Effect of Loxosceles gaucho venom on cell morphology and behaviour in vitro in the presence and absence of sphingomyelin.

This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-alpha release generated by sphingomyelinase D--a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-alpha release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-alpha release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-alpha release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-alpha resistant MRC-5 cells and TNF-alpha sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-alpha induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-alpha as an intermediary.     

血脂异常患者血清神经鞘磷脂的测定及其意义

目的:评价血脂异常患者血清神经鞘磷脂(SM)水平。方法:将血脂异常的患者分为总胆固醇(TC)升高组、三酯酰甘油(TG)升高组、低密度脂蛋白胆固醇(LDL-C)升高组和高密度脂蛋白胆固醇(HDL-C)降低组,与对照组SM水平比较。酶法测定SM、TC、TG、LDL-C、HDL-C。用单因素方差分析,Dunnett检验处理数据。结果:血脂异常患者SM水平与对照组比较有显著性差异(P<0.05);TC、LDL-C升高组SM水平与对照组比较有显著性差异(P<0.05),TG升高组、HDL-C降低组SM水平与对照组比较有极显著性差异(P<0.01)。结论:血清SM水平和已知的致动脉粥样硬化危险因子密切相关,提示SM可能是导致动脉粥样硬化发生的重要因素。     

Increased Acid Sphingomyelinase Activity in Peripheral Blood Cells of Acutely Intoxicated Patients With Alcohol Dependence

Background:  Acid sphingomyelinase (ASM; EC 3.1.4.12) hydrolyses membrane sphingomyelin into the bioactive lipid ceramide and is thus involved in different cellular processes such as differentiation, immunity, or cell death. Activation of ASM has been reported in particular in conjunction with the cellular stress response to several external stimuli, and increased ASM activity was observed in a variety of human diseases. Ethanol-induced activation of ASM has been observed in different cell culture systems, thus raising the question about the effect of alcohol intoxication in human subjects on ASM activity in vivo.
Methods:  We determined ASM activity in peripheral blood mononucleated cells of 27 patients suffering from alcohol dependence. Patients were classified according to their blood alcohol concentration at admission, and ASM activity was determined repeatedly from all patients during alcohol withdrawal.
Results:  Acutely intoxicated patients displayed significantly higher ASM activity than patients in early abstinence (Mann–Whitney U test: Z  = − 2.6, p  = 0.009). ASM activity declined in acutely intoxicated patients to normal values with the transition from the intoxicated state to early abstinence (Wilcoxon test: Z  = −2.7, p  = 0.007). At the end of withdrawal, ASM activity was significantly increased again compared to the early phase of abstinence in both patient groups (Wilcoxon test: Z  = −2.691, p  = 0.007 and Z  = −2.275, p  = 0.023, respectively).
Conclusions:  Alcohol-induced activation of ASM occurs in human subjects and might be responsible for deleterious effects of ethanol intoxication. Chronic alcohol abuse may induce deregulation of sphingomyelin metabolism in general, and this impairment may cause side effects during withdrawal from alcohol.     

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A₂ activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.     

Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann–Pick A disease

Niemann–Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS.     

LDL aggregation susceptibility is higher in healthy South Asian compared with white Caucasian men

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Comprehensive characterization of neutral and polar lipids of buttermilk from different sources and its milk fat globule membrane isolates

Buttermilk (BM) has recently received much attention as a source of milk fat globule membrane (MFGM) due to its high polar lipid (PL) content, which gives it functional properties and health benefits. Nevertheless BM can be obtained from two different technological processes, i) the BM from the butter oil industry (BM1) and ii) BM from the butter manufacture (BM2). Neutral lipids (NL) and PL characterization from both BM and their MFGM isolated fractions have been qualitatively and quantitatively characterized including the individual phospho- and sphingolipids by HPLC-ELSD as well as triacylglycerols (TAG) and cholesterol and FAME by GC-FID. The results revealed that while BM (either from BM1 or BM2) had a PL fraction of 12–16 %, the MFGM isolated fraction contained about 40 % of PL with major presence of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). Besides, MFGM showed a significant increase in the medium molecular weight TAG and cholesterol and almost two fold amount of PUFA content than BM due mainly to linoleic acid. The results of this study provide deeper information on the lipid composition of BM and MFGM isolated fractions of great importance for the design of dietary supplements with potential beneficial effects on human health.     

Visualization of sphingolipids and phospholipids in the fundic gland mucosa of human stomach using imaging mass spectrometry

AIM:To analyze the lipid distribution in gastric mucosae.METHODS:Imaging mass spectrometry(MS)is a useful tool to survey the distribution of biomolecules in surgical specimens.Here we used the imaging MS apparatus named i MScope to identify the dominant molecules present in the human gastric mucosa near the fundic glands.Five gastric specimens were subjected to iM Scope analysis.These specimens were also analyzed by immunohistochemistry using MUC5 AC,H(+)-K(+)-ATPaseβ Claudin18 antibodies.RESULTS:Three major molecules with m/z 725.5,780.5,and 782.5 detected in the gastric mucosa were identified as sphingomyelin(SM)(d18:1/16:0),phosphatidylcholine(PC)(16:0/18:2),and PC(16:0/18:1),respectively,through MS/MS analyses.Using immunohistological staining,SM(d18:1/16:0)signals were mainly colocalized with the foveolar epithelium marker MUC5 AC.In contrast,PC(16:0/18:2)signals were observed in the region testing positive for the fundic gland marker H(+)-K(+)-ATPaseβ.PC(16:0/18:1)signals were uniformly distributed throughout the mucosa.CONCLUSION:Our basic data will contribute to the studies of lipid species in physical and pathological conditions of the human stomach.