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Bcr‐abl1 oncogene causes a shift in the transcription start site of the SMS1 gene (SGMS1) encoding the sphingomyelin (SM) synthesizing enzyme, sphingomyelin synthase 1 (SMS1). This results in an mRNA with a significantly shorter 5′‐UTR, called 7‐SGMS1, which is translated more efficiently than another transcript (IIb‐SGMS1) with a longer 5′UTR in Bcr‐abl1‐positive cells. Here, we determine the effects of these alternative 5′UTRs on SMS1 translation and investigate the key features underlying such regulation. First, the presence of the longer IIb 5′UTR is sufficient to greatly impair translation of a reporter gene. Deletion of the upstream open reading frame (−164 nt) or of the predicted stem‐loops in the 5′UTR of IIb‐SGMS1 has minimal effects on SGMS1 translation. Conversely, deletion of nucleotides −310 to −132 enhanced transcription of IIb‐SGMS1 to reach that of 7‐SGMS1. We thus suggest that regulatory features within nucleotides −310 and −132 modulate IIb‐SGMS1 translation efficiency.

We developed a simple, rapid and cost-effective enzymatic-based cytometry platform to measure intracellular signaling pathway activity. Our single-cell microwell array platform quantifies protein phosphorylation using enzymatic signal amplification and exploiting Michaelis-Menten kinetics. Our method provides a two-fold increase in resolution compared to conventional flow cytometry.

Sphingolipid metabolism plays a critical role in regulating processes that control cellular fate. This dynamic pathway can generate and degrade the central players: ceramide, sphingosine and sphingosine-1-phosphate in almost any membrane in the cell, adding an unexpected level of complexity in deciphering signaling events. While in vitro assays have been developed for most enzymes in SL metabolism, these assays are setup for optimal activity conditions and can fail to take into account regulatory components such as compartmentalization, substrate limitations, and binding partners that can affect cellular enzymatic activity. Therefore, many in-cell assays have been developed to derive results that are authentic to the cellular situation which may give context to alteration in SL mass. This review will discuss approaches for utilizing probes for mammalian in-cell assays to interrogate most enzymatic steps central to SL metabolism. The use of inhibitors in conjunction with these probes can verify the specificity of cellular assays as well as provide valuable insight into flux in the SL network. The use of inhibitors specific to each of the central sphingolipid enzymes are also discussed to assist researchers in further interrogation of these pathways.

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.

Bcr-Abl (break-point cluster region-abelson), the oncogenic trigger of chronic myelogenous leukemia (CML), has previously been shown to up-regulate the expression and activity of sphingomyelin synthase 1 (SMS1), which contributes to the proliferation of CML cells; however, the mechanism by which this increased expression of SMS1 is mediated remains unknown. In the current study, we show that Bcr-Abl enhances the expression of SMS1 via a 30-fold up-regulation of its transcription. Of most interest, the Bcr-Abl-regulated transcription of SMS1 is initiated from a novel transcription start site (TSS) that is just upstream of the open reading frame. This shift in TSS utilization generates an SMS1 mRNA with a substantially shorter 5' UTR compared with its canonical mRNA. This shorter 5' UTR imparts a 20-fold greater translational efficiency to SMS1 mRNA, which further contributes to the increase of its expression in CML cells. Therefore, our study demonstrates that Bcr-Abl increases SMS1 protein levels via 2 concerted mechanisms: up-regulation of transcription and enhanced translation as a result of the shift in TSS utilization. Remarkably, this is the first time that an oncogene-Bcr-Abl-has been demonstrated to drive such a mechanism that up-regulates the expression of a functionally important target gene, SMS1.-Moorthi, S., Burns, T. A., Yu, G.-Q., Luberto, C. Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation.

Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.

Background/aim The aim of the present study was to assess the association between PM2.5, its sources, and preterm birth (PTB), low birth weight (LBW), and small for gestational age (SGA) in a large open residential cohort (Supersito Project in the Emilia-Romagna Region – Northern Italy). Methods We collected 2012–2014 pregnancy and childbirth data from Birth Assistance Certificates and selected the pregnancies of interest. PTBs (gestational age Results The study included 23,708 neonates born to 23,415 women, among whom 1,311 PTB, 424 LBW, and 1,354 SGA occurred. PTB was the only outcome associated with PM2.5 mass (OR 1.03, 95% CI 1.002-1.058 per 1 μg/m3). Traffic, oil combustion and secondary sulfates and organics showed independent effects on PTB. Exposure to secondary nitrates was associated with a lower risk of PTB. There was no association between LBW or SGA and source-specific PM2.5 components or the residual PM2.5 related to all other sources. Conclusion This study found an association between PTB and PM2.5. Traffic, secondary sulfates, and organic and oil combustion were the sources with most consistent association.

