Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes.

A 5'-nucleotidase with unique specificity has been identified in the soluble fraction of normal human erythrocytes. It mediates the hydrolytic dephosphorylation of pyrimidine 5'-ribosemonophosphates but is catalytically ineffective with purine nucleotides or with the 2'-, 3'-, or cyclic isomers of pyrimidine nucleotides. Activities at 37 degrees in dialyzed hemolysates of nromal human erythrocytes averaged 7.3 and 6.2 mumol of Pi liberated per hour per g of hemoglobin for the substrates UMP and CMP, respectively. Activity with TMP as substrate was approximately one-half as much as with UMP or CMP. Apparent Michaelis constants were 0.33 mM UMP, 0.15 mM CMP, and 1.0 mM TMP. Magnesium was required for optimal activity, and this cation could not be replaced by Mn2+. Maximum activity was obtained between pH 7.0 and 7.5 with rapid decreases in more alkaline media and moderate decreases with acidification. The enzyme was quite sensitive to heat and was strongly inhibited by AMP, by some purine bases, and by both purine and pyrimidine nucleosides. Divalent cations of heavy metals were also strongly inhibitory, as were agents active against sulfhydryl groups. The presence of substrates and/or 2-mercaptoethanol provided considerable protection against some of these deleterious agents and conditions. Pyrimidine 5'-nucleotidase activity in hemolysates was clearly distinguishable from erythrocyte acid phosphatase and from leukocyte and serum alkaline phosphatases and nucleotidases.     

Analysis of the genusZea (Poaceae) using polymorphic chloroplast simple sequence repeats

We have used polymorphic chloroplast simple sequence repeats (cpSSRs) to analyse levels of diversity and relationships within the genusZea. Between two and nine alleles were found at 15 polymorphic loci and combining the data from these loci gave 32 haplotypes in the 37 accessions studied. Genetic differentiation between the two sections within the genus was calculated using theST statistic which showed that 70% of the total variation was found to exist between the sections. A phylogenetic analysis based on the ² distance metric showed a large split between the two sections and suggested multiple origins of modern cultivated maizeZea mays subsp.mays. The agreement of the phylogenetic tree with other molecular, morphological and karyological studies suggests that cpSSRs may have value in phylogenetic studies in plants.     

Towards second-generation STS (sequence-tagged sites) linkage maps in conifers: a genetic map of Norway spruce (Picea abies K.)

Genetic linkage maps have been produced for a wide range of organisms during the last decade, thanks to the increasing availability of molecular markers. The use of microsatellites (or Simple Sequence Repeats, SSRs) as genetic markers has led to the construction of “second-generation” genetic maps for humans, mouse and other organisms of major importance. We constructed a second-generation single-tree genetic linkage map of Norway spruce (Picea abies K.) using a panel of 72 haploid megagametophytes with a total of 447 segregating bands [366 Amplified Fragment Length Polymorphisms (AFLPs), 20 Selective Amplification of Microsatellite Polymorphic Loci (SAMPLs) and 61 SSRs, each single band being treated initially as a dominant marker]. Four hundred and thirteen markers were mapped in 29 linkage groups (including triplets and doublets) covering a genetic length of 2198.3?cM, which represents 77.4% of the estimated genome length of Picea abies (approximately 2839?cM). The map is still far from coalescing into the expected 12 chromosomal linkage groups of Norway spruce (2n?=?2x?=?24). A?possible explanation for this comes from the observed non-random distribution of markers in the framework map. Thirty-eight SSR marker loci could be mapped onto 19 linkage groups. This set of highly informative Sequence Tagged Sites (STSs) can be used in many aspects of genetic analysis of forest trees, such as marker-assisted selection, QTL mapping, positional cloning, gene flow analysis, mating system analysis and genetic diversity studies.     

