Gentamicin Arrests Cancer Cell Growth: The Intriguing Involvement of Nuclear Sphingomyelin Metabolism

The use of gentamicin for the treatment of bacterial infection has always been an interesting and highly speculated issue for the scientific community. Conversely, its effect on cancer cells has been very little investigated. We studied the effect of high doses of gentamicin on non-Hodgkin’s T-cell human lymphoblastic lymphoma (SUP-T1). We showed that gentamicin delayed cell growth and induced cell death in lymphoma cells with a rather mild effect on lymphocytes. In SUP-T1 cells, GAPDH, B2M, CDKN1A and CDKN1B were down-expressed in comparison with lymphocytes. Gentamicin treatment in SUP-T1 cells restored the expression of GAPDH, B2M and CDKN1A to values similar to those of lymphocytes and caused overexpression of CDKN1B. The drug acted via sphingomyelin metabolism; in whole cells, sphingomyelinase activity was stimulated, whereas in purified nuclei, sphingomyelinase activity was inhibited and that of sphingomyelin-synthase was stimulated, with a consequent high level of nuclear sphingomyelin content. We suggest that the increase of nuclear sphingomyelin might enrich the nucleus of lipid microdomains that act as a platform for active chromatin and, thus, might be responsible for gene expression. It is possible that in lymphoblastic lymphoma, high doses of gentamicin induce a beneficial therapeutic outcome.     

Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells

The release of exosomes can lead to cell–cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.     

Involvement of the ceramide signaling pathway in modulating the differentiated state of porcine thyroid cells

Neutral sphingomyelinase (Smase) is a cell membrane-associated phospholipase that hydrolyzes sphingomyelin to phosphocholine and ceramide, a lipid second messenger involved in cell differentiation and/or apoptosis. We first evidenced that porcine cultured thyroid cells could express neutral Smase activity even if thyrotropin (TSH), an essential hormone in thyroid cell differentiation, was found to induce a 1.7-fold decrease in Smase activity. Triggering the ceramide pathway by exogenous addition of neutral bacterial Smase (0.1 U/mL for 48 h), which transiently increased ceramide level by fourfold, drastically modified thyroid cell morphology. The follicle-like structures generated by TSH were disrupted, and the Smase-induced cell spreading was accompanied by a parallel loss of cell ability to iodinate proteins as well as a decrease of the adenylate cyclase system response. These inhibitory effects have been reproduced using short-chain exogenous ceramide analogs (C₂-ceramides). Overall these data showed that ceramides emerged as potential mediators of dedifferentiation in thyroid cells.     

Development of intestinal alkaline sphingomyelinase in rat fetus and newborn rat

Sphingomyelin metabolism is a novel signal transduction pathway related to cell differentiation, proliferation, and apoptosis. Alkaline sphingomyelinase (alk-SMase) is specifically present in the intestinal tract of many species. The enzyme is important in digestion of dietary sphingomyelin. Milk is the ony exogenous source of sphingomyelin for an infant, and digestion of milk sphingomyelin may be important for development of intestinal mucosa. It is unknown whether alk-SMase is present before birth and whether it changes after birth and during the suckling period. We studied activities, expression, and distribution of alk-SMase in rat fetus and newborn. The changes of acid and neutral SMase as well as alkaline phosphatase were analyzed for comparison. Little activity of alk-SMase was identified up to gestation day 20, but increased 10 times during the following 2 d. After birth, the activity continused to increase during the following 4 wk. Western blot using IgY antibody against rat alk-SMase failed to identify the enzyme at gestation day 20 but clearly showed the protein at day 22. The distribution pattern of the enzyme along the intestinal tract in fetus was largely the same as in adult animals, but became more pronounced after birth. Short-term weaning had no effect on alk-SMase activity. The activities of acid and neutral SMase were high at gestation day 20 and decreased significantly before birth. The changes of alk-SMase also differed from those of alkaline phosphatase, another brush border enzyme. Thus, we conclude that alk-SMase is rapidly expressed during the last days of gestation and that the newborn rat acquires the ability to digest milk sphingomyelin early in life.     

