Transformation of phenylarsenic chemical warfare agents and their effect on bacterial communities in Baltic Sea sediment.

During the World Wars large quantities of phenylarsenic chemical warfare agents (CWAs) were dumped in the Baltic Sea. Many transformation products of these chemicals have been identified, but the pathways that produce the found chemicals has not been investigated. Here we studied the biotic and abiotic transformation of phenylarsenic CWAs under oxic and anoxic conditions and investigated how the sediment bacterial communities are affected by CWA exposure. By chemical analysis we were able to identify seventeen CWA-related phenylarsenicals, four of which (methylphenylarsinic acid (MPAA), phenylthioarsinic acid (PTAA), phenyldithioarsinic acid (PDTAA) and diphenyldithioarsinic acid (DPDTAA)) have not been reported for marine sediments before. For the first time PTAA was verified from environmental samples. We also observed equilibrium reactions between the found transformation products, which may explain the occurrence of the chemicals. 16S rRNA-analysis showed that bacterial communities in sediments are affected by exposure to phenylarsenic CWAs. We observed increases in the amounts of arsenic-resistant and sulphur-metabolising bacteria. Different transformation products were found in biotic and abiotic samples, which suggests that bacteria participate in the transformation of phenylarsenic CWAs. We propose that methylated phenylarsenicals are produced in microbial metabolism and that chemical reactions with microbially produced sulphur species form sulphur-containing transformation products.

Osmolal and anion gaps after acute self-poisoning with agricultural formulations of the organophosphorus insecticides profenofos and diazinon: A pilot study.

Self-poisoning with organophosphorus (OP) insecticides is an important means of global self-harm. The insecticides are formulated with solvents that may also contribute to toxicity. We set up a study to detect changes in osmolal and anion gaps following ingestion of OP insecticides. We recruited consecutive patients admitted to a Teaching Hospital, Sri Lanka, with a history of OP self-poisoning. The osmolal and anion gaps were calculated on admission and at 4, 24 and 72 h post-ingestion together with ethanol concentration. Forty-nine patients were recruited (28 profenofos, 10 diazinon, one coumaphos, one chlorpyrifos, one phenthoate and eight unknown OP). Only modest increases in osmolal and anion gaps were noted. Small rises in osmolal gap above the upper limit of normal were noted in 16/49 (32.7%) of all cases, 9/28 (32.1%) profenofos cases and 4/10 (40.0%) diazinon cases. The anion gap was raised in 24/49 (49.0%) of all cases, 15/28 (53.6%) profenofos cases and 5/10 (50.0%) diazinon cases. We observed a trend for a fall in osmolal gap during the first 24 h, followed by an increase up to 72 h. There was no correlation between the anion gap and serum lactate concentration, indicating that a lactic acidosis was not responsible for the anion gap. Formate, which could have explained the increased gap, was not detected in any of the samples; ketoacids (beta-hydroxybutyrate and acetoacetate) were not measured. This pilot study found that profenofos and diazinon poisoning caused only modest increases in the osmolal and anion gaps in a minority of cases.

Indirect Lipid Transfer Protein Activity Measurements Using Quantification of Glycosphingolipid Production.

Here we summarize how glycosphingolipid production can be followed using metabolic labeling with radiolabeled lipid precursors. No assays are available yet that directly would address the lipid transfer protein activity in vivo. Therefore, these approaches can serve as tools to indirectly study the lipid transfer protein activity in cells, by monitoring their impact on the glycosphingolipid homeostasis.

Osteoporosis and skeletal dysplasia caused by pathogenic variants in SGMS2.

Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane-resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.Ile62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.Ile62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasia. Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.Ile62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane-bound sphingomyelin metabolism in skeletal homeostasis.

Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

Purification and Validation of Lipid Transfer Proteins.

Understanding the holistic picture of lipid homeostasis not only involves the analysis of synthesis and breakdown of lipids but also requires a thorough understanding of their transport. The transport of lipid monomers in an aqueous environment is facilitated by different lipid transfer proteins. Their universal feature is the shielding or encapsulation of the hydrophobic part of the lipid, consequently overcoming the poor solubility of lipids in water. Here we describe a method to purify lipid transfer proteins using bacterial expression. We also present three methods to validate their transfer activity.

