Investigating genomic and phenotypic parallelism between piscivorous and planktivorous lake trout (Salvelinus namaycush) ecotypes by means of RADseq and morphometrics analyses

Repeated adaptive ecological diversification has commonly been reported in fish and has often been associated with trophic niche diversity. The main goal of this study was to investigate the extent of parallelism in the genomic and phenotypic divergence between piscivorous and planktivorous lake trout ecotypes from Laurentian Shield lakes, Canada. This was achieved by documenting the extent of morphological differentiation using geometric morphometrics and linear measurements as well as the pattern of genomic divergence by means of RADseq genotyping (3925 filtered SNPs) in 12 lakes. Our results indicate that the two ecotypes evolved distinct body shape and several linear measurements in parallel. Neutral genetic differentiation was pronounced between all isolated populations (Mean F_ST = 0.433), indicating no or very limited migration and pronounced genetic drift. Significant genetic differentiation also suggested partial reproductive isolation between ecotypes in the two lakes where they are found in sympatry. Combining different outlier detection methods, we identified 48 SNPs putatively under divergent selection between ecotypes, among which 10 could be annotated and related to functions such as developmental processes and ionic regulation. Finally, our results indicate that parallel morphological divergence is accompanied by both parallel and nonparallel genomic divergence, which is associated with the use of different trophic niches between ecotypes. The results are also discussed in the context of management and conservation of this highly exploited species throughout northern North America.     

Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations

The production of Leuconostoc mesenteroides sucrose phosphorylase has been studied in 10- and 20-L batch fermentations. A fermentation medium was devised combining rapid growth, high cell yield, and high enzyme levels. Overall fermentation dynamics and enzyme fermentation patterns are elucidated here in detail. Sucrose is phosphorolyzed into fructose and glucose-1-phosphate (G-1-P) with G-1-P preferentially utilized (thus saving ATP). Subsequently, fructose is gradually metabolized and is also converted to mannitol. Invertase activity is absent. Sucrose phosphorylase is formed transitorily with peak levels toward the end of active growth; a sharp decline in enzyme activity occurs upon further fermentation. The moment of cell (enzyme) harvest is thus critical in view of obtaining active cell or enzyme preparations for sucrose phosphorolysis. Microaerophilic and strictly anaerobic fermentations displayed no appreciable difference in sucrose phosphorylase formation profile. The enzyme is intracellularly located. It is constitutively formed in the absence of sucrose, contrary to that of Pseudomonas species; other disaccharide phosphorylases are not formed.     

Glycogen depletion produced in intrafusal nuclear bag muscle fibers by brief large-amplitude muscle stretches

The glycogen content of the three types of intrafusal muscle fibre was studied with histochemical techniques in cat muscle spindles of superficial lumbrical muscles after a very large number of brief large stretches. Zones of glycogen depletion were observed in a high proportion of nuclear bag fibres, notably in bag 1 fibres, but not in chain fibres. These observations suggest that stretching of bag fibres by itself may activate these fibres.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism.

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissue and mechanical dispersion of cells yielded about 650 x 10(6) viable cells. After centrifugal elutriation and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (approximately 99%) and purity (approximately 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continuous Percoll gradient. High-density splenic Kurloff cells (150 x 10(6) cells per spleen) were also obtained with high purity (approximately 99%) and viability (approximately 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 microM) in the absence or presence of 2 microM ionophore A23187. With 10 microM arachidonic acid the relative production of cyclooxygenase products was the following: TxB2 greater than PGE2 approximately PGI2. For an arachidonic acid concentration superior to 10 microM, the profile of release was PGE2 much greater than TxB2 greater than PGI2. Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 microM arachidonic acid in the absence or presence of 2 microM ionophore A23187, or in some experiments, with 2.5 microM leukotriene A4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A4 into leukotriene B4 suggesting the presence of LTA4 hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachidonic acid metabolism by these cells. The possible implications of Kurloff cells in various biological events are discussed.     

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.