Relationship between a Common Variant in the Fatty Acid Desaturase (FADS) Cluster and Eicosanoid Generation in Humans

Dramatic shifts in the Western diet have led to a marked increase in the dietary intake of the n-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA). Dietary LA can then be converted to arachidonic acid (ARA) utilizing three enzymatic steps. Two of these steps are encoded for by the fatty acid desaturase (FADS) cluster (chromosome 11, 11q12.2-q13) and certain genetic variants within the cluster are highly associated with ARA levels. However, no study to date has examined whether these variants further influence pro-inflammatory, cyclooxygenase and lipoxygenase eicosanoid products. This study examined the impact of a highly influential FADS SNP, rs174537 on leukotriene, HETE, prostaglandin, and thromboxane biosynthesis in stimulated whole blood. Thirty subjects were genotyped at rs174537 (GG, n = 11; GT, n = 13; TT, n = 6), a panel of fatty acids from whole serum was analyzed, and precursor-to-product PUFA ratios were calculated as a marker of the capacity of tissues (particularly the liver) to synthesize long chain PUFAs. Eicosanoids produced by stimulated human blood were measured by LC-MS/MS. We observed an association between rs174537 and the ratio of ARA/LA, leukotriene B₄, and 5-HETE but no effect on levels of cyclooxygenase products. Our results suggest that variation at rs174537 not only impacts the synthesis of ARA but the overall capacity of whole blood to synthesize 5-lipoxygenase products; these genotype-related changes in eicosanoid levels could have important implications in a variety of inflammatory diseases.     

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.     

Moose body mass variation revisited: disentangling effects of environmental conditions and genetics

Large-scale geographical variation in phenotypic traits within species is often correlated to local environmental conditions and population density. Such phenotypic variation has recently been shown to also be influenced by genetic structuring of populations. In ungulates, large-scale geographical variation in phenotypic traits, such as body mass, has been related to environmental conditions and population density, but little is known about the genetic influences. Research on the genetic structure of moose suggests two distinct genetic lineages in Norway, structured along a north-south gradient. This corresponds with many environmental gradients, thus genetic structuring provides an additional factor affecting geographical phenotypic variation in Norwegian moose. We investigated if genetic structure explained geographical variation in body mass in Norwegian moose while accounting for environmental conditions, age and sex, and if it captured some of the variance in body mass that previously was attributed to environmental factors. Genetic structuring of moose was the most important variable in explaining the geographic variation in body mass within age and sex classes. Several environmental variables also had strong explanatory power, related to habitat diversity, environmental seasonality and winter harshness. The results suggest that environmental conditions, landscape characteristics, and genetic structure should be evaluated together when explaining large-scale patterns in phenotypic characters or life history traits. However, to better understand the role of genetic and environmental effects on phenotypic traits in moose, an extended individual-based study of variation in fitness-related characters is needed, preferably in an area of convergence between different genetic lineages.     

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

Consensus Brain-derived Protein,Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.     

Temperature influence on Arctic charr (Salvelinus alpinus L.) antibody response to a cellular antigen

In order to elucidate the immune responses of Arctic charr in relation to temperature, groups were acclimated to a moderate (9°C) and a cold temperature regime (4°C), as well as subjected to a temperature decrease (from 9 to 4°C) immediately prior to an immunization with sheep red blood cells. The charr kept at 9°C responded with increased primary and secondary antibody titres, as seen by direct haemagglutination, while fish at 4°C, as well as the fish subjected to a temperature reduction, displayed lower and lowest antibody titres, respectively, and only after a second immunization. It is concluded that Arctic charr can respond to a cellular antigen with a humoral immune response typical for other teleosts, but that the immune response is delayed and diminished at low temperatures. This temperature-induced immune suppression is intensified if the fish have not been acclimated to cold water prior to immunization. Accepted: 10 October 1999     

Plant responses and herbivory following simulated browsing and stem cutting of Combretum apiculatum

Abstract. Time and mode of herbivory on savanna trees and their subsequent responses are dependent on, among other things, earlier herbivory and fire. We used clipping (simulated browsing) and stem cutting (simulated heavy browsing and to some extent simulated fire) to evaluate such interactions. Study organisms were a deciduous, broad‐leaved tree species, Combretum apiculatum (Combretaceae), browsing large herbivores and leaf‐eating insects. The treatments were done in the late dry season before bud break. Late in the following wet season, we recorded plant responses to treatment and browsing. The treated trees, especially the cut ones, responded by producing larger and fewer annual shoots. Compared to control trees, there was a slight increase in shoot biomass of clipped trees and a strong reduction of cut ones. Leaf area increased in clipped trees, but decreased in cut ones. A marked increase in the number of browsed trees was recorded amongst treated trees. Number of bites, consumption and utilization also increased with severity of treatment. In contrast, insect herbivory was reduced on both clipped and cut trees. The observed patterns are discussed in relation to current ideas on plant ‐ herbivore interactions.     

Iron and copper requirements for proliferation and differentiation of a human promyelocytic leukemia cell line (HL-60)

Trace mineral deficiencies tend to have profound effects on the integrity of formed blood elements. Anemia and neutropenia are commonly seen in copper (Cu) deficiency. We therefore developed a serum-free medium to examine the trace mineral requirements, in particular iron and Cu, for proliferation and retinoic acid (RA)-induced differentiation of HL-60 cells. This defined medium (DFM) was composed of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with insulin and human apo-transferrin (each at 5 μg/ml) and 1.4 μM FeSO₄. The iron concentration range for optimal cellular proliferation was narrow (2–3 μM). HL-60 cells could be maintained in DFM for 15 passages with a doubling time of 38–40 hr. The Cu content of IMDM was very low. Thus, by the fourth passage in DFM, the activity of cuproenzymes (cytochrome c oxidase, CCO; and copperzinc superoxide dismutase, CuZnSOD) began to decline. Supplementation of DFM with CuSO₄ (50 nM) restored enzyme activities. Treatment of cells with a Cu chelator (tetrathiomolybdate, 1 μM) rapidly reduced the activities of both CCO and CuZnSOD. Over the Cu concentration range examined (5–350 nM), Cu supplementation had little effect on HL-60 proliferation. Cell retained the ability to differentiate along the granulocytic pathway when treated with RA, but seemed to be less sensitive to the inducing agent except at the highest concentration tested (1 μM). This decreased sensitivity to RA did not seem to be related to the Cu status of the cells but rather to the absence of a component of serum. Indeed, cells grown in DFM regained their sensitivity to RA when allowed to differentiate in IMDM with 5% serum. These data indicate that the processes of growth and terminal differentiation in HL-60 cells are not greatly influenced by Cu. Thus, it seems likely that the insult resulting in neutropenia which is associated with Cu deficiency may occur earlier than the promyelocytic stage. However, the possibility that the mechanisms contributing to neutropenia may be unrelated to primary defects in the biochemistry of neutrophil maturation cannot be ruled out. © 1995 Wiley-Liss, Inc.