barrier breakdown accompanied by induction of apoptosis. Low FTY720 concentrations (0.1 mg/kg) reduced lung permeability in mechanically ventilated mice, but 2 mg/kg FTY720 increased pulmonary vascular permeability in ventilated mice accompanied by endothelial apoptosis, while not affecting permeability in non-ventilated mice. Moreover, hyperoxic mechanical ventilation sensitized the pulmonary vasculature to a barrier disrupting effect of FTY720, resulting in worsening of ventilator induced lung injury. In conclusion, the current data suggest FTY720 induced endothelial barrier dysfunction, which was probably caused by proapoptotic effects and enhanced by mechanical ventilation.

The immunosuppressant FTY720 (fingolimod) enhances glycosaminoglycan depletion in articular cartilage.

BACKGROUND: FTY720 (Fingolimod) is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P) receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction. METHODS: Bovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P) in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining. RESULTS: FTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner. CONCLUSIONS: Our results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.

Spinal mechanisms contributing to the development of pain hypersensitivity induced by sphingolipids in the rat.

BACKGROUND: Earlier studies show that endogenous sphingolipids can induce pain hypersensitivity, activation of spinal astrocytes, release of proinflammatory cytokines and activation of TRPM3 channel. Here we studied whether the development of pain hypersensitivity induced by sphingolipids in the spinal cord can be prevented by pharmacological inhibition of potential downstream mechanisms that we hypothesized to include TRPM3, σ1 and NMDA receptors, gap junctions and D-amino acid oxidase. METHODS: Experiments were performed in adult male rats with a chronic intrathecal catheter for spinal drug administrations. Mechanical nociception was assessed with monofilaments and heat nociception with radiant heat. N,N-dimethylsphingosine (DMS) was administered to induce pain hypersensitivity. Ononetin, isosakuranetin, naringenin (TRPM3 antagonists), BD-1047 (σ1 receptor antagonist), carbenoxolone (a gap junction decoupler), MK-801 (NMDA receptor antagonist) and AS-057278 (inhibitor of D-amino acid oxidase, DAAO) were used to prevent the DMS-induced hypersensitivity, and pregnenolone sulphate (TRPM3 agonist) to recapitulate hypersensitivity. RESULTS: DMS alone produced within 15 min a dose-related mechanical hypersensitivity that lasted at least 24 h, without effect on heat nociception. Preemptive treatments with ononetin, isosakuranetin, naringenin, BD-1047, carbenoxolone, MK-801 or AS-057278 attenuated the development of the DMS-induced hypersensitivity, but had no effects when administered alone. Pregnenolone sulphate (TRPM3 agonist) alone induced a dose-related mechanical hypersensitivity that was prevented by ononetin, isosakuranetin and naringenin. CONCLUSIONS: Among spinal pronociceptive mechanisms activated by DMS are TRPM3, gap junction coupling, the σ1 and NMDA receptors, and DAAO.

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery.

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel). We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina. The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

FTY720-induced suicidal erythrocyte death.

FTY720 is a potent anti-inflammatory drug known to trigger suicidal death or apoptosis of a variety of nucleated cells. Erythrocytes may similarly undergo suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis include cell membrane scrambling and cell shrinkage, which are triggered by increase in cytosolic Ca(2+) concentration and ceramide. The present study explored whether FTY720 stimulates eryptosis. Cell membrane scrambling was determined from annexin V-binding, cell shrinkage from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence, ceramide formation from fluorescence-labeled antibody binding and hemolysis from the hemoglobin concentration in the supernatant. Within 48 hours exposure to FTY720 (10 µM) significantly increased annexin V-binding, decreased forward scatter and increased cytosolic Ca(2+) concentration but did not significantly modify ceramide formation. The effects of FTY720 were significantly blunted in the nominal absence of extracelluar Ca(2+). In conclusion, at toxic concentrations, FTY720 stimulates suicidal cell death, an effect at least partially due to stimulation of Ca(2+) entry.

Alpha A-crystallin and alpha B-crystallin, newly identified interaction proteins of protease-activated receptor-2, rescue astrocytes from C2-ceramide- and staurosporine-induced cell death.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor activated by trypsin and other trypsin-like serine proteases. The widely expressed PAR-2 is involved in inflammation response but the physiological/pathological roles of PAR-2 in the nervous system are still uncertain. In the present study, we report novel PAR-2 interaction proteins, alphaA-crystallin and alphaB-crystallin. These 20 kDa proteins have been implicated in neurodegenerative diseases like Alexander's disease, Creutzfeldt-Jacob disease, Alzheimer's disease, and Parkinson's disease. Results from yeast two-hybrid assay using the cytoplasmic C-tail of PAR-2 as bait suggested that alphaA-crystallin interacts with PAR-2. We further demonstrate the in vitro and cellular in vivo interaction of C-tail of PAR-2 as well as of full-length PAR-2 with alphaA(alphaB)-crystallins. We use pull-down, co-immunoprecipitation, and co-localization assays. Analysis of alphaA-crystallin deletion mutants showed that amino acids 120-130 and 136-154 of alphaA-crystallin are required for the interaction with PAR-2. Co-immunoprecipitation experiments ruled out an interaction of alphaA(alphaB)-crystallins with PAR-1, PAR-3, and PAR-4. This demonstrates that alphaA(alphaB)-crystallins are PAR-2-specific interaction proteins. Moreover, we investigated the functional role of PAR-2 and alpha-crystallins in astrocytes. Evidence is presented to show that PAR-2 activation and increased expression of alpha-crystallins reduced C2-ceramide- and staurosporine-induced cell death in astrocytes. Thus, both PAR-2 and alpha-crystallins are involved in cytoprotection in astrocytes.

Susceptibility of Phelipanche and Orobanche species to AAL-toxin.

Fusarium and Alternaria spp. are phytopathogenic fungi which are known to be virulent on broomrapes and to produce sphinganine-analog mycotoxins (SAMs). AAL-toxin is a SAM produced by Alternaria alternata which causes the inhibition of sphinganine N-acyltransferase, a key enzyme in sphingolipid biosynthesis, leading to accumulation of sphingoid bases. These long chain bases (LCBs) are determinant in the occurrence of programmed cell death (PCD) in susceptible plants. We showed that broomrapes are sensitive to AAL-toxin, which is not common plant behavior, and that AAL-toxin triggers cell death at the apex of the radicle as well as LCB accumulation and DNA laddering. We also demonstrated that three Lag1 homologs, encoding components of sphinganine N-acyltransferase in yeast, are present in the Orobanche cumana genome and two of them are mutated leading to an enhanced susceptibility to AAL-toxin. We therefore propose a model for the molecular mechanism governing broomrape susceptibility to the fungus Alternaria alternata.