Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca²⁺-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.     

VEGF function in vascular pathogenesis

Vascular endothelial growth factor (VEGF) has proven to be the most critical angiogenic factor identified to date; its discovery has transformed the field of angiogenesis research. After two decades of intense studies by numerous laboratories, the complex biology of VEGF is slowly being elucidated. VEGF is a collection of several different isoforms with overlapping but also unique functions. Furthermore, there are at least four different VEGF receptors, adding to the complexity of the VEGF/receptor system. VEGF exerts its effects on endothelial cells, inflammatory cells and neuronal cells, and its expression may contribute to the pathologies of diseases ranging from retinopathies to cancer. This review focuses on the biochemistry and cell biology of VEGF, with particular emphasis on the different isoforms and receptors, thereby illustrating the multifunctional nature of this growth factor. Lastly, critical considerations based on the complex biology of VEGF for developing specific and more targeted anti-VEGF therapeutic agents will be discussed.     

Two new pycnidial members of the Atractiellales: Basidiopycnis hyalina and Proceropycnis pinicola

Two new genera, Basidiopycnis and Proceropycnis, and two new species, Basidiopycnis hyalina and Proceropycnis pinicola, are described. An integrated analysis of morphological, ecological, ultrastructural and molecular data indicates that the new taxa belong to the Atractiellales. Relevant characteristics of the new genera and species are discussed and compared with those of related taxa.     

An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences

In this study we provide a phylogenetically based introduction to the classes and orders of Pucciniomycotina (= Urediniomycetes), one of three subphyla of Basidiomycota. More than 8000 species of Pucciniomycotina have been described including putative saprotrophs and parasites of plants, animals and fungi. The overwhelming majority of these (approximately 90%) belong to a single order of obligate plant pathogens, the Pucciniales (= Uredinales), or rust fungi. We have assembled a dataset of previously published and newly generated sequence data from two nuclear rDNA genes (large subunit and small subunit) including exemplars from all known major groups in order to test hypotheses about evolutionary relationships among the Pucciniomycotina. The utility of combining nuc-lsu sequences spanning the entire D1-D3 region with complete nuc-ssu sequences for resolution and support of nodes is discussed. Our study confirms Pucciniomycotina as a monophyletic group of Basidiomycota. In total our results support eight major clades ranked as classes (Agaricostilbomycetes, Atractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes and Pucciniomycetes) and 18 orders.     

Oncostatin-M up-regulates VCAM-1 and synergizes with IL-4 in eotaxin expression: involvement of STAT6

Oncostatin-M (OSM) is an IL-6/gp130 family member that can stimulate the eosinophil-selective CC chemokine eotaxin-1 in vitro and eosinophil accumulation in mouse lung in vivo. The adhesion molecule VCAM-1 and eotaxin have been implicated in extravasation and accumulation of eosinophils into tissue in animal models of asthma. In this study, we investigated the role of OSM in regulation of VCAM-1 expression, and STAT6 tyrosine 641 phosphorylation in murine fibroblasts. OSM induced VCAM-1 expression in C57BL/6 mouse lung fibroblasts (MLF) and NIH 3T3 fibroblasts at the protein and mRNA level in vitro. OSM also induced STAT6 Y641 phosphorylation in MLF and NIH 3T3 fibroblasts, an activity not observed with other IL-6/gp130 cytokine family members (IL-6, leukemia inhibitory factor, cardiotropin-1, and IL-11) nor in cells derived from STAT6(-/-) mice (STAT6(-/-) MLF). STAT6 was not essential for OSM-induced VCAM-1 or eotaxin-1 as assessed in STAT6(-/-) MLF. Combination of IL-4 and OSM synergistically enhanced eotaxin-1 expression in MLF. IL-4 induction and the IL-4/OSM synergistic induction of eotaxin-1 was abrogated in STAT6(-/-) MLF, however, regulation of IL-6 was similar in -/- or wild-type MLF. Induction of VCAM-1 by OSM was diminished by pharmacological inhibitors of PI3K (LY294002) but not inhibitors of ERK1/2 (PD98059) or p38 MAPK (SB203580). These data support the role of OSM in eosinophil accumulation into lung tissue through eotaxin-1 and VCAM-1 expression and the notion that OSM is able to induce unique signal transduction events through its receptor complex of OSMR beta-chain and gp130.     

