Formation and hydrolysis of triacylglycerol and sterols epoxides: role of unsaturated triacylglycerol peroxyl radicals

Epoxidation of unsaturated pure triacylglycerols (TAGs), cholesterol, and phytosterols was investigated using air and 18O2 oxidation experiments. Oxidized lipids were analyzed using both triple quadrupole mass spectrometry (MS), ion-trap MS in the direct infusion mode, and triple quadrupole MS in tandem with a liquid chromatograph (LC-MS/MS). Pure 1,2-distearoyl-3-oleoyl-glycerol (SSO) samples were heated in sealed vials under air or 18O2 atmosphere at 160 degrees C for 1 h. LC-MS/MS analysis of 18O-labeled oxidized TAGs revealed that hydroperoxides and epoxide TAGs are formed mainly during this first step. Then, oxidized TAGs were incubated under an inert atmosphere, separately with 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) at 160 degrees C for 90 min, and with cholesterol and stigmasterol at 100 degrees C for 10 min. Subsequent LC-MS/MS analysis revealed the occurrence of epoxidation products of PPO, cholesterol, and sitosterol. Therefore, we showed the epoxidation of unsaturated lipids proceeds readily in contact with hydroperoxide TAGs, in the absence of molecular oxygen. Dual oxidation experiments using both air and 18O2 allowed investigation of oxygen atom transfer during epoxidation of lipids. Moreover, the experimental oxidation design presented can be used to study fragmentation pathways, as illustrated for 5,6-epoxycholesterol (CE) on both triple quadrupole and ion-trap MS. We report for the first time the occurrence of 5,6;22,23-diepoxystigmasterol (StDE) and 5,6;22,23-diepoxybrassicasterol (BDE) in autoxidized vegetable oils. Additionally, acid-catalyzed hydrolysis of epoxidized lipids, with emphasis on phytosterol polyol formation, was investigated using a model gastric medium. For confirmation, almost all identified products were synthesized and characterized by MS.     

Test System for Measurement of Translational Activity in Vivo

Abstract

A new plasmid which makes it possible to measure formation of a mature protein, as well as prematurely terminated gene products, has been constructed. It can be used for the analysis of translational efficiency of specified codon sequences in vivo.     

Population genetic structure and connectivity in the endangered Ethiopian mountain Nyala (Tragelaphus buxtoni): recommending dispersal corridors for future conservation

Habitat fragmentation is an increasing threat to wildlife species across the globe and it has been predicted that future biodiversity will decrease rapidly without the intervention of scientifically-based management. In this study we have applied a landscape genetics approach to suggest a network design that will maintain connectivity among populations of the endangered mountain Nyala (Tragelaphus buxtoni) in the fragmented highlands of Ethiopia. DNA was obtained non-invasively from 328 individuals and genetic population structure and gene flow were estimated using 12 microsatellite markers. In addition, a 475-bp segment of the mitochondrial control region was sequenced for 132 individuals. Potential dispersal corridors were determined from least-cost path analysis based on a habitat suitability map. The genetic data indicated limited gene flow between the sampled mountain Nyala populations of the Bale Massif and the Arsi Massif. The genetic differentiation observed among five sampling areas of the Bale Massif followed a pattern of isolation by distance. We detected no impact of habitat resistance on the gene flow. In the future, however, the current expanding human population in the highlands of Ethiopia may reduce the current mountain Nyala habitat and further limit migration. Hence, maintaining habitat connectivity and facilitating survival of stepping-stone populations will be important for the future conservation of the species. The approach used here may also be useful for the study and conservation of other wildlife species inhabiting areas of increasing human encroachment.     

Extensive MHC class II DRB3 diversity in African and European cattle

Genetic deversity at the highly polymorphic BoLA-DRB3 locus was investigated by DNA sequence analyses of 18 African cattle from two breeds representing the two subspecies of cattle, Bos primigenius indicus and Bos primigenius taurus. Yhe polymorphism was compared with that found in a sample ofd 32 European cattle from four breeds, all classified as B. p. taurus. Particularly extensive genetic diversity was found among African cattle, in which as many as 18 alleles were recognized in this small random sample of animals from two breeds. The observed similarity in allele frequency distribution between the two African populations, N'Dama and Zebu cattle, is consistent with the recent recognition of gene flow between B. p. indicus and B. P taurus cattle in Africa. A total of 30 DRB3 alleles were documented and as many as 26 of these were classified as major allelic types showing at least five amino acid substitutions compared with other major types. The observation of extensive genetic diversity at MHC loci in cattle, as well as in other farm animals, provides a compelling argument against matin-type preferences as a primary cause in maintaining major histocompatibility complex diversity, since the reproduction of these animals has been controlled by humans for many generations.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been given the accession numbers X87641-X87670     

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.     

Congruent responses to weather variability in high arctic herbivores

Assessing the role of weather in the dynamics of wildlife populations is a pressing task in the face of rapid environmental change. Rodents and ruminants are abundant herbivore species in most Arctic ecosystems, many of which are experiencing particularly rapid climate change. Their different life-history characteristics, with the exception of their trophic position, suggest that they should show different responses to environmental variation. Here we show that the only mammalian herbivores on the Arctic islands of Svalbard, reindeer (Rangifer tarandus) and sibling voles (Microtus levis), exhibit strong synchrony in population parameters. This synchrony is due to rain-on-snow events that cause ground ice and demonstrates that climate impacts can be similarly integrated and expressed in species with highly contrasting life histories. The finding suggests that responses of wildlife populations to climate variability and change might be more consistent in Polar regions than elsewhere owing to the strength of the climate impact and the simplicity of the ecosystem.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.     

A long‐term record of Quercus decline,logging and fires in a southern Swedish Fagus‐Picea forest

Abstract. We reconstructed forest development and disturbance events (fire and logging) during the last 1000 yr with tree‐ring data, pollen and charcoal analysis from a semi‐natural Fagus sylvatica‐Picea abies forest (ca. 1 km²) in the hemiboreal zone. According to pollen analysis, Quercus robur together with Pinus sylvestris was abundant in the forest until the turn of the 18th/19th centuries when these species disappeared completely (Quercus) or nearly completely (Pinus) and were replaced by Fagus and Picea. The disappearance of Quercus was corroborated by the remarkable discovery of a single Quercus stump that had been cut in the 18th century and had become overgrown and preserved by a very old Picea. In total 11 fires were dated from 1555 to 1748 from fire scars in several Pinus stumps cut 100 ‐ 200 yr ago. Since the last fire in 1748, no Quercus or Pinus have regenerated in the core of the reserve apart from single pines in neighbouring managed forest (80 yr ago). During the period of documented fires Fagus was protected from fires in a refuge made up of large boulders. Picea colonized the region at the time when the fires ceased 250 yr ago. We hypothesize that most of the fires were probably of human origin because of their patchiness and high frequency compared to the natural background levels of lightning ignitions in the region. On a 300‐yr time scale, logging and fire suppression seem to strongly overshadow the effect of climate change on forest composition and dynamics.     

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A′ model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.