Inhibition of Endothelial Lipase Activity by Sphingomyelin in the Lipoproteins

Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration‐dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn‐2 position, generating the corresponding PUFA‐lyso PtdCho. This specificity for PUFA‐PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.     

Ceramide in Pseudomonas aeruginosa infections

Cystic fibrosis (CF), the most common autosomal recessive disorder, at least in western countries, is caused by mutations of the cystic fibrosis transmembranous conductance regulator (CFTR) molecule and affects approximately 80,000 patients in Europe and the USA. Most, if not all, CF patients develop a chronic pulmonary infection with Pseudomonas aeruginosa. At present it is unknown why CF patients are highly sensitive to P. aeruginosa infections, and most importantly, no curative treatment for CF is available. P. aeruginosa infection results in an activation of the enzyme acid sphingomyelinase which catalyzes the release of ceramide from sphingomyelin in the cell membrane. Ceramide forms large ceramide‐enriched membrane domains that are required for internalization of bacteria, induction of cell death in infected cells and a controlled release of cytokines from infected cells. Ceramide‐enriched membrane platforms seem to serve the reorganization of receptors and intracellular signaling molecules involved in the infection of mammalian cells with P. aeruginosa. The significance of the acid sphingomyelinase and ceramide for the infection of mammalian cells with P. aeruginosa was demonstrated on mice genetically deficient for the acid sphingomyelinase. Further studies with N. gonorrhoeae, S. aureus and rhinoviruses indicate that ceramide‐enriched membrane domains are also important for the infection of mammalian cells with other bacterial and viral pathogens, suggesting a general role of these membrane domains in infectious biology.     

Acid Sphingomyelinase Deficiency: A Clinical and Immunological Perspective

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disease caused by deficient activity of acid sphingomyelinase (ASM) enzyme, leading to the accumulation of varying degrees of sphingomyelin. Lipid storage leads to foam cell infiltration in tissues, and clinical features including hepatosplenomegaly, pulmonary insufficiency and in some cases central nervous system involvement. ASM enzyme replacement therapy is currently in clinical trial being the first treatment addressing the underlying pathology of the disease. Therefore, presently, it is critical to better comprehend ASMD to improve its diagnose and monitoring. Lung disease, including recurrent pulmonary infections, are common in ASMD patients. Along with lung disease, several immune system alterations have been described both in patients and in ASMD animal models, thus highlighting the role of ASM enzyme in the immune system. In this review, we summarized the pivotal roles of ASM in several immune system cells namely on macrophages, Natural Killer (NK) cells, NKT cells, B cells and T cells. In addition, an overview of diagnose, monitoring and treatment of ASMD is provided highlighting the new enzyme replacement therapy available.     

Ursolic acid and other pentacyclic triterpenoids stimulate intestinal alkaline sphingomyelinase in vitro

Purpose: Alkaline sphingomyelinase (alk‐SMase) is an enzyme that hydrolyses sphingomyelin in a bile salt‐dependent manner in the gastrointestinal tract, and has been proposed as an inhibitor of colon carcinogenesis. Ursolic acid (UA) is a plant‐derived pentacyclic triterpenoid that has been shown to have anti‐proliferative and apoptotic effects on HT29 human colon adenocarcinoma cells, with activation of alk‐SMase as an early event. The aim of this study was to study the in vitro effects of UA and its analogues on the activity of purified rat intestinal alk‐SMase. Methods: Rat intestinal alk‐SMase activity was determined after incubation with UA in the presence and absence of taurocholate (TC). The effect was compared with boswellic acids, another group of pentacyclic triterpenoids. Results: UA enhanced the activity of rat intestinal alk‐SMase in a dose‐dependent manner, without a similar effect on bacterial neutral SMase. Four types of boswellic acid also increased the enzyme activity, with the effect of acetyl‐keto‐β‐boswellic acid being most potent. Activation of alk‐SMase by TC at a low concentration (0.4 mM), but not at a high concentration, was enhanced by UA. Conclusions: Ursolic acid and four types of boswellic acid, all pentacyclic triterpenoids, have a stimulatory effect on the activity of intestinal alk‐SMase.     