In the last ten years, mass spectrometry (MS) has become the favored analytical technique for sphingolipid (SPL) analysis and measurements. Indeed MS has the unique ability to both acquire sensitive and quantitative measurements and to resolve the molecular complexity characteristic of SPL molecules, both across the different SPL families and within the same SPL family. Currently, two complementary MS-based approaches are used for lipid research: analysis of lipid extracts, mainly by infusion electrospray ionization (ESI), and mass spectrometry imaging (MSI) from a sample surface (i.e. intact tissue sections, cells, model membranes, thin layer chromatography plates) (Fig. 1). The first allows for sensitive and quantitative information about total lipid molecular species from a given specimen from which lipids have been extracted and chromatographically separated prior to the analysis; the second, albeit generally less quantitative and less specific in the identification of molecular species due to the complexity of the sample, allows for spatial information of lipid molecules from biological specimens. In the field of SPL research, MS analysis of lipid extracts from biological samples has been commonly utilized to implicate the role of these lipids in specific biological functions. On the other hand, the utilization of MSI in SPL research represents a more recent development that has started to provide interesting descriptive observations regarding the distribution of specific classes of SPLs within tissues. Thus, it is the aim of this review to discuss how MSI technology has been employed to extend the study of SPL metabolism and the type of information that has been obtained from model membranes, single cells and tissues. We envision this discussion as a complementary compendium to the excellent technical reviews recently published about the specifics of MSI technologies, including their application to SPL analysis (Fuchs et al., 2010; Berry et al., 2011; Ellis et al., 2013; Eberlin et al., 2011; Kraft and Klitzing, 2014).

Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells.

Selenium (Se) is a metalloid of considerable nutritional and toxicological importance in humans. To date, limited epidemiologic evidence exists about the health effects of exposure to this trace element in drinking water. We investigated the relationship between Se levels in water and mortality in the municipality of Reggio Emilia, Italy, where high levels of Se were previously observed in drinking water. From 1974 to 1985, 2065 residents consumed drinking water with Se levels close to the European standard of 10μg/l, in its inorganic hexavalent form (selenate). Follow-up was conducted for the years 1986-2012 in Reggio Emilia and a lesser exposed comparison group of around 100,000 municipal residents, with comparable socio-demographic characteristics. Overall mortality from all causes, cardiovascular disease and cancer showed little evidence of differences. However, excess rate ratios were seen for some site specific cancers such as neoplasms of buccal cavity and pharynx, urinary tract, lymphohematopoietic tissue, melanoma, and two neurodegenerative diseases, Parkinson's disease and amyotrophic lateral sclerosis. Excess mortality in the exposed cohort for specific outcomes was concentrated in the first period of follow-up (1986-1997), and waned starting 10 years after the high exposure ended. We also found lower mortality from breast cancer in females during the first period of follow-up. When we extended the analysis to include residents who had been consuming the high-selenium drinking water for a shorter period, mortality rate ratios were also increased, but to a lesser extent. Overall, we found that the mortality patterns related to long-term exposure to inorganic hexavalent selenium through drinking water were elevated for several site-specific cancers and neurodegenerative disease.

Summary In previous studies we showed that the replication of Cryptococcus neoformans in the lung environ- ment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltrans- ferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceram- ide. The C. neoformans Dsmt1 mutant does not make C9-methyl ceramide and, thus, any methy- lated GlcCer. However, it accumulates demethy- lated ceramide and demethylated GlcCer. The Dsmt1 mutant loses more than 80% of its virulence compared with the wild type and the reconstituted strain. Interestingly, growth of C. neoformans Dsmt1 in the lung was decreased and C. neofor- mans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild type and Dsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organiza- tion and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids rep- resents a critical factor for the ability of the fungus to establish virulence.

