Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Fish or n3-PUFA intake and body composition: a systematic review and meta-analysis.

Obesity is a major public health issue and an important contributor to the global burden of chronic disease and disability. Studies indicate that fish and omega-3 polyunsaturated fatty acids (n3-PUFA) supplements may help prevent cardiovascular and metabolic diseases. However, the effect of fish oil on body composition is still uncertain, so we performed a systematic review of randomized controlled trials and the first meta-analysis on the association between fish or fish oil intake and body composition measures. We found evidence that participants taking fish or fish oil lost 0.59 kg more body weight than controls (95% confidence interval [CI]: -0.96 to -0.21). Treatment groups lost 0.24 kg m(-2) (body mass index) more than controls (-0.40 to -0.08), and 0.49 % more body fat than controls (-0.97 to -0.01). Fish or fish oil reduced waist circumference by 0.81 cm (-1.34 to -0.28) compared with control. There was no difference for fat mass and lean body mass. Further research is needed to confirm or refute our findings and to reveal possible mechanisms by which n3-PUFAs might reduce weight.

Antioxidant supplementation attenuates oxidative stress in patients undergoing coronary artery bypass graft surgery.

Ischemia-reperfusion has been reported to be associated with augmented oxidative stress in the course of surgery, which might be causally involved in the onset of atrial fibrillation (AF), the most common arrhythmia after cardiac surgery. We hypothesized that supplementation of antioxidants and n-3 polyunsaturated fatty acids (n-3 PUFAs) might lower the incidence of AF following coronary artery bypass graft (CABG) surgery. In the present study, by monitoring oxidative stress in the course of CABG surgery, we analyzed the efficacy of vitamins (ascorbic acid and α-tocopherol) and/or n-3 PUFAs (eicosapentaenoic acid and docosahexaenoic acid). Subjects (n = 75) were divided into 4 subgroups: control, vitamins, n-3 PUFAs, and a combination of vitamins and n-3 PUFAs. Fluorescent techniques were used to measure the antioxidative capacity, i.e. ability to inhibit oxidation. Total peroxides, endogenous peroxidase activity, and antibodies against oxidized LDL (oLAb) were used as serum oxidative stress biomarkers. Post-operative increase in oxidative stress was associated with the consumption of antioxidants and a simultaneous onset of AF. This was confirmed through an increased peroxide level and a decreased oLAb titer in control and n-3 PUFAs groups, indicating the binding of antibodies to oxidative modified epitopes. In both subgroups that were supplemented with vitamins, total peroxides decreased, and the maintenance of a constant IgG antibody titer was facilitated. However, treatment with vitamins or n-3 PUFAs was inefficient with respect to AF onset and its duration. We conclude that the administration of vitamins attenuates post-operative oxidative stress in the course of CABG surgery.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight {HSPs} families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the {HSP60} family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.

Lipids and nitrogen isotopes of deep-water corals

The lipid and organic nitrogen isotopic (delta15N) compositions of two common deep-water corals (Lophelia pertusa and Madrepora oculata) collected from selected locations of the NE Atlantic are compared to the composition of suspended particulate organic matter, in order to determine their principle food source. Initial results suggest that they may feed primarily on zooplankton. This is based on the increased abundances of mono-unsaturated fatty acids and alcohols and the different ratios of the polyunsaturated fatty acids, 22:6/20:5 of the corals when compared to those of the suspended particulate organic matter. There is enrichment in L. pertusa of mono-unsaturated fatty acids and of delta15N relative to M. oculata. It is unclear whether this reflects different feeding strategies or assimilation/storage efficiencies of zooplankton tissue or different metabolism in the two coral species.

Element contents, sulfur and oxygen isotopes, and molecular biomarkers of phosphatic crust samples from the Chimbote Platform of the shelf off Peru

