Determination of cis,cis-methylene interrupted polyunsaturated fatty acids in aqueous solutions by lipoxygenase chemiluminescence

The chemiluminescent reaction of luminol during lipoxygenase-catalyzed oxygenations was studied with the purpose of developing a specific luminometric assay for cis,cis-1,4-pentadiene fatty acids directly in aqueous solutions. The addition of picomole levels of either linoleic or arachidonic acids to reaction systems containing 0.04 mM luminol and 40 micrograms/ml of purified soybean lipoxygenase-1 gave light emission curves with a single sharp maximum. Under these conditions the peak heights were linearly dependent on the fatty acid concentration and the detection limit for both of the fatty acids was 2 pmol with a signal to noise ratio of 2. For maximum reproducibility of the assays a procedure for the proper quantitation of the enzyme was developed. The fact that the assay proved to be relatively interference-free was ascribed to the high molar enzyme/substrate ratio (above 1).     

Time course changes to structural,mechanical and material properties of bone in rats after complete spinal cord injury

Objective:Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats.Methods:8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy.Results:The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone.Conclusion:Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.     

Aquatic bryophytes play a key role in sediment-stressed boreal headwater streams

Hydrobiologia - Forestry-related land use can cause increasing instream sedimentation, burying and eradicating stream bryophytes, with severe ecological consequences. However, there is limited...     

Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.     

Adenovirus-mediated gene transfer of Lp-PLA2 reduces LDL degradation and foam cell formation in vitro

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.