The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

Understanding the mechanisms underlying the abnormal differentiation of human acute myeloid leukemia (AML) may reveal novel therapies to eradicate leukemic stem cells (LSC), which are often resistant to standard treatments and contribute to relapse. Cellular metabolism is recognized as a hallmark of cancer and is known to be distinct between hematopoietic stem cells (HSC) and downstream progenitors. In particular, sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that regulates proliferation and survival and is implicated in HSC egress, lymphocyte trafficking and mouse lymphoid lineage determination primarily through binding to S1PR1, one of five S1P G-protein coupled receptors. However, sphingolipids are understudied in human LSC and HSC. We recently found that sphingolipid perturbation governs HSC self-renewal and influences lineage outcome. Here, we show that S1PR3 governs myeloid commitment of LSC in a subset of human AML and is thus an attractive therapeutic target. Lipidomic analysis of LSC+ and LSC‒ fractions derived from AML patient samples and validated by xenotransplantation assays showed distinct sphingolipid alterations when compared to each other and to normal human cord blood (CB) CD34+CD38‒ stem cells and CD34+CD38+ progenitor populations. Interestingly, LSC+ fractions have increased S1P levels, prompting us to wonder if S1P signaling contributes to LSC maintenance. To gain a better understanding of the role of S1P in stemness, we examined S1P signaling in normal CB. Analysis of gene expression of S1PR1-5 and S1P transporters (SPNS2 and MFSD2B) within a comprehensive transcriptional roadmap of human hematopoiesis comprising thirteen normal populations from HSC to mature blood lineages demonstrated distinct expression patterns in specific blood lineages. S1PR1 and MFSD2B were most highly expressed in lymphoid and erythroid lineages, respectively, consistent with murine data. Notably, S1PR3 expression is specific to mature monocytes and granulocytes in human CB. S1PR3 protein was absent from the surface of long-term (LT) and short-term (ST)-HSC as determined by flow cytometry. Remarkably, lentiviral overexpression (OE) of S1PR3 was sufficient to promote myelopoiesis at the expense of erythropoiesis from LT- and ST-HSC in vitro in liquid culture, single cell assays and colony forming assays. To ascertain the mechanisms promoting myeloid commitment, we performed gene expression profiling by RNA-seq of LT- and ST-HSC following S1PR3 OE. This yielded a subset of shared genes similarly upregulated following S1PR3 OE relative to controls, including the known myeloid differentiation and AML-associated factors Early Growth Response 1 and 2 (EGR1/2) and Tribbles 1 (TRIB1). Thus, promiscuous expression of S1PR3 promotes a myeloid fate program in human HSC. S1PR3 protein expression was higher in AML patient samples relative to human CB and bone marrow cells by flow cytometry. Bioinformatic analysis of three independent AML cohorts revealed that AML patient samples with high S1PR3 gene expression also had high EGR2 and TRIB1 expression. Limiting dilution xenotransplantation assays of LSC-containing fractions sorted based on surface expression of S1PR3 demonstrated lower LSC frequency in S1PR3high versus S1PR3low/- LSC fractions. Moreover, S1PR3 OE in LSC+ fractions virtually abolished leukemic engraftment in xenotranplantation assays. These results suggest that higher S1PR3 levels in LSC are associated with a more mature myeloid state and that further increasing S1PR3 levels disrupts LSC maintenance. Treatment of mice bearing primary AML xenografts with FTY720, a S1P mimetic that targets S1P receptors including S1PR3, decreased leukemia burden in a subset of patient samples tested, including those obtained from relapsed and treatment-refractory patients. 70% of AML samples tested showed decreased LSC frequency in serial repopulation assays following FTY720 treatment. Importantly, treatment with FTY720 did not alter normal hematopoietic xenografts, demonstrating the existence of a therapeutic window. Collectively, our results provide the first direct evidence that sphingolipids govern myeloid commitment in human HSC and LSC, and demonstrate the potential of S1PR3 as a novel therapeutic target in AML for eradication of LSC while sparing HSC. Disclosures No relevant conflicts of interest to declare.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMaseWT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C16-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMaseWT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMaseS508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C16-ceramide. Taken together, these data support a crucial role for Ser508 in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.