Authigenic phosphatic laminites enclosed in phosphorite crusts from the shelf off Peru (10°01' S and 10°24' S) consist of carbonate fluorapatite layers, which contain abundant sulfide minerals including pyrite (FeS2) and sphalerite (ZnS). Low d34Spyrite values (average -28.8 per mill) agree with bacterial sulfate reduction and subsequent pyrite formation. Stable sulfur isotopic compositions of sulfate bound in carbonate fluorapatite are lower than that of sulfate from ambient sea water, suggesting bacterial reoxidation of sulfide by sulfide-oxidizing bacteria. The release of phosphorus and subsequent formation of the autochthonous phosphatic laminites are apparently caused by the activity of sulfate-reducing bacteria and associated sulfide-oxidizing bacteria. Following an extraction-phosphorite dissolution-extraction procedure, molecular fossils of sulfate-reducing bacteria (mono-O-alkyl glycerol ethers, di-O-alkyl glycerol ethers, as well as the short-chain branched fatty acids i/ai-C15:0, i/ai-C17:0 and 10MeC16:0) are found to be among the most abundant compounds. The fact that these molecular fossils of sulfate-reducing bacteria are distinctly more abundant after dissolution of the phosphatic laminite reveals that the lipids are tightly bound to the mineral lattice of carbonate fluorapatite. Moreover, compared with the autochthonous laminite, molecular fossils of sulfate-reducing bacteria are: (1) significantly less abundant and (2) not as tightly bound to the mineral lattice in the other, allochthonous facies of the Peruvian crusts consisting of phosphatic coated grains. These observations confirm the importance of sulfate-reducing bacteria in the formation of the phosphatic laminite. Model calculations highlight that organic matter degradation by sulfate-reducing bacteria has the potential to liberate sufficient phosphorus for phosphogenesis.

Compilation of scientific results of the OASIS project

Seamounts are of great interest to science, industry and conservation because of their potential role as 'stirring rods' of the oceans, their enhanced productivity, their high local biodiversity, and the growing exploitation of their natural resources. This is accompanied by rising concern about the threats to seamount ecosystems, e.g. through over-fishing and the impact of trawling. OASIS described the functioning characteristics of seamount ecosystems. OASIS' integrated hydrographic, biogeochemical and biological information. Based on two case studies. The scientific results, condensed in conceptual and mass balanced ecosystem models, were applied to outline a model management plan as well as site-specific management plans for the seamounts investigated. OASIS addressed five main objectives: Objective 1: To identify and describe the physical forcing mechanisms effecting seamount systems Objective 2: To assess the origin, quality and dynamics of particulate organic material within the water column and surface sediment at seamounts. Objective 3: To describe aspects of the biodiversity and the ecology of seamount biota, to assess their dynamics and the maintenance of their production. Objective 4: Modelling the trophic ecology of seamount ecosystems. Objective 5: Application of scientific knowledge to practical conservation.

Biogeochemistry of the Darwin Mounds, NE Atlantic

The Darwin Mounds are a series of small (<=5 m high, 75-100 m diameter) sandy features located in the northern Rockall Trough. They provide a habitat for communities of Lophelia pertusa and associated fauna. Suspended particulate organic matter (sPOM) reaching the deep-sea floor, which could potentially fuel this deep-water coral (DWC) ecosystem, was collected during summer 2000. This was relatively "fresh" (i.e. dominated by labile lipids such as polyunsaturated fatty acids) and was derived largely from phytoplankton remains and faecal pellets, with contributions from bacteria and microzooplankton. Labile sPOM components were enriched in the benthic boundary layer (~10 m above bottom (mab)) relative to 150 mab. The action of certain benthic fauna that are exclusively associated with the DWC ecosystem (e.g. echiuran worms) leads to the subduction of fresh organic material into the sediments. The mound surface sediments are enriched in organic carbon, relative to off-mound sites. There is no evidence for hydrocarbon venting at this location.

Particle analysis in the Kara Sea

In September 1999 two short-term moorings with cylindrical sediment traps were deployed to collect sinking particles in bottom waters off the Ob and Yenisei river mouths. Samples were studied for their bulk composition, pigments, phytoplankton, microzooplankton, fecal material, amino acids, hexosamines, fatty acids and sterols and compared to suspended matter and surface sediments in order to collect information about the nature and cycling of particulate matter in the water column. Results of all measured components in sinking particles point to an ongoing seasonality in the pelagic system from blooming diatoms in the first phase to a more retention system in the second half of trap deployment. Due to a phytoplankton bloom observed north of the Ob estuary, flux rates were generally higher in the trap deployed off the Ob than off the Yenisei. The Ob trap collected fresh surface-derived particulate matter. Particles from the Yenisei trap were more degraded and resembled deep water suspension. This material may partly have been derived from resuspended sediments.