Inhibitory effects of some long-chain unsaturated fatty acids on mitochondrial beta-oxidation. Effects of streptozotocin-induced diabetes on mitochondrial beta-oxidation of polyunsaturated fatty acids.

Evidence showing that some unsaturated fatty acids, and in particular docosahexaenoic acid, can be powerful inhibitors of mitochondrial beta-oxidation is presented. This inhibitory property is, however, also observed with the cis- and trans-isomers of the C18:1(16) acid. Hence it is probably the position of the double bond(s), and not the degree of unsaturation, which confers the inhibitory property. It is suggested that the inhibitory effect is caused by accumulation of 2,4-di- or 2,4,7-tri-enoyl-CoA esters in the mitochondrial matrix. This has previously been shown to occur with these fatty acids, in particular when the supply of NADPH was limiting 2,4-dienoyl-CoA reductase (EC 1.3.1.-) activity [Hiltunen, Osmundsen & Bremer (1983) Biochim. Biophys. Acta 752, 223-232]. Liver mitochondria from streptozotocin-diabetic rats showed an increased ability to beta-oxidize 2,4-dienoyl-CoA-requiring acylcarnitines. Docosahexaenoylcarnitine was also found to be less inhibitory at lower concentrations with incubation under coupled conditions. With uncoupling conditions there was little difference between mitochondria from normal and diabetic rats in these respects. This correlates with a 5-fold stimulation of 2,4-dienoyl-CoA reductase activity found in mitochondria from streptozotocin-diabetic rats.     

Dating the arthropod tree based on large-scale transcriptome data

Molecular sequences do not only allow the reconstruction of phylogenetic relationships among species, but also provide information on the approximate divergence times. Whereas the fossil record dates the origin of most multicellular animal phyla during the Cambrian explosion less than 540 million years ago(mya), molecular clock calculations usually suggest much older dates. Here we used a large multiple sequence alignment derived from Expressed Sequence Tags and genomes comprising 129genes (37,476 amino acid positions) and 117 taxa, including 101 arthropods. We obtained consistent divergence time estimates applying relaxed Bayesian clock models with different priors and multiple calibration points. While the influence of substitution rates, missing data, and model priors were negligible, the clock model had significant effect. A log-normal autocorrelated model was selected on basis of cross-validation. We calculated that arthropods emerged ~600 mya. Onychophorans (velvet worms) and euarthropods split ~590 mya, Pancrustacea and Myriochelata ~560 mya, Myriapoda and Chelicerata ~555 mya, and 'Crustacea' and Hexapoda ~510 mya. Endopterygote insects appeared ~390 mya. These dates are considerably younger than most previous molecular clock estimates and in better agreement with the fossil record. Nevertheless, a Precambrian origin of arthropods and other metazoan phyla is still supported. Our results also demonstrate the applicability of large datasets of random nuclear sequences for approximating the timing of multicellular animal evolution.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

The dynamics of food chains under climate change and nutrient enrichment

Warming has profound effects on biological rates such as metabolism, growth, feeding and death of organisms, eventually affecting their ability to survive. Using a nonlinear bioenergetic population-dynamic model that accounts for temperature and body-mass dependencies of biological rates, we analysed the individual and interactive effects of increasing temperature and nutrient enrichment on the dynamics of a three-species food chain. At low temperatures, warming counteracts the destabilizing effects of enrichment by both bottom-up (via the carrying capacity) and top-down (via biological rates) mechanisms. Together with increasing consumer body masses, warming increases the system tolerance to fertilization. Simultaneously, warming increases the risk of starvation for large species in low-fertility systems. This effect can be counteracted by increased fertilization. In combination, therefore, two main drivers of global change and biodiversity loss can have positive and negative effects on food chain stability. Our model incorporates the most recent empirical data and may thus be used as the basis for more complex forecasting models incorporating food-web structure.     

Methane emission,digestive characteristics and faecal archaeol in heifers fed diets based on silage from brown midrib maize as compared to conventional maize

The aim of the present experiment was to compare silage prepared from maize having a brown midrib (BMR) mutation with control (CTR) maize to identify their effects on enteric methane emission, digesta mean retention time (MRT), ruminal fermentation and digestibility. In addition, the utility of archaeol present in faecal samples was validated as a proxy for methane production. Seven German Holstein heifers were fed total mixed rations with a maize-silage proportion (either BMR or CTR) of 920 g/kg dry matter (DM) in a change-over design. Heifers were fed boluses with markers to measure MRT; faeces were collected for 7 days and rumen fluid was collected on the penultimate day. Methane emission was measured in respiration chambers on one day. Data were analysed by t-test and regression analysis. DM intake did not differ between the two diets. The apparent digestibility of DM and most nutrients was unaffected by diet type, but apparent digestibility of neutral and acid detergent-fibre was higher in those heifers fed BMR than in those fed CTR. Comparisons between diets revealed no difference in particle or solute MRT in the gastro-intestinal tract and the reticulorumen. Concentrations of short-chain fatty acid and ammonia in rumen fluid and its pH were not affected by silage type. Independent of the mode of expression [l/d, l/kg DM intake, l/kg digested organic matter], methane emissions were not affected by maize-silage type, but with BMR, there was a trend towards lower methane production per unit of digested neutral detergent fibre than there was with CTR silage. Results of the present study show that feeding heifers BMR silage does not increase methane emissions despite a higher fibre digestibility as compared to CTR silage. Therefore, it is assumed that improvements in animal productivity achieved by feeding BMR silage, as some studies have reported, can be obtained without extra environmental cost per unit of milk or meat. Neither faecal archaeol content [µg/g] nor daily amount excreted [mg/d] is suitable to predict methane production in absolute terms [l per day]. However, faecal archaeol content has a certain potential for predicting the methane yield [l per kg DM intake] of individual animals.     

Do ice nucleating lipoproteins protect frozen insects against toxic chemical agents?

As the body fluid of freeze-tolerant organisms freezes, solutes become concentrated in the gradually smaller unfrozen fluid fraction, and dissolved trace metals may reach toxic levels. A dialysis technique was used to investigate the metal binding capacity of the low density fraction of the hemolymph from the freeze tolerant beetle Phyto depressus. The low density fraction, assumed to contain the ice nucleating lipoproteins, showed approximately 100 times greater capacity to bind metals (Cd ²⁺, Cu ²⁺ and Zn ²⁺) than the proteins albumin, hemoglobin and similar to metallothionein. The high metal binding capacity in the low density fraction raises the question if the ice nucleating lipoproteins might assist in detoxification of potentially toxic concentrations of metals that may occur when a large fraction of the bodyfluids of freeze tolerant insects freeze. This hypotheis is consistent with the fact that the lipoprotein ice nucleators are present in far greater amounts than required for ice nucleation, and also with the fact that the lipoprotein ice nucleators have a remarkably high content of amino acids with negatively charged residues that may act as metal binding sites.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.