Phosphoenolpyruvate carboxylase genes in C3, crassulacean acid metabolism (CAM) and C3/CAM intermediate species of the genus Clusia: rapid reversible C3/CAM switches are based on the C3 housekeeping gene

The genus Clusia includes species that exhibit either the C3 or crassulacean acid metabolism (CAM) mode of photosynthesis, or those that are able to switch between both modes according to water availability. In order to screen for species-specific genetic variability, we investigated the key carboxylase for CAM, phosphoenolpyruvate carboxylase (PEPC). Sequence analysis of DNA isolated from the obligate CAM species, Clusia hilariana, the obligate C3 species, Clusia multiflora, and an intermediate species that can switch between C3 and CAM photosynthesis, Clusia minor, revealed three different isoforms for C. hilariana and one each for the other two species. Sequence alignments indicated that PEPC from the intermediate species had high homology with the C3 protein and with one of CAM plant proteins. These were assumed to constitute 'housekeeping' proteins, which can also support CAM in intermediate species. The other two isoforms of the CAM plant C. hilariana were either CAM-specific or showed homologies with PEPC from roots. Phylogenetic trees derived from neighbour-joining analysis of amino acid sequences from 13 different Clusia species resulted in two distinct groups of plants with either 'housekeeping' PEPC only, or additionally CAM-related isoforms. Only C. hilariana showed the third, probably root-specific isoform. The high homology of the PEPC from the intermediate species with the C3 protein indicates that for the reversible transition from the C3 to CAM mode of photosynthesis, the C3 type of PEPC is sufficient. Its expression, however, is strongly increased under CAM-inducing conditions. The use of the C3 isoform could have facilitated the evolution of CAM within the genus, which occurred independently for several times.     

Free fatty acids normalize a rosiglitazone-induced visfatin release

The detrimental effect of elevated free fatty acids (FFAs) on insulin sensitivity can be improved by thiazolidinediones (TZDs) in patients with type 2 diabetes mellitus. It is unknown whether this salutary action of TZD is associated with altered release of the insulin-mimetic adipocytokine visfatin. In this study, we investigated whether visfatin concentrations are altered by FFA and TZD treatment. In a randomized, double-blind, placebo-controlled, parallel-group study 16 healthy volunteers received an infusion of triglycerides/heparin to increase plasma FFA after 3 wk of treatment with rosiglitazone (8 mg/day, n = 8) or placebo (n = 8), and circulating plasma visfatin was measured. As a corollary, human adipocytes were incubated with synthetic fatty acids and rosiglitazone to assess visfatin release in vitro. The results were that rosiglitazone treatment increased systemic plasma visfatin concentrations from 0.6 +/- 0.1 to 1.7 +/- 0.2 ng/ml (P < 0.01). Lipid infusion caused a marked elevation of plasma FFA but had no effect on circulating visfatin in controls. In contrast, elevated visfatin concentrations in subjects receiving rosiglitazone were normalized by lipid infusion. In isolated adipocytes, visfatin was released into supernatant medium by acute addition and long-term treatment of rosiglitazone. This secretion was blocked by synthetic fatty acids and by inhibition of phosphatidylinositol 3-kinase or Akt. In conclusion, release of the insulin-mimetic visfatin may represent a major mechanism of metabolic TZD action. The presence of FFA antagonizes this action, which may have implications for visfatin bioactivity.     

Matter-tag: A universal immobilization platform for enzymes on polymers,metals, and silicon-based materials

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag–based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag–based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (K_D) of 2.9·10⁻⁸ M and a maximal surface coverage of 504 ng/cm².     

Prediction of Co-Receptor Usage of HIV-1 from Genotype

Human Immunodeficiency Virus 1 uses for entry into host cells a receptor (CD4) and one of two co-receptors (CCR5 or CXCR4). Recently, a new class of antiretroviral drugs has entered clinical practice that specifically bind to the co-receptor CCR5, and thus inhibit virus entry. Accurate prediction of the co-receptor used by the virus in the patient is important as it allows for personalized selection of effective drugs and prognosis of disease progression. We have investigated whether it is possible to predict co-receptor usage accurately by analyzing the amino acid sequence of the main determinant of co-receptor usage, i.e., the third variable loop V3 of the gp120 protein. We developed a two-level machine learning approach that in the first level considers two different properties important for protein-protein binding derived from structural models of V3 and V3 sequences. The second level combines the two predictions of the first level. The two-level method predicts usage of CXCR4 co-receptor for new V3 sequences within seconds, with an area under the ROC curve of 0.937±0.004. Moreover, it is relatively robust against insertions and deletions, which frequently occur in V3. The approach could help clinicians to find optimal personalized treatments, and it offers new insights into the molecular basis of co-receptor usage. For instance, it quantifies the importance for co-receptor usage of a pocket that probably is responsible for binding sulfated tyrosine.