Metabolic Conversion of Ceramides in HeLa Cells - A Cholesteryl Phosphocholine Delivery Approach.

Ceramides can be delivered to cultured cells without solvents in the form of complexes with cholesteryl phosphocholine. We have analysed the delivery of three different radiolabeled D-erythro-ceramides (C6-Cer, C10-Cer and C16-Cer) to HeLa cells, and followed their metabolism as well as the cell viability. We found that all three ceramides were successfully taken up by HeLa cells when complexed to CholPC in an equimolar ratio, and show that the ceramides show different rates of cellular uptake and metabolic fate. The C6-Cer had the highest incorporation rate, followed by C10-Cer and C16-Cer, respectively. The subsequent effect on cell viability strongly correlated with the rate of incorporation, where C6-Cer had the strongest apoptotic effects. Low-dose (1 µM) treatment with C6-Cer favoured conversion of the precursor to sphingomyelin, whereas higher concentrations (25-100 µM) yielded increased conversion to C6-glucosylceramide. Similar results were obtained for C10-Cer. In the lower-dose C16-Cer experiments, most of the precursor was degraded, whereas at high-dose concentrations the precursor remained un-metabolized. Using this method, we demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake. The cellular fate of the externally delivered ceramides are clearly connected to their rate of incorporation and their subsequent effects on cell viability may be in part determined by their chain length.

Alternation in the glycolipid transfer protein expression causes changes in the cellular lipidome.

The glycolipid transfer protein (GLTP) catalyzes the binding and transport of glycolipids, but not phospholipids or neutral lipids. With its all-alpha helical fold, it is the founding member for a new superfamily, however its biological role still remains unclear. We have analyzed changes in the HeLa cell lipidome in response to down- and up-regulation of GLTP expression. We used metabolic labeling and thin layer chromatography analysis, complemented with a lipidomics mass spectroscopic approach. HeLa cells were treated with GLTP siRNA or were transiently overexpressing the GLTP gene. We identified eight different lipid classes that changed as a result of the GLTP down- or up-regulation treatments; glucosylceramide, lactosylceramide, globotriaosylceramide, ceramide, sphingomyelin, cholesterol-esters, diacylglycerol and phosphatidylserine. We discovered that the amount of globotriaosylceramide (Gb3) was extensively lowered after down-regulation of GLTP. Further, an up-regulation of GLTP caused a substantial increase in both the Gb3 and glucosylceramide levels compared to the controls. Total galactosylceramide levels remained unchanged. Both lactosylceramide and ceramide showed small changes, an increase with increasing GLTP and a decrease in the HeLa cell GLTP knockdowns. The cholesterol-esters and diacylglycerol masses increased in cells that had upregulated GLTP protein levels, wheras down-regulation did not affect their amounts. For the glycerophospholipids, phosphatidylserine was the only species that was lower in GLTP overexpressing cells. Phosphatidylethanolamine, phosphatidylglyerol and phosphatidylinositol remained unaltered. A total of 142 lipid species were profiled and quantified using shotgun lipidomics analyses. This work provides for the first time insights into how alternations in the levels of a protein that binds and transfers glycolipids affects the cellular lipid metabolism. We discuss the observed changes in the lipidome and how these relate to GLTP. We suggest, that GLTP not only could be a significant player in cellular sphingolipid metabolism, but also could have a much broader role in the overall lipid metabolism.

Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

Correction: Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis.

[This corrects the article DOI: 10.1371/journal.pone.0070283.].

The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment.

The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity. To examine this we have used an anthrylvinyl-labeled ceramide analogue. We found that if ceramide is in a tightly packed environment such as in sphingomyelin or dipalmitoylphosphatidylcholine containing membranes, the CERT transfer activity is markedly reduced. Ceramide in fluid membranes on the other hand are available for CERT mediated transfer. CERT also favors membranes that contain phosphatidylinositol 4-monophospate, due to its binding capacity of the pleckstrin homology domain towards phosphatidylinositol 4-monophospate. From this study we conclude that the membrane matrix surrounding ceramide, that is ceramide miscibility, is largely affecting the transfer activity of CERT.

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles.

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC