Longan Polysaccharides with Covalent Selenylation Combat the Fumonisin B1-Induced Cell Toxicity and Barrier Disruption in Intestinal Epithelial (IEC-6) Cells.

In this study, the soluble, but non-digestible, longan (Dimocarpus longan Lour.) polysaccharides (LP) were extracted from dried longan fruits and then chemically selenylated to produce two selenylated products, namely SeLP1 and SeLP2, with different selenylation extents. The aim was to investigate their protective effects on rat intestinal epithelial (IEC-6) cells exposed to the food toxin fumonisin B1 (FB1). LP only contained total Se content of less than 0.01 g/kg, while SeLP1 and SeLP2 were measured with respective total Se content of up to 1.46 and 4.79 g/kg. The cell viability results showed that these two selenylated products were more efficient than LP in the IEC-6 cells in alleviating FB1-induced cell toxicity, suppressing lactate dehydrogenase (LDH) release, and decreasing the generation of intracellular reactive oxygen species (ROS). These two selenylated products were also more effective than LP in combating FB1-induced barrier disruption via increasing the transepithelial electric resistance (TEER), reducing the paracellular permeability, decreasing the mitochondrial membrane potential (MMP) loss, and maintaining cell barrier integrity by upregulating the tight-junction-related genes and proteins. FB1 caused cell oxidative stress and barrier dysfunction by activating the MAPK and mitochondrial apoptosis signaling pathways, while SeLP1 and SeLP2 could regulate the tMAPK- and apoptosis-related proteins to suppress the FB1-mediated activation of the two pathways. Overall, SeLP2 was observed to be more active than SeLP1 in the IEC-6 cells. In conclusion, the chemical selenylation of LP caused an activity enhancement to ameliorate the FB1-induced cell cytotoxicity and intestinal barrier disruption. Meanwhile, the increased selenylation of LP would endow the selenylated product SeLP2 with more activity.

Plant Metabolites Affect Fusarium proliferatum Metabolism and In Vitro Fumonisin Biosynthesis.

Fusarium proliferatum is a common hemi-biotrophic pathogen that infect a wide range of host plants, often leading to substantial crop loss and yield reduction. F. proliferatum synthesizes various mycotoxins, and fumonisins B are the most prevalent. They act as virulence factors and specific effectors that elicit host resistance. The effects of selected plant metabolites on the metabolism of the F. proliferatum strain were analyzed in this study. Quercetin-3-glucoside (Q-3-Glc) and kaempferol-3-rutinoside (K-3-Rut) induced the pathogen's growth, while DIMBOA, isorhamnetin-3-O-rutinoside (Iso-3-Rut), ferulic acid (FA), protodioscin, and neochlorogenic acid (NClA) inhibited fungal growth. The expression of seven F. proliferatum genes related to primary metabolism and four FUM genes was measured using RT-qPCR upon plant metabolite addition to liquid cultures. The expression of CPR6 and SSC1 genes was induced 24 h after the addition of chlorogenic acid (ClA), while DIMBOA and protodioscin reduced their expression. The transcription of FUM1 on the third day of the experiment was increased by all metabolites except for Q-3-Glc when compared to the control culture. The expression of FUM6 was induced by protodioscin, K-3-Rut, and ClA, while FA and DIMBOA inhibited its expression. FUM19 was induced by all metabolites except FA. The highest concentration of fumonisin B1 (FB1) in control culture was 6.21 µg/mL. Protodioscin did not affect the FB content, while DIMBOA delayed their synthesis/secretion. Flavonoids and phenolic acids displayed similar effects. The results suggest that sole metabolites can have lower impacts on pathogen metabolism and mycotoxin synthesis than when combined with other compounds present in plant extracts. These synergistic effects require additional studies to reveal the mechanisms behind them.

Dietary fumonisin may compromise the nutritive value of feed and distort copper and zinc digestibility and retention in weaned piglets.

