A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-β glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.     

Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Characterization of type II thioesterases involved in natamycin biosynthesis in Streptomyces chattanoogensis L10

The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.     

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C₃₀ carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a “dehydrosqualene synthase.” All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.     

Substrate recognition of holocytochrome c synthase: N-terminal region and CXXCH motif of mitochondrial cytochrome c

Holocytochrome c synthase (HCCS) attaches heme covalently to mitochondrial respiratory cytochromes c. Little is known about the reaction of heme attachment to apocytochromes c by HCCS, although recently it has been established that the CXXCH motif and the N-terminus of the apocytochrome polypeptide are important protein–protein recognition motifs. Here, we explore further the important features of the N-terminal sequence and investigate what variations in the CXXCH residues are productively recognised by HCCS in its substrate.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

Arginase inhibition restores endothelial function in diet-induced obesity

Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor N^ω-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40 mg kg^-1/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction.     

Accumulation of nano-sized particles in a murine model of angiogenesis

Purpose

To evaluate the ability of nm-scaled iron oxide particles conjugated with Azure A, a classic histological dye, to accumulate in areas of angiogenesis in a recently developed murine angiogenesis model.

Materials and methods

We characterised the Azure A particles with regard to their hydrodynamic size, zeta potential, and blood circulation half-life. The particles were then investigated by Magnetic Resonance Imaging (MRI) in a recently developed murine angiogenesis model along with reference particles (Ferumoxtran-10) and saline injections.

Results

The Azure A particles had a mean hydrodynamic diameter of 51.8 ± 43.2 nm, a zeta potential of −17.2 ± 2.8 mV, and a blood circulation half-life of 127.8 ± 74.7 min. Comparison of MR images taken pre- and 24-h post-injection revealed a significant increase in R₂^* relaxation rates for both Azure A and Ferumoxtran-10 particles. No significant difference was found for the saline injections. The relative increase was calculated for the three groups, and showed a significant difference between the saline group and the Azure A group, and between the saline group and the Ferumoxtran-10 group. However, no significant difference was found between the two particle groups.

Conclusion

Ultrahigh-field MRI revealed localisation of both types of iron oxide particles to areas of neovasculature. However, the Azure A particles did not show any enhanced accumulation relative to Ferumoxtran-10, suggesting the accumulation in both cases to be passive.