Depletion of sphingolipids facilitates endosome to Golgi transport of ricin

It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.     

Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.     

Self-assembly of HEK cell-secreted ApoE particles resembles ApoE enrichment of lipoproteins as a ligand for the LDL receptor-related protein

Recent studies have shown that the lipidation and assembly state of apolipoprotein E (apoE) determine receptor recognition and amyloid-beta peptide (Abeta) binding. We previously demonstrated that apoE secreted by HEK cells stably expressing apoE3 or apoE4 (HEK-apoE) binds Abeta and inhibits Abeta-induced neurotoxicity by an isoform-specific process that requires apoE receptors. Here we characterized the structure of HEK-apoE assemblies and determined their receptor binding specificity. By chromatography, HEK-apoE elutes in high molecular mass fractions and is the size of plasma HDL, consistent with a multiprotein assembly. No lipid was associated with these apoE assemblies. Several methods for analyzing receptor binding indicate that HEK-apoE is a ligand for low-density lipoprotein (LDL) receptor-related protein (LRP) but not the LDL receptor. This suggests that self-assembly of apoE may induce a functional conformation necessary for binding to LRP. Our results indicate that, in addition to lipid content, the assembly state of apoE influences Abeta binding and receptor recognition.     

Does dietary fat affect inflammatory markers in overweight and obese individuals?—a review of randomized controlled trials from 2010 to 2016

Background

Obesity, a major cause of death and disability, is increasing worldwide. Obesity is characterized by a chronic, low-grade inflammatory state which is suggested to play a critical role in the development of obesity-related diseases like cardiovascular diseases and type 2 diabetes. In fact, in the hours following consumption of a meal, a transient increase in inflammatory markers occurs, a response that is exaggerated in obese subjects. Dietary composition, including content of dietary fatty acids, may affect this inflammatory response both acutely and chronically, and thereby be predictive of progression of disease. The aim of the review was to summarize the literature from 2010 to 2016 regarding the effects of dietary fat intake on levels of inflammatory markers in overweight and obesity in human randomized controlled trials.

Methods and results

We performed a literature search in MEDLINE, EMBASE, and PubMed databases. The literature search included human randomized controlled trials, both postprandial and long-term interventions, from January 2010 to September 2016. In total, 37 articles were included. Interventions with dairy products, vegetable oils, or nuts showed minor effects on inflammatory markers. The most consistent inflammatory-mediating effects were found in intervention with whole diets, which suggests that many components of the diet reduce inflammation synergistically. Furthermore, interventions with weight reduction and different fatty acids did not clearly show beneficial effects on inflammatory markers.

Conclusion

Most interventions showed either no or minor effects of dietary fat intake on inflammatory markers in overweight and obese subjects. To progress our understanding on how diet and dietary components affect our health, mechanistic studies are required. Hence, future studies should include whole diets and characterization of obese phenotypes at a molecular level, including omics data and gut microbiota.

     

HAI‐2 stabilizes,inhibits and regulates SEA‐cleavage‐dependent secretory transport of matriptase

It has recently been shown that hepatocyte growth factor activator inhibitor‐2 (HAI‐2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI‐2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI‐2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non‐SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto‐cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI‐2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI‐2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI‐2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.      

Metabolite profiling of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) plants transformed with an antisense chalcone synthase gene

A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and ¹H NMR are discussed. The glucosinolate composition in rosette leaves was shown to vary naturally within this ecotype. Relatively modest environmental changes had a strong effect on the wild type level of flavonoids and some sinapate esters but much less effect on the glucosinolates. Potentially a reduction in the level of flavonoids could be expected in the transgenic lines relative to the wild type. In practice the reductions achieved were highly variable both between lines and within a given line on different occasions when the plants were grown. Possible reasons for this variability are discussed with reference to current models of gene silencing. The metabolite profiles of the transgenic lines were examined for unintended effects of the modification. An apparently major effect on the glucosinolate composition was shown to result from an unusual genetic variation in the ecotype and not from the modification. The modification did produce a different but much more subtle change in the levels of certain glucosinolates.Funding: EU 5th Framework Programme (Project QLK1-1999-00765, GMOCARE) and the BBSRC (UK) Core Strategic Grant.     

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different CD133 antibody clones possibly recognizing different CD133 splice variants with epitopes of different glycosylation status confuses the field. The aim was to investigate if current inconsistent CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Ten glioblastomas were immunohistochemically stained with four different CD133 antibody clones (AC133, W6B3C1, C24B9, and ab19898) and analyzed by quantitative stereology. Moreover, the CD133 staining pattern of each antibody clone was investigated in kidney, pancreas, and placenta tissue as well as in glioblastoma and retinoblastoma cultures and cell lines. All antibody clones revealed CD133+ niches and single cells in glioblastomas, but when using different clones, their distribution rarely corresponded. Morphology of identified single cells varied, and staining of various tissues, cultures, and cells lines was also inconsistent among the clones. In conclusion, the authors report inconsistent CD133 detection when using different primary CD133 antibody clones. Thus, direct comparison of studies using different antibody clones and conclusions based on CD133 immunohistochemistry should be performed with caution.