Fumonisins (FUM) have been reported to impede gut functioning in pigs. However, investigations into the possible effect on mineral metabolism are limited. Thus, the trial studied the apparent total tract digestibility (ATTD) and retention of dietary nitrogen and minerals, intestinal architecture, digestive enzymes activity and heat-shock protein 70 (Hsp70) activity. Eighteen weaned piglets of 7 weeks old were assigned to three groups and their feed either contained 0, 15 or 30 mg FUM/kg for 21 days. ATTD and retention of dietary N and minerals were measured in a 5- day long balance trial between Day 17 and Day 21. The digestible and metabolisable energy (DE and ME) content of the feeds were also determined. The body weights, cumulative feed intake, relative organ weights, digestive enzymes activity and intestinal morphology were not affected (p > 0.05) by dietary treatments. The DE content was significantly lower (p < 0.05) when the feed contained 15 mg/kg FUM, but no statistically reliable treatment effect was confirmed for ME content. Dietary FUM significantly lowered (p < 0.05) the ATTD of Ca and P but not (p > 0.05) N, K, Mg and Na. The relative retention rate of N, Ca, P, K, Mg and Na in all groups were not impacted (p > 0.05) by treatments. The ATTD and relative retention of Cu and Zn were remarkably (p < 0.05) lower in piglets fed FUM-contaminated feed. In addition, the expression of Hsp70 activity in the liver was significantly elevated (p < 0.05) in the highest treatment group. These findings suggest that a dietary dose of 15 or 30 mg FUM/kg diet distorts the nutritive value of the mixed feed, results in poor ATTD and retention rates of Zn and Cu, and elevate Hsp70 activity in the liver without altering intestinal architecture or digestive enzymes' activity in weaned piglets.

Fumonisin B1 as a Tool to Explore Sphingolipid Roles in Arabidopsis Primary Root Development.

Fumonisin B1 is a mycotoxin that is structurally analogous to sphinganine and sphingosine and inhibits the biosynthesis of complex sphingolipids by repressing ceramide synthase. Based on the connection between FB1 and sphingolipid metabolism, FB1 has been widely used as a tool to explore the multiple functions of sphingolipids in mammalian and plant cells. The aim of this work was to determine the effect of sphingolipids on primary root development by exposing Arabidopsis (Arabidopsis thaliana) seedlings to FB1. We show that FB1 decreases the expression levels of several PIN-FORMED (PIN) genes and the key stem cell niche (SCN)-defining transcription factor genes WUSCHEL-LIKE HOMEOBOX5 (WOX5) and PLETHORAs (PLTs), resulting in the loss of quiescent center (QC) identity and SCN maintenance, as well as stunted root growth. In addition, FB1 induces cell death at the root apical meristem in a non-cell-type-specific manner. We propose that sphingolipids play a key role in primary root growth through the maintenance of the root SCN and the amelioration of cell death in Arabidopsis.

Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

Research Progress on Fumonisin B1 Contamination and Toxicity: A Review.

Fumonisin B1 (FB1), belonging to the member of fumonisins, is one of the most toxic mycotoxins produced mainly by Fusarium proliferatum and Fusarium verticillioide. FB1 has caused extensive contamination worldwide, mainly in corn, rice, wheat, and their products, while it also poses a health risk and is toxic to animals and human. It has been shown to cause oxidative stress, endoplasmic reticulum stress, cellular autophagy, and apoptosis. This review focuses on the current stage of FB1 contamination, its toxic effects of acute toxicity, immunotoxicity, organ toxicity, and reproductive toxicity on animals and humans. The potential toxic mechanisms of FB1 are discussed. One of the main aims of the work is to provide a reliable reference strategy for understanding the occurrence and toxicity of FB1.

Epigenetic effect of the mycotoxin fumonisin B1 on DNA methylation.

Mycotoxin fumonisin B1 (FB1) is a secondary metabolite that is produced by certain Fusarium species. Although numerous studies demonstrate toxic and carcinogenic effects of FB1, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the epigenetic effects of FB1 for the first time using FLO assays, which detect epigenetic changes that affect the flocculation gene (FLO1) promoter activity in budding yeast. FLO assays showed increased reporter activities of the FLO1 promoter in the presence of 10 and 20 µM FB1. FB1 (20 µM) treatments also promoted flocculation. In subsequent in vitro methylation assays of a bacterial DNA methyltransferase (DNMT), FB1 treatments increased DNMT activities. Moreover, global DNA methylation was significantly increased in HEK293 cells treated with 100 µM FB1. Taken together, these results suggest that FB1 exposure leads to unique epigenetic alterations due to increased DNMT activities and demonstrate that FB1 may be an important risk factor for epigenetic dysfunction-associated human diseases including cancer.