Ceramide synthases (CerS1–CerS6), which catalyze the N-acylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide in both the de novo and the salvage or recycling pathway of ceramide generation, have been implicated in the control of programmed cell death. However, the regulation of the de novo pathway compared with the salvage pathway is not fully understood. In the current study, we have found that late accumulation of multiple ceramide and dihydroceramide species in MCF-7 cells treated with TNFα occurred by up-regulation of both pathways of ceramide synthesis. Nevertheless, fumonisin B1 but not myriocin was able to protect from TNFα-induced cell death, suggesting that ceramide synthase activity is crucial for the progression of cell death and that the pool of ceramide involved derives from the salvage pathway rather than de novo biosynthesis. Furthermore, compared with control cells, TNFα-treated cells exhibited reduced focal adhesion kinase and subsequent plasma membrane permeabilization, which was blocked exclusively by fumonisin B1. In addition, exogenously added C6-ceramide mimicked the effects of TNFα that lead to cell death, which were inhibited by fumonisin B1. Knockdown of individual ceramide synthases identified CerS6 and its product C16-ceramide as the ceramide synthase isoform essential for the regulation of cell death. In summary, our data suggest a novel role for CerS6/C16-ceramide as an upstream effector of the loss of focal adhesion protein and plasma membrane permeabilization, via the activation of caspase-7, and identify the salvage pathway as the critical mechanism of ceramide generation that controls cell death.

Sphingolipids are a class of bioactive lipids, which are key modulators of an increasing number of physiologic and pathophysiologic processes that include cell cycle, apoptosis, angiogenesis, stress and inflammatory responses. Sphingomyelin is an important structural component of biological membranes, and one of the end-points in the synthesis of sphingolipids. Mainly synthetized in the Golgi apparatus, sphingomyelin is transported to all other biological membranes. Upon stimulation, sphingomyelin can be hydrolyzed to ceramide by 5 different sphingomyelinases. The diversity and cellular topology of ceramide allow it to exert multiple biologies. Furthermore, ceramide can be metabolized to many other bioactive sphingolipids. Ceramide, coming from sphingomyelin or other complex sphingolipids, can be hydrolyzed to sphingosine, which can easily change cellular localization. In turn, sphingosine can be recycled to ceramide and to sphingomyelin in the endoplasmic reticulum, completing the sphingomyelin cycle. Our understanding of the roles of various sphingolipids in the regulation of different cellular processes has come from studying the enzymes that regulate these sphingolipids, and their manipulation. The use of pharmacologic inhibitors has been critical for their study, as well as being promising bullets for disease treatment. Some of these diseases involving the sphingomyelin cycle include cancer, inflammation, atherosclerosis, diabetes and some rare diseases such as Niemann–Pick disease. This review will focus on the enzymes involved in the sphingomyelin cycle, their history, and their involvement in pathophysiological processes. Finally, it will describe in details all the small molecules that are being used to inhibit these enzymes and their use in therapeutics.

The authors inadvertently omitted a phosphate group from the chemical structure of Inositol phosphorylceramide (IPC) in Fig 2. The authors have provided a corrected version here. Fig 2 Glycosphingolipid pathway in fungi.

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients.

Inositol-phosphorylceramide synthase 1 (Ipc1) is a fungal-specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG). In previous studies, we demonstrated that Ipc1 regulates the expression of the antiphagocytic protein 1 (App1), a novel fungal factor involved in pathogenicity of Cryptococcus neoformans. Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in the C. neofor-mans GAL7:IPC1 strain, in which the Ipc1 expression can be modulated, and found that the luciferase activity was indeed regulated when Ipc1 was modulated. Next, using the luciferase reporter assay in both C. neoformans wild-type and GAL7:IPC1 strains, we investigated the role of DAG and sphingolipids in the activation of the APP1 promoter and found that treatment with 1,2-dioctanoylglycerol does increase APP1 transcription, whereas treatment with phytosphingosine or ceramides does not. Two putative consensus sequences were found in the APP1 promoter for ATF and AP-2 transcription factors. Mutagenesis analysis of these sequences revealed that they play a key role in the regulation of APP1 transcription: ATF is an activator, whereas AP-2 in a negative regulator. Finally, we identified a putative Atf2 transcription factor, which is required for APP1 transcription and under the control of Ipc1-DAG pathway. These studies provide novel regulatory mechanisms of the sphingolipid pathway involved in the regulation of gene transcription of C. neoformans.

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of ∼75 kDa and one of 30–35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

One of the characteristics of ceramide-mediated biology is the variety of biological outcomes observed in response to its intracellular accumulation. The molecular mechanisms that govern the cell "decision-making" in response to ceramide remain largely unclear. In this perspective, the study of neural models has begun to provide important insight into the understanding of these mechanisms that regulate differentiation and cell death. Indeed, differentiation and cell death are among the most common effects elicited by ceramide in most cell types and in neural cells, too. Therefore, the lessons we may learn from the study of ceramide regulation of neurobiology would also shed light on the regulation of ceramide-mediated biology in other cellular models. Since increasing evidence links aberrant metabolism of ceramide to different pathologies, the understanding of the mechanisms underlying these events may represent the key to the design of novel therapeutic approaches.

