Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.     

Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.     

Detection of phages carrying the Shiga toxin 1 and 2 genes in waste water and river water samples

AIMS: To evaluate the occurrence and abundance of phages that carry the stx(1) and stx(2) gene in water samples of different quality. METHODS AND RESULTS: Phages growing on the Shiga toxin-negative Escherichia coli O157:H7 (ATCC 43,888) strain were enumerated by a plaque assay in concentrated raw and treated waste water samples and river water samples. Plaques were investigated for the presence of stx(1) and stx(2) genes by a multiplex/nested PCR procedure. An overall number of 805 plaques were tested for the presence of stx-carrying phages. Stx genes could be demonstrated in 2% (stx(1)) and 16% (stx(2)) of the plaques. Stx-phages were eliminated with approximately the same efficiency in comparison with somatic coliphages during the waste water treatment process. CONCLUSIONS: Due to the low numbers of phages carrying the stx genes 1 and 2 in treated waste water and river water, the dilution and inactivation of host bacteria and the unsuitable conditions for the transduction of host organisms in aquatic environments, it is difficult to derive from the data the direct evidence for a public health problem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show the quantitative occurrence of stx-carrying phages in waste and river water and confirm the frequent circulation of these viruses in the aquatic environment.     

Biomass adaptation over anaerobic co-digestion of sewage sludge and trapped grease waste

The feasibility of sewage sludge co-digestion using intermediate waste generated inside a wastewater treatment plant, i.e. trapped grease waste from the dissolved air flotation unit, has been assessed in a continuous stirred lab reactor operating at 35 °C with a hydraulic retention time of 20 days. Three different periods of co-digestion were carried out as the grease waste dose was increased. When the grease waste addition was 23% of the volatile solids fed (organic loading rate 3.0 kg_COD m⁻³ d⁻¹), an increase in methane yield of 138% was reported. Specific activity tests suggested that anaerobic biomass had adapted to the co-substrate. The adapted inoculum showed higher acetoclastic methanogenic and β-oxidation synthrophic acetogenic activities but lower hydrogenotrophic methanogenic activity. The results indicate that a slow increase in the grease waste dose could be a strategy that favours biomass acclimation to fat-rich co-substrate, increases long chain fatty acid degradation and reduces the latter’s inhibitory effect.     

Sphingolipid homeostasis in the web of metabolic routes

Sphingolipids play a key role in cells as structural components of membrane lipid bilayers and signaling molecules implicated in important physiological and pathological processes. Their metabolism is tightly regulated. Mechanisms controlling sphingolipid metabolism are far from being completely understood. However, they already reveal the integration of sphingolipids in the whole metabolic network as signaling devices that coordinate different metabolic pathways. A picture of sphingolipids integrated into metabolic networks might help to understand sphingolipid homeostasis. This review describes recent advances in the regulation of de novo sphingolipid synthesis with a focus on the bridges that exist with other metabolic pathways and the importance of this crosstalk in the control of sphingolipid homeostasis. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Glyco- and sphingophosphonolipids from the medusa Phyllorhiza punctata: NMR and ESI-MS/MS fingerprints

The medusa Phyllorhiza punctata has been found in Brazilian waters where it is an exotic species, having arrived in ballasts from the Indo-Pacific Ocean in the general region of North Australia and Indonesia. Fatty acids of the intact animal and its component umbrella, oral arms, and mucus were identified. Two different groups of glycolipids and a sphingolipid were isolated by silica-gel column chromatography and characterized using GC-MS, ESI-MS, 1D, 2D (13)C, (1)H and (31)P NMR spectroscopy. They were sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and ceramide aminoethylphosphonate (CAEP). The CAEP long chain base (LCB) and its polar head group (PHG) formed by partial hydrolysis, were analyzed by ESI-MS/MS. The probable origin of MGDG and SQDG in the jellyfish is the result of an endosymbiotic association with a microalga of the Dinoflagellate group, since these lipids are commonly found in photosynthetic membranes.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Dominance of mixed ether/ester,intact polar membrane lipids in five species of the order Rubrobacterales: Another group of bacteria not obeying the “lipid divide”

