Distinct cardiotoxic effects by venoms of a spitting cobra (Naja pallida) and a rattlesnake (Crotalus atrox) revealed using an ex vivo Langendorff heart model.

Here we describe the acute myocardial effects of an elapid (red spitting cobra, Naja pallida) and a viper (western diamondback rattlesnake, Crotalus atrox) venom using an ex vivo heart model. Our results reveal two different pathophysiological trajectories that influence heart function and morphology. While cobra venom causes a drop in contractile force, rattlesnake venom causes enhanced contractility and frequency that coincides with differences in myocellular morphology. This highlights the medical complexity of snake venom-induced cardiotoxicity.

Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions-Ara h2 and Ara h6 Are Readily Bio-Accessible.

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (>8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity.

Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors.

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.

AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency.

BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.

ABCA1-dependent but apoA-I-independent cholesterol efflux mediated by fatty acid-bile acid conjugates (FABACs).

FABACs (fatty acid-bile acid conjugates) are synthetic molecules that are designed to treat a range of lipid disorders. The compounds prevent cholesterol gallstone formation and diet-induced fatty liver, and increase reverse cholesterol transport in rodents. The aim of the present study was to investigate the effect of FABACs on cholesterol efflux in human cells. Aramchol (3beta-arachidylamido-7alpha,12alpha,5beta-cholan-24-oic acid) increased cholesterol efflux from human skin fibroblasts in a dose-dependent manner in the absence of known efflux mediators such as apoA-I (apolipoprotein A-I), but had little effect on phospholipid efflux. An LXR (liver X receptor) agonist strongly increased Aramchol-induced cholesterol efflux; however, in ABCA1 (ATP-binding cassette transporter A1)-deficient cells from Tangier disease patients, the Aramchol effect was absent, indicating that activity of ABCA1 was required. Aramchol did not affect ABCA1 expression, but plasma membrane levels of the transporter increased 2-fold. Aramchol is the first small molecule that induces ABCA1-dependent cholesterol efflux without affecting transcriptional control. These findings may explain the beneficial effect of the compound on atherosclerosis.

Enhanced conversion of triglyceride-rich lipoproteins and increased low-density lipoprotein removal in LPLS447X carriers.

OBJECTIVE: Lipoprotein lipase (LPL) exerts 2 principal actions, comprising enzymatic hydrolysis of triglyceride-rich lipoproteins (TRLs) and nonenzymatic ligand capacity for enhancing lipoprotein removal. The common LPLS447X variant has been associated with cardiovascular protection, for which the mechanism is unknown. We therefore evaluated enzymatic and nonenzymatic consequences of this LPL variant on TRL metabolism. METHODS AND RESULTS: TRL apolipoprotein B100 (apoB100) metabolism was determined in 5 homozygous LPLS447X carriers and 5 controls. Subjects were continuously fed and received infusion of stable isotope l-[1-(13C)]-valine. Results were analyzed by SAAMII modeling. Also, preheparin and postheparin LPL concentration and activity were measured. Compared with controls, carriers presented increased very low-density lipoprotein 1 (VLDL1) to VLDL2 apoB100 flux (P=0.04), increased VLDL2 to intermediate-density lipoprotein (IDL) apoB100 flux (P=0.02), increased IDL to low-density lipoprotein (LDL) apoB100 flux (P=0.049), as well as an increased LDL clearance (P=0.04). Additionally, IDL apoB100 synthesis was attenuated (P=0.05). Preheparin LPL concentration was 4-fold higher compared with controls (P=0.01), and a correlation was observed between preheparin LPL concentration and LDL clearance (r2=0.92; P=0.01). CONCLUSIONS: Enhanced TRL conversion and enhanced LDL removal combined with increased preheparin LPL concentration suggest increased enzymatic consequences as well as increased nonenzymatic consequences of LPL in LPLS447X carriers, which might both contribute to the cardiovascular benefit of this LPL variant.

Coformulated N-octanoyl-glucosylceramide improves cellular delivery and cytotoxicity of liposomal doxorubicin.

