Glycosphingolipids and drug resistance

Drug resistance, an all too frequent characteristic of cancer, represents a serious barrier to successful treatment. Although many resistance mechanisms have been described, those that involve membrane-resident proteins belonging to the ABC (ATP binding cassette) transporter superfamily are of particular interest. In addition to cancer, the ABC transporter proteins are active in diseases such as malaria and leishmaniasis. A recent renaissance in lipid metabolism, specifically ceramide and sphingolipids, has fueled research and provided insight into the role of glycosphingolipids in multidrug resistance. This article reviews current knowledge on ceramide, glucosylceramide synthase and cerebrosides, and the relationship of these lipids to cellular response to anticancer agents.     

Glycosphingolipids and insulin resistance

Obesity is associated with an increased risk for insulin resistance, a state characterized by impaired responsiveness of liver, muscle and adipose tissue to insulin. One class of lipids involved in the development of insulin resistance are the (glyco)sphingolipids. Ceramide, the most simple sphingolipid, directly inhibits phosphorylation of the insulin signaling mediator Akt/Protein Kinase B. More complex glycosphingolipids, so-called gangliosides, block phosphorylation of the insulin receptor and down-stream signaling, possibly by exclusion of the insulin receptor from specific membrane domains. Pharmacological inhibition of glycosphingolipid synthesis is found to markedly improve insulin sensitivity in rodent models of insulin resistance. Partial glycosphingolipid reduction is well tolerated and may thus offer an attractive new treatment modality for obesity-induced insulin resistance and type II diabetes.     

Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases.     

Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

Metronidazole and drug resistance

In recent years, the basis of metronidozole resistance has been examined in anaerobic protozoa, such as Trichomonas and Giardia, as well as anaerobic bacteria, such as Bacteroides and Clostridium. In this review, Patricia Johnson looks at a variety of mechanisms that lead to reduced susceptibility of these pathogens to the drug. The frequent correlation between metronidozole resistance and inefficient drug activation suggests that this is the level at which drug resistance operates.     

Ethics and drug resistance

This paper reviews the dynamics behind, and ethical issues associated with, the phenomenon of drug resistance. Drug resistance is an important ethical issue partly because of the severe consequences likely to result from the increase in drug resistant pathogens if more is not done to control them. Drug resistance is also an ethical issue because, rather than being a mere quirk of nature, the problem is largely a product of drug distribution. Drug resistance results from the over-consumption of antibiotics by the wealthy; and it, ironically, results from the under-consumption of antibiotics, usually by the poor or otherwise marginalized. In both kinds of cases the phenomenon of drug resistance illustrates why health (care)--at least in the context of infectious disease--should be treated as a (global) public good. The point is that drug resistance involves 'externalities' affecting third parties. When one patient develops a resistant strain of disease because of her over- or under-consumption of medication, this more dangerous malady poses increased risk to others. The propriety of free-market distribution of goods subject to externalities is famously dubious--given that the 'efficiency' rationale behind markets assumes an absence of externalities. Market failure in the context of drug resistance is partly revealed by the fact that no new classes of antibiotics have been developed since 1970. I conclude by arguing that the case of drug resistance reveals additional reasons--to those traditionally appealed to by bioethicists--for treating health care as something special when making policy decisions about its distribution.     

Glycosphingolipids as toxin receptors

A number of proteins produced by certain bacteria and plants are potently toxic to mammalian cells. This toxicity results from their ability to catalytically modify macromolecules that are required for essential cellular functions such as vesicular trafficking, cytoskeletal assembly, signalling or protein synthesis. To reach their targets, these proteins bind specific surface receptors before endocytosis and translocation across an internal membrane. The surface receptors exploited by different toxins include a range of proteins and lipids. Here we focus on specific glycosphingolipid receptors and two well-characterised subsets of toxins that exploit them for surface binding, intracellular trafficking, and signalling events.     

Glycosphingolipids govern gene expression

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.     

Glycosphingolipids of porcine pancreas

Neutral and acidic glycosphingolipids were purified from porcine pancreas by chromatography on columns of DEAE-Sephadex and Iatrobeads. The chemical structures of the purified glycolipids were determined by carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, NMR and IR. The major glycolipid of porcine pancreas was Gal(alpha,1-4)Gal(beta,1-)ceramide. Gangliosides GM3 and GD3 were major acidic components and galactosylceramide 3-sulfate was also found.     

Glycosphingolipids govern gene expression

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of β1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGFα receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Galectin-3 Interactions with Glycosphingolipids

Galectins have essential roles in pathological states including cancer, inflammation, angiogenesis and microbial infections. Endogenous receptors include members of the lacto- and neolacto-series glycosphingolipids present on mammalian cells and contain the tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) that form their core structural components and also ganglio-series glycosphingolipids. We present crystallographic structures of the carbohydrate recognition domain of human galectin-3, both wild type and a mutant (K176L) that influenced ligand affinity, in complex with LNT, LNnT and acetamido ganglioside a-G_M3 (α2,3-sialyllactose). Key structural features revealed include galectin-3's demonstration of a binding mode towards gangliosides distinct from that to the lacto/neolacto-glycosphingolipids, with its capacity for recognising the core β-galactoside region being challenged when the core oligosaccharide epitope of ganglio-series glycosphingolipids (G_M3) is embedded within particular higher-molecular-weight glycans. The lacto- and neolacto- glycosphingolipids revealed different orientations of their terminal galactose in the galectin-3-bound LNT and LNnT structures that has significant ramifications for the capacity of galectin-3 to interact with higher-order lacto/neolacto-series glycosphingolipids such as ABH blood group antigens and the HNK-1 antigen that is common on leukocytes. LNnT also presents an important model for poly-N-acetyllactosamine-containing glycans and provides insight into galectin-3's accommodation of extended oligosaccharides such as the poly-N-acetyllactosamine-modified N- and O-glycans that, via galectin-3 interaction, facilitate progression of lung and bladder cancers, respectively. These findings provide the first atomic detail of galectin-3's interactions with the core structures of mammalian glycosphingolipids, providing information important in understanding the capacity of galectin-3 to engage with receptors identified as facilitators of major disease.     

念珠菌阴道炎及其耐药性分析

目的探索女性念珠菌阴道炎发病情况及耐药性。方法白带常规采用直接盐水涂片法;阴道分泌物培养按细菌培养常规法进行培养。结果白带常规检查中念珠菌阳性率为12．9％,滴虫阳性率为2．0％,线索细胞阳性率为10．3％,清洁度（Ⅲ～Ⅳ）为13．0％。阴道分泌物培养情况如下：念珠菌阳性率为26％,细菌感染阳性率为45％．今珠菌培养阳性的标本中：白色念珠菌占90％,光滑念珠菌占7．8％,热带念珠菌占2．2％。念珠菌药敏结果分析：制霉素敏感度大于80％,是治疗念珠菌阴道炎最理想的药物。结论念珠菌阴道炎处于逐年上升的趋势：医生应根据药敏结果合理选择抗真菌药物,以免抗生素的滥用。