Tools and methodologies to support more sustainable biofuel feedstock production

Increasingly, government regulations, voluntary standards, and company guidelines require that biofuel production complies with sustainability criteria. For some stakeholders, however, compliance with these criteria may seem complex, costly, or unfeasible. What exiting tools, then, might facilitate compliance with a variety of biofuel-related sustainability criteria? This paper presents four existing tools and methodologies that can help stakeholders assess (and mitigate) potential risks associated with feedstock production, and can thus facilitate compliance with requirements under different requirement systems. These include the Integrated Biodiversity Assessment Tool (IBAT), the ARtificial Intelligence for Ecosystem Services (ARIES) tool, the Responsible Cultivation Areas (RCA) methodology, and the related Biofuels + Forest Carbon (Biofuel + FC) methodology.     

Detection of Helicobacter Species in the Gastrointestinal Tract of Wild Rodents From Brazil

Since we have limited knowledge about the occurrence of Helicobacter in wild animals, we searched for Helicobacter species in the gastrointestinal tract of 75 rodents captured in forest remnants of Minas Gerais, Brazil. Fragments from the antrum and corpus of the stomach and from the colon were taken for PCR assays for Helicobacter detection. Although gastric mucosa was Helicobacter-positive in only one animal, the bacterium was detected in the colonic mucosa of 23 rodents (30.7%). Helicobacter detection was more frequent in the colon of terraced rice rat (56%) and house rat (30%) in contrast to punare and Spix’s yellow-toothed cavy, in which the presence of the bacterium was not detected. Helicobacter rodentium, H. marmotae, H. cinaedi, and other species closely related to the murine helicobacters were presumptively identified by DNA sequencing. Wild rodents may serve as a reservoir of these Helicobacter species in nature.     

PERMANENT GENETIC RESOURCES: Characterization of eight microsatellite loci in the woolly mouse opossum, Micoureus paraguayanus, isolated from Micoureus demerarae

Eight novel microsatellite markers were isolated from the woolly mouse opossum from the Amazon Forest in Peru, Micoureus demerarae, using a partial genomic DNA library and an enrichment protocol. These loci were polymorphic in M. demerarae and Micoureus paraguayanus populations from the Atlantic Forest in Brazil with the number of alleles ranging from two to 23. Those eight loci plus another five already described for M. paraguayanus will allow for the evaluation of genetic diversity of populations from the 'Rio Doce' Park, one of the last Atlantic Forest fragments in Minas Gerais state, Brazil.     

Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated.

Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35). Phosphorylation of Pgamma at either residue caused a minor decrease--not the previously reported increase--in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits. The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527-27534). In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work. In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only approximately 10% of the endogenous Pgamma was phosphorylated. This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6. However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802-37808) in photoreceptor cells.     

miRTissue: a web application for the analysis of miRNA-target interactions in human tissues

Background

microRNAs act as regulators of gene expression interacting with their gene targets. Current bioinformatics services, such as databases of validated miRNA-target interactions and prediction tools, usually provide interactions without any information about what tissue that interaction is more likely to appear nor information about the type of interactions, causing mRNA degradation or translation inhibition respectively.

Results

In this work, we introduce miRTissue, a web application that combines validated miRNA-target interactions with statistical correlation among expression profiles of miRNAs, genes and proteins in 15 different human tissues. Validated interactions are taken from the miRTarBase database, while expression profiles are downloaded from The Cancer Genome Atlas repository. As a result, the service provides a tissue-specific characterisation of each couple of miRNA and gene together with its statistical significance (p-value). The inclusion of protein data also allows providing the type of interaction. Moreover, miRTissue offers several views for analysing interactions, focusing for example on the comparison between different cancer types or different tissue conditions. All the results are freely downloadable in the most common formats.

Conclusions

miRTissue fills a gap concerning current bioinformatics services related to miRNA-target interactions because it provides a tissue-specific context to each validated interaction and the type of interaction itself. miRTissue is easily browsable allowing the user to select miRNAs, genes, cancer types and tissue conditions. The results can be sorted according to p-values to immediately identify those interactions that are more likely to occur in a given tissue. miRTissue is available at http://tblab.pa.icar.cnr.it/mirtissue.html.