燃料电池用质子交换膜电渗拖曳的研究进展

质子交换膜的电渗拖曳直接影响燃料电池的水管理和电池系统复杂性。本文对质子交换膜电渗拖曳系数的测量方法进行了综述,并指出了各种方法的优缺点。经比较发现：电渗拖曳池、氢泵、电泳核磁共振法可测量质子交换膜与液态水接触时的电渗拖曳系数,其值一般在2～5;活度梯度法和电泳核磁共振法可测量质子交换膜与气态水接触时的电渗拖曳系数。随着复合质子交换膜的发展,亟需普遍性的测量方法测定复合膜的电渗拖曳系数,为燃料电池模型提供相关的参数,以利于数学模型对质子交换膜的准确描述。     

Aerobic Training in Rats Increases Skeletal Muscle Sphingomyelinase and Serine Palmitoyltransferase Activity,While Decreasing Ceramidase Activity

Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of fat,FA, and cholesterol on sphingomyelin hydrolysis induced by rat intestinal alkaline sphingomyelinase

Dietary sphingomyelin (SM) may have regulatory effects on cell proliferation and tumorigenesis in the colon. Alkaline sphingomyelinase (SMase) is the major enzyme responsible for hydrolysis of SM in the gut. Previously we purified the enzyme and showed that the presence of glycerophospholipids inhibited SM hydrolysis induced by alkaline SMase in vitro. In the present work, we studied the effects of TG, DG, FA, ceramide, and cholesterol on SM hydrolysis catalyzed by purified alkaline SMase. The results showed that both TG (triolein and tristearin) and DG (1,2-dioleoyl-sn-glycerol and 1,2-distearoyl-rac-glycerol) inhibited the activity of alkaline SMase. 1-Mono-oleoyl-rac-glycerol, 1-monostearoyl-rac-glycerol, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid stimulated the activity of alkaline SMase at 0.4–0.8 mM concentrations but inhibited the enzyme at higher concentrations. There was no difference between the effects induced by saturated and unsaturated FA. A short-chain FA such as lauric acid had a stronger stimulatory effect at low concentrations and weaker inhibitory effect at high concentrations than long-chain FA. Choosing linoleic acid as an example, we found that FA had similar effects on both alkaline SMase and neutral SMase. Cholesterol and ceramide when mixed with FA to increase its solubility in bile salt micelles inhibited SMase activity. In conclusion, glycerides, FA, ceramide, and cholesterol influence SM hydrolysis catalyzed by intestinal alkaline SMase. The presence of lipids in the diet may thus influence the course of SM digestion in the gut and thereby the exposure of colon to SM metabolites.     

Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion

To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary. bile acid, phospholipid, and cholesterol and in increased cholesterol 7α-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, ∼45%; apoB-100, ∼32%) and lipids (∼55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol out-put pathways in hepatocytes.     

Effect of Sterol Carrier Protein-2 Expression on Sphingolipid Distribution in Plasma Membrane Lipid Rafts/Caveolae

Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.     

Acid Sphingomyelinase,a Lysosomal and Secretory Phospholipase C,Is Key for Cellular Phospholipid Catabolism

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.     