The composition of the core lipids and intact polar lipids (IPLs) of five Rubrobacter species was examined. Methylated (ω-4) fatty acids (FAs) characterized the core lipids of Rubrobacter radiotolerans, R. xylanophilus and R. bracarensis. In contrast, R. calidifluminis and R. naiadicus lacked ω-4 methyl FAs but instead contained abundant (i.e., 34–41 % of the core lipids) ω-cyclohexyl FAs not reported before in the order Rubrobacterales. Their genomes contained an almost complete operon encoding proteins enabling production of cyclohexane carboxylic acid CoA thioester, which acts as a building block for ω-cyclohexyl FAs in other bacteria. Hence, the most plausible explanation for the biosynthesis of these cyclic FAs in R. calidifluminis and R. naiadicus is a recent acquisition of this operon. All strains contained 1-O-alkyl glycerol ether lipids in abundance (up to 46 % of the core lipids), in line with the dominance (>90 %) of mixed ether/ester IPLs with a variety of polar headgroups. The IPL head group distribution of R. calidifluminis and R. naiadicus differed, e.g. they lacked a novel IPL tentatively assigned as phosphothreoninol. The genomes of all five Rubrobacter species contained a putative operon encoding the synthesis of the 1-O-alkyl glycerol phosphate, the presumed building block of mixed ether/ester IPLs, which shows some resemblance with an operon enabling ether lipid production in various other aerobic bacteria but requires more study. The uncommon dominance of mixed ether/ester IPLs in Rubrobacter species exemplifies our recent growing awareness that the lipid divide between archaea and bacteria/eukaryotes is not as clear cut as previously thought.     

Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimer's disease

Multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) is a well-developed technology for global lipid analysis, which identifies and quantifies individual lipid molecular species directly from lipid extracts of biological samples. By using this technology, we have revealed three marked changes of lipids in brain samples of subjects with mild cognitive impairment of Alzheimer's disease including sulfatides, ceramides, and plasmalogens. Further studies using MDMS-SL lead us to the identification of the potential biochemical mechanisms responsible for the altered lipids at the disease state, which are thoroughly discussed in this minireview. Specifically, in studies to identify the causes responsible for sulfatide depletion at the mild cognitive impairment stage of Alzheimer's disease, we have found that apolipoprotein E is associated with sulfatide transport and mediates sulfatide homeostasis in the nervous system through lipoprotein metabolism pathways and that alterations in apolipoprotein E-mediated sulfatide trafficking can lead to sulfatide depletion in the brain. Collectively, the results obtained from lipidomic analyses of brain samples provide important insights into the biochemical mechanisms underlying the pathogenesis of Alzheimer's disease.     

Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review

The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by a variety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attempts based on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering of microorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops, however, appears to be a realistic goal for the future.     

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [¹⁴C]oleic acid (OA) and [¹⁴C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO₂, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.     

Omega-3 fatty acids and metabolic syndrome: Effects and emerging mechanisms of action

Epidemiological, human, animal, and cell culture studies show that n−3 fatty acids, especially α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), reduce the risk factors of cardiovascular diseases. EPA and DHA, rather than ALA, have been the focus of research on the n−3 fatty acids, probably due to the relatively inefficient conversion of ALA to EPA and DHA in rodents and humans. This review will assess our current understanding of the effects and potential mechanisms of actions of individual n−3 fatty acids on multiple risk factors of metabolic syndrome. Evidence for pharmacological responses and the mechanism of action of each of the n−3 fatty acid trio will be discussed for the major risk factors of metabolic syndrome, especially adiposity, dyslipidemia, insulin resistance and diabetes, hypertension, oxidative stress, and inflammation. Metabolism of n−3 and n−6 fatty acids as well as the interactions of n−3 fatty acids with nutrients, gene expression, and disease states will be addressed to provide a rationale for the use of n−3 fatty acids to reduce the risk factors of metabolic syndrome.