Fumonisin B1 induces poly (ADP-ribose) (PAR) polymer-mediated cell death (parthanatos) in neuroblastoma.

Fumonisin B1 (FB1) is a well-known mycotoxin produced by Fusarium spp. and has a wide range of dose-dependent toxic effects, including nephrotoxicity, hepatotoxicity, and neurotoxicity. This research illustrated that FB1 exerts its toxicity in the neuroblastoma cell line through a distinct cell-death pathway called parthanatos. FB1 can cause excessive DNA strand breaks, leading to poly (ADP-ribose) polymerase-1 (PARP-1) overactivation and cell death. In this study, we used 50 µM FB1-treated SH-SY5Y neuroblastoma cells to elucidate the signaling pathway of FB1-induced parthanatos. We observed that FB1-induced cell death is caspase-independent and accompanied by rapid activation of PARP-1, c-Jun N-terminal kinase activation, reactive oxygen species (ROS) generation, and intracellular calcium increase. FB1 treatment also increased endoplasmic reticulum stress due to the rapid increase of calcium ions and ROS levels. In addition, FB1 induced massive DNA damage and chromatin decondensation. We also observed that apoptosis-inducing factor nuclear translocation and PAR accumulation were associated with the necroptosis signal.

Sphingosine Promotes Embryo Biomass in Upland Cotton: A Biochemical and Transcriptomic Analysis.

Sphingolipids are essential membrane components and signal molecules, but their regulatory role in cotton embryo growth is largely unclear. In this study, we evaluated the effects of treatment with the sphingolipid synthesis inhibitor fumonisin B1 (FB1), the serine palmityl transferase (SPT) inhibitor myriocin, the SPT sphingolipid product DHS (d18:0 dihydrosphingosine), and the post-hydroxylation DHS product PHS (t18:0 phytosphingosine) on embryo growth in culture, and performed comparative transcriptomic analysis on control and PHS-treated samples. We found that FB1 could inhibit cotton embryo development. At the five-day ovule/embryo developmental stage, PHS was the most abundant sphingolipid. An SPT enzyme inhibitor reduced the fresh weight of embryos, while PHS had the opposite effect. The transcriptomic analysis identified 2769 differentially expressed genes (1983 upregulated and 786 downregulated) in the PHS samples. A large number of transcription factors were highly upregulated, such as zinc finger, MYB, NAC, bHLH, WRKY, MADS, and GRF in PHS-treated samples compared to controls. The lipid metabolism and plant hormone (auxin, brassinosteroid, and zeatin) related genes were also altered. Our findings provide target metabolites and genes for cotton seed improvement.

The Effect of Fusarium verticillioides Fumonisins on Fatty Acids, Sphingolipids, and Oxylipins in Maize Germlings.

Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

The SiaABC threonine phosphorylation pathway controls biofilm formation in response to carbon availability in Pseudomonas aeruginosa.

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

Fumonisin B1-Induced Changes in Cotton Fiber Elongation Revealed by Sphingolipidomics and Proteomics.

Sphingolipids are essential biomolecules and membrane components, but their regulatory role in cotton fiber development is poorly understood. Here, we found that fumonisin B1 (FB1)-a sphingolipid synthesis inhibitor-could block fiber elongation severely. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we detected 95 sphingolipids that were altered by FB1 treatment; of these, 29 (mainly simple sphingolipids) were significantly increased, while 33 (mostly complex sphingolipids) were significantly decreased. A quantitative analysis of the global proteome, using an integrated quantitative approach with tandem mass tag (TMT) labeling and LC-MS/MS, indicated the upregulation of 633 and the downregulation of 672 proteins after FB1 treatment. Most differentially expressed proteins (DEPs) were involved in processes related to phenylpropanoid and flavonoid biosynthesis. In addition, up to 20 peroxidases (POD) were found to be upregulated, and POD activity was also increased by the inhibitor. To our knowledge, this is the first report on the effects of FB1 treatment on cotton fiber and ovule sphingolipidomics and proteomics. Our findings provide target metabolites and biological pathways for cotton fiber improvement.

Organized Disassembly of Photosynthesis During Programmed Cell Death Mediated By Long Chain Bases.

In plants, pathogen triggered programmed cell death (PCD) is frequently mediated by polar lipid molecules referred as long chain bases (LCBs) or ceramides. PCD interceded by LCBs is a well-organized process where several cell organelles play important roles. In fact, light-dependent reactions in the chloroplast have been proposed as major players during PCD, however, the functional aspects of the chloroplast during PCD are largely unknown. For this reason, we investigated events that lead to disassembly of the chloroplast during PCD mediated by LCBs. To do so, LCB elevation was induced with Pseudomonas syringae pv. tomato (a non-host pathogen) or Fumonisin B1 in Phaseolus vulgaris. Then, we performed biochemical tests to detect PCD triggering events (phytosphingosine rises, MPK activation and H2O2 generation) followed by chloroplast structural and functional tests. Observations of the chloroplast, via optical phenotyping methods combined with microscopy, indicated that the loss of photosynthetic linear electron transport coincides with the organized ultrastructure disassembly. In addition, structural changes occurred in parallel with accumulation of H2O2 inside the chloroplast. These features revealed the collapse of chloroplast integrity and function as a mechanism leading to the irreversible execution of the PCD promoted by LCBs.

Fumonisin B1 Affects the Biophysical Properties, Migration and Cytoskeletal Structure of Human Umbilical Vein Endothelial Cells.

Fumonisin B1 (FB1) is an important mycotoxin in nature and is a serious threat to human and animal health, but its specific target and molecular mechanism of the toxicity and potential carcinogenicity remain unclear. In this study, we first detected the effects of FB1 on the cell viability, biophysical properties, migration ability, and reactive oxygen species (ROS) of human umbilical vein endothelial cells (HUVECs). Subsequently, changes in the cytoskeletal structure and its binding proteins were analyzed by immunofluorescence and real-time PCR, respectively. The results showed that FB1 could inhibit the viability of HUVECs in a dose-dependent manner. After treatment of HUVECs with FB1, the hypotonic resistance, cell surface charges, cell membrane fluidity, and migration ability were weakened, whereas the ROS levels were significantly increased. Moreover, the cytoskeletal structure of the HUVECs was significantly changed, and the mRNA expression of some important actin-binding proteins was altered. Therefore, this study revealed that FB1 can affect the migration and cytoskeletal structure of HUVECs, which provides a new perspective for further understanding the molecular mechanisms of FB1 toxicity.

Curcumin stimulates exosome/microvesicle release in an in vitro model of intracellular lipid accumulation by increasing ceramide synthesis.

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin.

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling.

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.

Exercise-induced changes of gene expression in the cerebellum of aged mice.

PURPOSE: Exercise has been prescribed to the elderly based on its effect on increasing muscle strength and protein synthesis that prevent sense of balance and/or cognitive functions. However, a few molecular mechanism researches has been conducted on how the vestibular organs, cerebellum, and hippocampus, which are responsible for the deterioration and balance of spatial learning memory due to aging, are affected by exercise. METHODS: The 9-week old and 84-week old C57Bl/6 were assigned randomly to Young-Control (YC), Young-Exercise (YE), Old-Control (OC) and Old-Exercise (OE) groups for 4 -week treadmill running. A Rotarod test was used to evaluate motor coordination function. Moreover, a high-throughput whole transcript expression RNA array approach was applied to the cerebellum of aged mice to explain the novel molecular mechanism of beneficial effect of exercise. RESULTS: As results, the motor coordination function was significantly improved in exercise-aged mice. The RNA sequencing analysis showed that the expression of cerebellar genes was significantly changed by aging rather than exercise. Especially, Cers1 was up-regulated in sedentary aged mice and down-regulated in exercise aged mice. Fumonisin B1, inhibition of Cers1, mitigates neuronal cell death induced by doxorubicin. CONCLUSION: These results provide unraveling specific transcripts and understanding of the exercise-related cerebellum transcriptome in aged mice. Well-designed exercise program might prevent the motor coordination defect in aged model, which development of the exercise protocol for elderly population based on these markers.

Fumonisin B1 damages the barrier functions of porcine intestinal epithelial cells in vitro.

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums-contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B1 (FB1 ) on barrier functions of porcine intestinal epithelial cells, IPEC-J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB1 significantly decreased the viability of IPEC-J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden-1 (ZO-1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB1 treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB1 also enhanced the monolayer cell permeability of IPEC-J2. Importantly, FB1 promoted the expression of phosphorylated extracellular regulated protein kinase (p-ERK1/2 ). These data suggest that long-term treatment of FB1 can suppress IPEC-J2 proliferation, damage tight junctions of IPEC-J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK1/2 phosphorylation pathway.

Ceramide synthase inhibition by fumonisins: a perfect storm of perturbed sphingolipid metabolism, signaling, and disease.

Fumonisins are mycotoxins that cause diseases of plants and, when consumed by animals, can damage liver, kidney, lung, brain, and other organs, alter immune function, and cause developmental defects and cancer. They structurally resemble sphingolipids (SLs), and studies nearly 30 years ago discovered that the most prevalent fumonisin [fumonisin B1 (FB1)] potently inhibits ceramide synthases (CerSs), enzymes that use fatty acyl-CoAs to N-acylate sphinganine (Sa), sphingosine (So), and other sphingoid bases. CerS inhibition by FB1 triggers a "perfect storm" of perturbations in structural and signaling SLs that include: reduced formation of dihydroceramides, ceramides, and complex SLs; elevated Sa and So and their 1-phosphates, novel 1-deoxy-sphingoid bases; and alteration of additional lipid metabolites from interrelated pathways. Moreover, because the initial enzyme of sphingoid base biosynthesis remains active (sometimes with increased activity), the impact is multiplied by the continued production of damaging metabolites. Evidence from many studies, including characterization of knockout mice for specific CerSs and analyses of human blood (which found that FB1 intake is associated with elevated Sa 1-phosphate), has consistently pointed to CerS as the proximate target of FB1 It is also apparent that the changes in multiple bioactive lipids and related biologic processes account for the ensuing spectrum of animal and plant disease. Thus, the diseases caused by fumonisins can be categorized as "sphingolipidoses" (in these cases, due to defective SL biosynthesis), and the lessons learned about the consequences of CerS inhibition should be borne in mind when contemplating other naturally occurring and synthetic compounds (and genetic manipulations) that interfere with SL metabolism.

Elevated Levels of Phosphorylated Sphingobases Do Not Antagonize Sphingobase- or Fumonisin B1-Induced Plant Cell Death.

Long-chain bases (LCBs), also termed sphingobases, are building blocks of sphingolipids, which make up a significant proportion of the cellular membrane system. They are also bioactive molecules regulating intracellular processes. Elevated levels of LCBs like phytosphingosine and dihydrosphingosine can induce cell death in plants and correlate with programmed cell death (PCD) reactions after pathogen recognition. We investigated the previously hypothesized antagonism between phosphorylated and nonphosphorylated LCBs with respect to cell death in Arabidopsis thaliana. Using HPLC-MS/MS, we determined levels of phosphorylated and nonphosphorylated LCBs after cell death induction by LCB application or by Fumonisin B1 (FB1) treatment. We show that previously reported antagonistic effects of phosphorylated LCBs after simultaneous application with nonphosphorylated LCBs are linked to reduced uptake of nonphosphorylated LCBs into the tissue. Furthermore, phosphorylated LCBs did not antagonize PCD induced by avirulence protein recognition. In a functional approach, we used Arabidopsis lines with perturbed levels of phosphorylated LCBs. In these plants, the degree of FB1-induced cell death did not consistently correlate negatively with levels of phosphorylated LCBs, but positively with levels of major nonphosphorylated LCBs phytosphingosine and dihydrosphingosine. As treatment with phosphorylated LCBs did not antagonize cell death, and elevated in vivo levels of these LCB species did not reduce FB1-induced cell death, we conclude that the hypothesized general cell death-antagonizing effect of phosphorylated LCBs in plant cell death reactions should be rejected. Instead, our time-course analysis of LCB levels during cell death reactions showed a positive correlation between levels of nonphosphorylated LCBs and cell death.