Acid-Sphingomyelinase Triggered Fluorescently Labeled Sphingomyelin Containing Liposomes in Tumor Diagnosis after Radiation-Induced Stress

In liposomal delivery, a big question is how to release the loaded material into the correct place. Here, we will test the targeting and release abilities of our sphingomyelin-consisting liposome. A change in release parameters can be observed when sphingomyelin-containing liposome is treated with sphingomyelinase enzyme. Sphingomyelinase is known to be endogenously released from the different cells in stress situations. We assume the effective enzyme treatment will weaken the liposome making it also leakier. To test the release abilities of the SM-liposome, we developed several fluorescence-based experiments. In in vitro studies, we used molecular quenching to study the sphingomyelinase enzyme-based release from the liposomes. We could show that the enzyme treatment releases loaded fluorescent markers from sphingomyelin-containing liposomes. Moreover, the release correlated with used enzymatic activities. We studied whether the stress-related enzyme expression is increased if the cells are treated with radiation as a stress inducer. It appeared that the radiation caused increased enzymatic activity. We studied our liposomes’ biodistribution in the animal tumor model when the tumor was under radiation stress. Increased targeting of the fluorescent marker loaded to our liposomes could be found on the site of cancer. The liposomal targeting in vivo could be improved by radiation. Based on our studies, we propose sphingomyelin-containing liposomes can be used as a controlled release system sensitive to cell stress.     

Synthesis and antiapoptotic activity of a novel analogue of the neutral sphingomyelinase inhibitor scyphostatin

The enantioselective synthesis of an analogue of scyphostatin, a potent inhibitor of the neutral sphingomyelinase, is described. The synthesis starts with cyclohexanone and a protected D-serine derivative. The key step is an asymmetric hydroxylation to access a hydroxycyclohexanone, which is transformed into a substituted hydroxycyclohexenone. This is converted into the scyphostatin analogue 14, a chemically and metabolically stabilised compound lacking the epoxy function of the natural congener and carrying a palmitic acid group instead of the native trienoyl residue. An evaluation of the biological activity of 14 revealed neutral sphingomyelinase inhibition in several in vivo test systems (monocytes, macrophages, hepatocytes) monitoring antiapoptotic effects and the inversion of phorbolester-induced translocation of green fluorescent protein labelled kinase (protein kinase C-alpha).     

Potent Inhibition of Acid Sphingomyelinase by Phosphoinositide Analogues

The different mammalian sphingomyelinases are involved in cell regulation, apoptosis and inflammatory events. Recent reports suggest pharmacological potential especially for inhibitors of the acid sphingomyelinase. Phosphatidyl inositol‐3,5bisphosphate (PtdIns3,5P₂) is the most potent selective acid sphingomyelinase inhibitor known to date. In the present study, we synthesized analogues of PtdIns3,5P₂ for initial structure–activity‐relationship (SAR) studies. We identified an inhibitor that is easy to synthesize, that has superior chemical and biophysical properties when compared to PtdIns3,5P₂ and that should be stable against virtually all phospholipases. Last but not least, the new inhibitor partially protected cells from dexamethasone‐induced cell death.     

Reliability Evaluation of an Open‐Cathode PEMFC at Operating State and Longtime Vibration by Mechanical Loads

Reliability and durability of proton exchange membrane fuel cells (PEMFCs) are one of the most complex issues in real applications' environment. Especially, when it subjects to the various mechanical loads and vibrations. This topic requires more attention for research and experimental works. In this study, the effect of mechanical loads was investigated on an open‐cathode PEMFC in operating state. Several long time vibration tests have been applied on non‐operating PEMFC, and the performance of the fuel cell was evaluated during the test. Hydrogen leakage as a key parameter was investigated in performance monitoring instead of measuring torque on compression bolts. The vibration tests were consisted of sine, shock and random for X, Y and Z axes in operating state and random vibration in non‐operating state of PEMFC. The experimental results in operating state were indicated that the fuel cell performance has not been affected by the proposed vibrations. Furthermore, the test results of non‐operating state have been shown that the performance of PEMFC reduces about 0.6% in each four‐hour step of the vibration test. In addition, the experiments reveal that if the mechanical loads and vibrations cause physical damage on the fuel cell components, they can change the performance and reliability of the fuel cell.