Background A novel murine mitochondria-associated neutral sphingomyelinase (MA-nSMase) has been recently cloned and partially characterized. The subcellular localization of the enzyme was found to be predominant in mitochondria. In this work, the determinants of mitochondrial localization and its topology were investigated. Methods MA-nSMase mutants lacking consecutive regions and fusion proteins of GFP with truncated MA-nSMase regions were constructed and expressed in MCF-7 cells. Its localization was analyzed using confocal microscopy and sub-cellular fractionation methods. The sub-mitochondrial localization of MA-nSMase was determined using protease protection assay on isolated mitochondria. Results The results initially showed that a putative mitochondrial localization signal (MLS), homologous to an MLS in the zebra-fish mitochondrial SMase is not necessary for the mitochondrial localization of the murine MA-nSMase. Evidence is provided to the presence of two regions in MA-nSMase that are sufficient for mitochondrial localization: a signal sequence (amino acids 24–56) that is responsible for the mitochondrial localization and an additional ‘signal-anchor’ sequence (amino acids 77–99) that anchors the protein to the mitochondrial membrane. This protein is topologically located in the outer mitochondrial membrane where both the C and N-termini remain exposed to the cytosol. Conclusions MA-nSMase is a membrane anchored protein with a MLS and a signal-anchor sequence at its N-terminal to localize it to the outer mitochondrial membrane. General significance Mitochondrial sphingolipids have been reported to play a critical role in cellular viability. This study opens a new window to investigate their cellular functions, and to define novel therapeutic targets.

Sphingolipids have recently emerged as key regulators of pathogenicity in a variety of fungi. Glucosylceramide is a sphingolipid important for fungal cell division, alkaline tolerance, hyphal formation, and spore germination and, thus, for the regulation of fungal virulence. Present in many fungi, including yeasts, molds, and in dimorphic fungi, fungal glucosylceramide exhibits a characteristic structure synthesized by fungal specific enzymes. Thus, it is envisioned as an important fungal target for compounds with specific and broad-spectrum activity.

Protein kinase C (PKC) is known to activate NF-kappaB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-kappaB by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCbeta2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-kappaB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.

Cryptococcus neoformansis a leading cause of life-threatening fungal infection in immunocompromised patients. Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungus-specific enzyme, encoded by the essentialIPC1gene, that catalyzes the formation of complex sphingolipids and may also regulate the levels of phytoceramide and diacylglycerol. Here, we investigated the functions of this essential gene by modulating its expression inC. neoformansusing a galactose-inducible promoter. Down-regulation ofIPC1significantly lowers the expression of certain virulence traits such as melanin pigmentation and, remarkably, impairs pathogenicity ofC. neoformansin an established rabbit model. Interestingly, we found thatIPC1down-regulation significantly decreases the intracellular growth ofC. neoformansin the J774.16 murine macrophage-like cells. Finally, we studied the effect ofIPC1expression under different stress conditions and found that down-regulation ofIPC1confers a defect on in vitro growth at low pH. Because this environment is similar to that in the phagolysosome of J774.16 macrophage-like cells, our findings indicate that down-regulation ofIPC1confers a growth defect in vivo through a pH-dependent mechanism. In conclusion, our study is the first to define a novel and crucial function of Ipc1 in fungal pathogenesis.

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show that ISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression of ISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates of ISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [(3)H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.

In order to study the in vivo function of the phosphatidylinositol transfer protein β (PI-TPβ), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPβ. Two stable cell lines were isolated (SPIβ2 and SPIβ8) in which the levels of PI-TPβ were increased 16- and 11-fold respectively. The doubling time of the SPIβ cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPβ expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-3H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [3H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [3H]SM in wt NIH3T3 cells was degraded, whereas the levels of [3H]SM in SPIβ cells appeared to be unaffected. Since the release of [3H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIβ cells, these results strongly suggest that breakdown of SM in SPIβ cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [3H]SM in SPIβ cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIβ cells than in 50% [3H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIβ cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIβ cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPβ plays an important role in restoring the steady-state levels of SM.

