The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).     

Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.     

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

RepeatAround: a software tool for finding and visualizing repeats in circular genomes and its application to a human mtDNA database

RepeatAround is a Windows based software tool designed to find "direct repeats", "inverted repeats", "mirror repeats" and "complementary repeats", from 3 to 64bp length, in circular genomes. It processes input files directly extracted from GenBank database, providing visualisation of the repeats location in the genomic structure, so that for instance, in most mtDNAs the user can check if the repeats are located in coding or non-coding region (and in the first case in which gene), and how far apart the repeat pair(s) are. Besides the visual tool, it provides other outputs in a spreadsheet containing information on the number and location of the repeats, facilitating graphic analyses. Several genomes can be inputed simultaneously, for phylogenetic comparison purposes. Other capabilities of the software are the generation of random circular genomes, for statistical evaluation of comparison between observed repeats distributions with their shuffled counterparts, as well as the search for specific motifs, allowing an easy confirmation of repeats flanking a newly detected rearrangement. As an example of the programme's applications we analysed the Direct Repeats distribution in a large human mtDNA database. Results showed that Direct Repeats, even the larger ones, are evenly distributed among the human mtDNA haplogroups, enabling us to state that, based only on the repetitive motifs, no haplogroup is particularly more or less prone to mtDNA macrodeletions.     

Different modelling tools of aquatic ecosystems: A proposal for a unified approach

Over the last few decades, several modelling tools have been developed for the simulation of hydrodynamic and biogeochemical processes in aquatic ecosystems. Until late 70's, coupling hydrodynamic models to biogeochemical models was not common and today, problems linked to the different scales of interest remain. The time scale of hydrodynamic phenomena in coastal zone (minutes to hours) is much lower than that of biogeochemistry (few days). Over the last years, there has been an increasing tendency to couple hydrodynamic and biogeochemical models in a clear recognition of the importance of incorporating in one model the feedbacks between physical, chemical and biological processes. However, different modelling teams tend to adopt different modelling tools, with the result that benchmarking exercises are sometimes difficult to achieve in projects involving several institutions. Therefore, the objectives of this paper are to provide a quick overview of available modelling approaches for hydrodynamic and biogeochemical modelling, to help people choose among the diversity of available models, as a function of their particular needs, and to propose a unified approach to allow modellers to share software code, based on the object oriented programming potentiality. This approach is based on having object dynamic link libraries that may be linked to different model shells. Each object represents different processes and respective variables, e.g. hydrodynamic, phytoplankton and zooplankton objects. Some simple rules are proposed to link available objects to programs written in different source codes.     

A novel polymorphic Alu insertion embedded in a LINE 1 retrotransposon in the human X chromosome (DXS225): identification and worldwide population study

We describe a novel polymorphic Alu insertion (DXS225) on the human X chromosome (Xq21.3) embedded into an L1 retrotransposon. The DXS225 polymorphism was genotyped in 684 males from the CEPH Human Genome Diversity Panel. This insertion was found in all regions of the globe, suggesting that it took place before modern humans spread from Africa ca. 100,000 years ago. However, only one Amerindian population (Karitiana) showed this insertion allele, which may have been introduced by European admixture. Thus, it appears likely that the Alu insertion was absent from pre-Columbian America. Analysis of molecular variance worldwide demonstrated that 92.2% of the genetic variance was concentrated within populations. DXS225 is flanked by two microsatellites (DXS8114 and DXS1002), which are 86 kb apart and are in very strong linkage disequilibrium. The combination of a unique event polymorphism on the X chromosome in linkage disequilibrium with two rapidly evolving microsatellites should provide a useful tool for studies of human evolution.     

Reproductive ecology of Brazilian freshwater fishes

We used metadata on nine reproductive traits of 67 species of Brazilian iteroparous, oviparous, teleost freshwater fishes to test phenotypes associations to discriminate species that only spawn in large rivers (lotic fishes) from those capable to spawn in lentic habitats (lentic fishes). We tested the hypothesis that lotic fishes present spawning migration, shorter spawning season, single spawning, no parental care, free eggs, higher relative fecundity, faster embryogenesis, and larger size, while lentic fishes present no spawning migration, longer spawning season, multiple spawning, parental care, adhesive eggs, lower relative fecundity, slower embryogenesis, and smaller size. Our analyses supported the hypothesis but not all phenotypic associations satisfied it, specifically with regard to lentic fishes or to pairs of phenotypes typical of lentic fishes. We also concluded that spawning in large rivers is a better predictor of bionomic characters than spawning in lentic habitats, and lotic fishes are specialists compared to lentic fishes.