Isolation and characterization of seven tetranucleotide microsatellite loci from Atlantic northern bluefin tuna Thunnus thynnus thynnus

Microsatellite‐enriched genomic libraries were obtained from the Atlantic northern bluefin tuna, Thunnus thynnus thynnus and seven tetranucleotide markers were successfully isolated and characterized from this library. These markers were found to have between 1 and 17 alleles in Atlantic northern bluefin tuna and heterozygosity ranged from 0 to 0.85. No deviations from the expectation of Hardy‐Weinburg equilibrium were found for any marker. Several of these markers amplify reliably in other tuna species.     

Microsatellite DNA markers for population‐genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus

Twenty‐five microsatellites from Atlantic bluefin tuna (Thunnus thynnus thynnus) were characterized. All 25 microsatellites were polymorphic; the number of alleles among up to 56 individuals surveyed ranged from two to 23. Atlantic bluefin tuna are highly exploited and major questions remain as to stock structure and abundance in the eastern and western North Atlantic. The microsatellites will be useful in testing stock‐structure hypotheses and in generating estimates of effective population size. The polymerase chain reaction primer sets developed also amplified identifiable alleles in three other species of genus Thunnus: T. albacares (yellowfin tuna), T. alalunga (albacore tuna) and T. obesus (bigeye tuna).     

Rapid simulation and analysis of isotopomer distributions using constraints based on enzyme mechanisms: an example from HT29 cancer cells

MOTIVATION: Addition of labeled substrates and the measurement of the subsequent distribution of the labels in isotopomers in reaction networks provide a unique method for assessing metabolic fluxes in whole cells. However, owing to insufficiency of information, attempts to quantify the fluxes often yield multiple possible sets of solutions that are consistent with a given experimental pattern of isotopomers. In the study of the pentose phosphate pathways, the need to consider isotope exchange reactions of transketolase (TK) and transaldolase (TA) (which in past analyses have often been ignored) magnifies this problem; but accounting for the interrelation between the fluxes known from biochemical studies and kinetic modeling solves it. The mathematical relationships between kinetic and equilibrium constants restrict the domain of estimated fluxes to the ones compatible not only with a given set of experimental data, but also with other biochemical information. METHOD: We present software that integrates kinetic modeling with isotopomer distribution analysis. It solves the ordinary differential equations for total concentrations (accounting for the kinetic mechanisms) as well as for all isotopomers in glycolysis and the pentose phosphate pathway (PPP). In the PPP the fluxes created in the TK and TA reactions are expressed through unitary rate constants. The algorithms that account for all the kinetic and equilbrium constant constraints are integrated with the previously developed algorithms, which have been further optimized. The most time-consuming calculations were programmed directly in assembly language; this gave an order of magnitude decrease in the computation time, thus allowing analysis of more complex systems. The software was developed as C-code linked to a program written in Mathematica (Wolfram Research, Champaign, IL), and also as a C++ program independent from Mathematica. RESULTS: Implementing constraints imposed by kinetic and equilibrium constants in the isotopomer distribution analysis in the data from the cancer cells eliminated estimates of fluxes that were inconsistent with the kinetic mechanisms of TK and TA. Fluxes measured experimentally in cells can be used to estimate better the kinetics of TK and TA as they operate in situ. Thus, our approach of integrating various methods for in situ flux analysis opens up the possibility of designing new types of experiments to probe metabolic interrelationships, including the incorporation of additional biochemical information. AVAILABILITY: Software is available freely at: http://www.bq.ub.es/bioqint/selivanov.htm CONTACT: martacascante@ub.edu     

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

Although protein kinase F_A/GSK-3 (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase F_A/GSK-3 is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32M sucrose, approximately 40% of the total F_A/GSK-3 was found in the cytosol. However, when brain was homogenized in buffer containing 0.32M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total F_A/GSK-3 was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase F_A/GSK-3 in high recovery and in a substantial amount from brain tissue. The data explain why kinase F_A/GSK-3 cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase F_A/GSK-3 from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase F_A/GSK-3 in the regulation of brain functions especially in the brain particulate membrane fractions.