In conclusion, the study of signaling and cell regulation through ceramide has now evolved to the biochemical level by focusing on specific enzymes of ceramide metabolism. A general hypothesis can be presented whereby individual enzymes of ceramide metabolism serve as input points in the regulation of ceramide levels (Scheme 1). For example, activation of SMases or ceramide synthase would elevate ceramide levels and activate ceramide-induced responses. On the other hand, activation of enzymes of ceramide degradation or incorporation such as ceramidases or SM synthase decreases and attenuates ceramide levels. Also, as an important corollary, some of these enzymes may play an additional fundamental role in interconverting lipid signals. For example, SM synthase has the capacity of interconverting a ceramide signal into a DAG signal, whereas ceramidases can transform a ceramide signal into a sphingosine or sphingosine-phosphate signal. This area of research promises great future insight into important areas of cell studies.

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. In this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). The addition of [3H]C2-ceramide to cells resulted in a time-dependent formation of [3H]C2-SM. At 24 h after treatment, normal WI38 cells cleared 17% of [3H]C2-ceramide producing [3H]C2-SM, which accounted for 13% of total radioactivity. On the other hand, SV40-transformed cells cleared 45% of [3H]C2-ceramide and produced C2-SM, which accounted for 24% of total radioactivity. This enhanced production of C2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cells had SMS activity of 222 pmol/mg of protein/h, whereas wild type cells had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasma membrane. Treatment of cells with exogenous bacterial sphingomyelinase (SMase) for 25 min resulted in cleavage of 90-95% of total SM and the concomitant generation of ceramide. After bacterial SMase treatment, wild type WI38 cells cleared ceramide very slowly (19.2 pmol of ceramide/nmol of phosholipid Pi after 6 h of incubation) and hardly regenerated any SM. On the other hand, SV40-transformed cells cleared ceramide much faster (41.1 pmol/nmol of Pi after 6 h of incubation) and regenerated approximately 80% of the original SM. These results show that the enhanced SMS activity of transformed cells is particularly pronounced when ceramide is produced in the plasma membrane. Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC). We, therefore, tested the effects of D609, a purported PC-PLC-specific inhibitor on the activity of SMS. D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10-50 microg/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.

The effects of the partial pressure of oxygen (pO2) on antioxidant efficiency of β-carotene in inhibiting radical-initiated lipid peroxidation were studied in murine normal and tumor thymocytes. At 150 mm Hg pO2 (the pressure of oxygen in normal air), β-carotene acted as an antioxidant, inhibiting radical-induced lipid peroxidation in both normal and tumor thymocytes. At 760 mm Hg pO2, β-carotene lost its antioxidant activity in normal thymocytes and exhibited a dose-dependent prooxidant effect in tumor thymocytes. In these cells, the prooxidant effect of β-carotene was also accompanied by an increase of endogenous α-tocopherol loss. β-Carotene radical-trapping and autooxidation reactions were faster at 760 mm Hg pO2 than at 150 mm Hg pO2 in both normal and tumor thymocytes and the carotenoid was more rapidly consumed in tumor cells. These data point out a key role of the oxygen tension on the antioxidant effectiveness of β-carotene. They also show a selective prooxidant effect of β-carotene under 100% oxygen in tumor cells. Copyright © 1997 Elsevier Science Inc.

Just as non-lipidologists were becoming reconciled to the complexities of networks of ceramide metabolism and the diverse roles of ceramide in cell regulation and membrane structure, a new study reveals a new layer of regulation of ceramide through the actions of a ceramide binding/transfer protein.

Subjects at high risk for colon cancer received different doses of fish oil on a 30-day randomized double-blind trial to evaluate the chemopreventive effect of n-3 fatty acids against colorectal cancer. Using rectal mucosal proliferation, assessed with 3 H-thymidine autoradiography, fish oil induced in the treated groups but not in the placebo group a change in the proliferative pattern, which resulted similar to that observed in low risk polulation; in the same groups rectal mucosal n-3 fatty acid content increased, where arachidonic acid level decreased. Moreover, n-3 PUFA treatment induced modifications of Vitamin E status. The results suggest that n-3 PUFA could protect high-risk subjects from colon cancer by a mechanism involving a modulation of Vitamin E.

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-β cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

In the last five years tremendous progress has been made toward the understanding of the mechanisms that govern sphingomyelin (SM) synthesis in animal cells. In line with the complexity of most biological processes, also in the case of SM biosynthesis, the more we learn the more enigmatic and finely tuned the system appears. Therefore with this review we aim first, at highlighting the most significant discoveries that advanced our knowledge and understanding of SM biosynthesis, starting from the discovery of SM to the identification of the enzymes responsible for its production; and second, at discussing old and new riddles that such discoveries pose to current investigators.