The anticancer agent doxorubicin is in certain cases administered as a long-circulating liposomal formulation. Due to angiogenesis-related structural abnormalities in the endothelial lining of many neoplasms, these complexes tend to extravasate and accumulate in the tumor stroma. However, delivery of doxorubicin is still not optimal since liposomes are not taken up directly by tumor cells. Instead, doxorubicin is gradually released into the interstitial space, and the subsequent uptake by surrounding cells is a limiting step in the delivery process. We recently demonstrated that plasma membrane-inserted short-chain sphingomyelin facilitates the cellular uptake of free doxorubicin. Here, we report that N-octanoyl-glucosylceramide acts equally potent but is itself less toxic. When coformulated with liposomal doxorubicin, this short-chain glycosphingolipid administered to cultured A431 epidermoid carcinoma cells led to superior (up to 4-fold) cellular doxorubicin accumulation and cytotoxicity, compared with control doxorubicin liposomes. These results were fully reproducible when N-octanoyl-glucosylceramide was postinserted into Caelyx, a commercial liposomal doxorubicin preparation. The doxorubicin-potentiating effect of N-octanoyl-glucosylceramide-enriched liposomes proved relatively insensitive to high serum concentrations, indicating that in vivo application is a feasible option. N-Octanoyl-glucosylceramide enrichment might thus represent a major improvement of conventional liposomal doxorubicin formulations.

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport.

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.

The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs.

Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short-chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival. Furthermore, N-(n-butyl)deoxynojirimycin, a potent and non-toxic GCS inhibitor, had no chemosensitizing effect on wild-type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug-resistance mechanisms.

Ceramide: second messenger or modulator of membrane structure and dynamics?

The physiological role of ceramide formation in response to cell stimulation remains controversial. Here, we emphasize that ceramide is not a priori an apoptotic signalling molecule. Recent work points out that the conversion of sphingomyelin into ceramide can play a membrane structural (physical) role, with consequences for membrane microdomain function, membrane vesiculation, fusion/fission and vesicular trafficking. These processes contribute to cellular signalling. At the Golgi, ceramide takes part in a metabolic flux towards sphingomyelin, diacylglycerol and glycosphingolipids, which drives lipid raft formation and vesicular transport towards the plasma membrane. At the cell surface, receptor clustering in lipid rafts and the formation of endosomes can be facilitated by transient ceramide formation. Also, signalling towards mitochondria may involve glycosphingolipid-containing vesicles. Ceramide may affect the permeability of the mitochondrial outer membrane and the release of cytochrome c. In the effector phase of apoptosis, the breakdown of plasma membrane sphingomyelin to ceramide is a consequence of lipid scrambling, and may regulate apoptotic body formation. Thus ceramide formation serves many different functions at distinct locations in the cell. Given the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of natural ceramide species, the topology and membrane sidedness of ceramide generation are crucial determinants of its impact on cell biology.

Ara-C- and daunorubicin-induced recruitment of Lyn in sphingomyelinase-enriched membrane rafts.

Induction of apoptosis by DNA-damaging agents such as 1-beta-D-arabinofuranosylcytosine (Ara-C) includes the activation of Lyn protein tyrosine kinase. We have previously established that Ara-C-induced activation of Lyn results in its binding to a neutral sphingomyelinase (SMase) and is requisite for its stimulation and the induction of apoptosis in U937 cells. However, the spacio-temporal organization of these events is unclear. This study demonstrates that part of the total cellular SMase activity is sequestered in sphingomyelin-enriched plasma membrane microdomains (rafts). Under Ara-C and daunorubicin (DNR) treatment, Lyn is rapidly activated and translocated into rafts. The compartmentalization of Lyn (as well as neutral SMase activation and apoptosis) induced by these drugs was blocked by the tyrosine kinase inhibitor herbimycin A and raft disruption. In conclusion, this study establishes that DNA-damaging agents such as Ara-C and DNR rapidly induce Lyn activation and its translocation into membrane rafts. This, in turn leads to neutral SMase activation and raft-associated sphingomyelin hydrolysis with the concomitant generation of the proapoptotic lipid second messenger, ceramide. The apparent topological partitioning between DNA damage and apoptosis signaling (integrated into specialized plasma membrane domains) is discussed.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.