Avanti Polar Lipids (United States)

The discovery of a novel sphingolipid subclass, the (1-deoxy)sphingolipids, which lack the 1-hydroxy group, attracted considerable attention in the last decade, mainly due to their involvement in disease. They differed in their physico-chemical properties from the canonical (or 1-hydroxy) sphingolipids and they were more toxic when accumulated in cells, inducing neurodegeneration and other dysfunctions. (1-Deoxy)ceramides, (1-deoxy)dihydroceramides, and (1- deoxymethyl)dihydroceramides, the latter two containing a saturated sphingoid chain, have been studied in this work using differential scanning calorimetry, confocal fluorescence and atomic force microscopy, to evaluate their behavior in bilayers composed of mixtures of three or four lipids. When compared to canonical ceramides (Cer), a C16:0 (1-deoxy)Cer shows a lower miscibility in mixtures of the kind C16:0 sphingomyelin/cholesterol/XCer, where XCer is any (1-deoxy)ceramide, giving rise to the coexistence of a liquid-ordered phase and a gel phase. The latter resembles, in terms of thermotropic behavior and nanomechanical resistance, the gel phase of the C16:0 sphingomyelin/cholesterol/C16:0 Cer mixture [Busto et al., Biophys. J. 2014, 106, 621–630]. Differences are seen between the various C16:0 XCer under study in terms of nanomechanical resistance, bilayer thickness and bilayer topography. When examined in a more fluid environment (bilayers based on C24:1 SM), segregated gel phases are still present. Probably related to such lateral separation, XCer preserve the capacity for membrane permeation, but their effects are significantly lower than those of canonical ceramides. Moreover, C24:1 XCer show significantly lower membrane permeation capacity than their C16:0 counterparts. The above data may be relevant in the pathogenesis of certain sphingolipid-related diseases, including certain neuropathies, diabetes, and glycogen storage diseases.

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.

The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids, and ceramides (Cers) in an equimolar ratio. The huge class of skin Cers consists of three groups: group I, "classical" long and very long chain Cers; group II, ultra-long chain Cers; and group III, ω-esterified ultra-long chain Cers, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acylceramides as a new class of epidermal Cers in humans and mice. These Cers contain, in both the N- and 1-O-position, long to very long acyl chains. They derive from the group I of classical Cers and make up 5% of all esterified Cers. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A2. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified Cers. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at endoplasmic reticulum-related sites.

While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acylphosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1% of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, monounsaturated, and polyunsaturated species. N-Acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells, and yeast, but not Escherichia coli. N-Acyl-PSs may be biosynthetic precursors of N-acylserine molecules, such as the recently reported signaling lipid N-arachidonoylserine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acylserine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoylethanolamine (anadamide) from N-arachidonoylphosphatidylethanolamine.

Intracellular nucleoside reverse transcriptase inhibitor triphosphate (NRTI-TP) concentrations are crucial in suppressing HIV replication. Little is known about how commonly used dual-NRTI regimens affect the intracellular levels of NRTI-TPs, the active form of these drugs. This study investigates the effect of dual-NRTI therapy in intracellular NRTI-TP levels. NRTI and NRTI-TP concentrations were evaluated in HIV-infected patients receiving either lamivudine (3TC) and stavudine (d4T) or lamivudine with zidovudine (ZDV); NRTI and NRTI-TP concentrations were determined using a validated HPLC/MS/MS method. Plasma HIV-1 RNA levels were determined at baseline and monthly to examine the relationship between NRTI-TP concentrations and plasma HIV-1 RNA. Forty-one subjects completed the study. 3TC-TP significantly increased between day 1 and week 28 from 1.48 to 5.00 pmol/10(6) peripheral blood mononuclear cells (PBMC; P < 0.0001). NRTI-TP concentrations for d4T and ZDV did not significantly increase, with values at week 28 of 0.011 and 0.02 pmol/10(6) PBMC, respectively. Mean NRTI-TP/plasma ratios were 3%, 0.007% and 0.05% for 3TC, d4T and ZDV, respectively. Linear relationships were observed between ZDV- and 3TC-TP and changes in plasma HIV-1 RNA. Of the three drugs studied, only 3TC-TP levels increased significantly between day 1 and week 28. ZDV-TP and 3TC-TP levels were unaffected by dual-NRTI therapy relative to monotherapy, regardless of the combination (3TC-ZDV or 3TC-d4T). Intracellular levels of d4T-TP were similar to previous reports for dual-NRTI therapy; however, in the case of d4T, these values appear lower than those achieved with d4T monotherapy.

The Venereal Disease Research Laboratory (VDRL) test is a microflocculation test for syphilis that uses an antigen containing cardiolipin, lecithin, and cholesterol. For more than 50 years, the preparation of natural cardiolipin and lecithin for this test has been based on the Pangborn method which involves isolating and purifying these components from beef hearts. This process is tedious and time-consuming and results in a variable purity range. In our studies, we found that a VDRL antigen using synthetic tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (lecithin) was as specific in detecting syphilis as a VDRL antigen made with natural components. In 85% of the cases, we obtained an endpoint titer of 1/2 or 1 dilution more than a titer obtained with a VDRL antigen made with natural components. The use of these pure synthetic compounds, with a purity of 99%, would offer advantages in the standardization and stability of the VDRL antigen. Because this antigen is the basic ingredient in the preparation of nontreponemal reagents such as the rapid plasma reagin, toluidine red unheated serum test, and the unheated serum reagin, the use of this synthetic VDRL antigen should also increase the reactivity of these reagents.

Gene therapy, the newest and mostly unexplored frontier of medical research, offers the promise of treatment and perhaps cures for many dreadful afflictions of humanity, many of which are inflicted upon the most innocent of victims, infants and children who are powerless to defend themselves from the devastating effects of disease and illness. While the technology exists to determine the affected gene and manufacture a replacement, a suitable delivery vehicle for the genetic material which allows incorporation into the cell and utilization by the cellular machinery to manufacture the gene product has yet to be developed. Currently there are two approaches to this problem, commonly grouped into the classes referred to as viral vectors, modified viral particles capable of infecting cells and delivering genetic material, and non-viral vectors, primarily cationic amphiphilic lipid-like molecules which complex naked genetic material and facilitate its transport across the cell membrane. Viral vectors, while successful at delivering genetic material to cells, presents a number of severe limitations. Many of these limitations can be circumvented by the use of non-viral vectors, although the efficiency of gene delivery is dramatically decreased. This article will concentrate on discussing the factors affecting the handling and use of surfactant and lipid vectors, as well as the preparation of lipid suspensions for the delivery of proteins and genetic material to cells. We will conclude with a brief discussion of an alternative to the classical approaches to protein and gene delivery.