Phylogenetic shadowing and computational identification of human microRNA genes

We sequenced 122 miRNAs in 10 primate species to reveal conservation characteristics of miRNA genes. Strong conservation is observed in stems of miRNA hairpins and increased variation in loop sequences. Interestingly, a striking drop in conservation was found for sequences immediately flanking the miRNA hairpins. This characteristic profile was employed to predict novel miRNAs using cross-species comparisons. Nine hundred and seventy-six candidate miRNAs were identified by scanning whole-genome human/mouse and human/rat alignments. Most of the novel candidates are conserved also in other vertebrates (dog, cow, chicken, opossum, zebrafish). Northern blot analysis confirmed the expression of mature miRNAs for 16 out of 69 representative candidates. Additional support for the expression of 179 novel candidates can be found in public databases, their presence in gene clusters, and literature that appeared after these predictions were made. Taken together, these results suggest the presence of significantly higher numbers of miRNAs in the human genome than previously estimated.     

Tissue-specific expression of the acetylcholinesterase gene in Drosophila melanogaster embryos

Summary Acetylcholinesterase activity is present in both particulate and soluble forms in wild-type Drosophila melanogaster embryos. The particulate form of the enzyme is localized in the CNS, while the soluble forms are non-CNS-specific. Deletion mapping studies show that all AChE activity is abolished if the cytological region between 87E1-2 and 87E4 is missing. An additional region mapping to the proximal part of the 87E4 band is needed for CNS-specific AChE activityAbbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - ChE pseudocholinesterase (acetylcholine acylhydrolase, EC 3.1.1.8) - BAP 1,5-bis(allyldimethylammoniumphenyl)-pentan-3-one dibromide - i-OMPA tetraisopropylpyrophosphoramide - CNS central nervous system     

Continuous monitoring of fermentation outlet gas using a computer coupled MS

Summary Using a quadrupole mass spectrometer (QMS), we continuously monitored N₂, O₂, CO₂, and ethanol in the outlet gas of a batch fermentor. A computer was used to control the QMS, perform correction calculations and account for interferences between some ion peaks. The ability to continously scan multiple peaks and to use N₂ as an internal standard provided rapid and accurate monitoring of fermentation performance. Over the range of interest, a linear response was obtained for all the compounds measured by the QMS.     

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.     

Ectodysplasin regulates activator-inhibitor balance in murine tooth development through Fgf20 signaling

Uncovering the origin and nature of phenotypic variation within species is the first step in understanding variation between species. Mouse models with altered activities of crucial signal pathways have highlighted many important genes and signal networks regulating the morphogenesis of complex structures, such as teeth. The detailed analyses of these models have indicated that the balanced actions of a few pathways regulating cell behavior modulate the shape and number of teeth. Currently, however, most mouse models studied have had gross alteration of morphology, whereas analyses of more subtle modification of morphology are required to link developmental studies to evolutionary change. Here, we have analyzed a signaling network involving ectodysplasin (Eda) and fibroblast growth factor 20 (Fgf20) that subtly affects tooth morphogenesis. We found that Fgf20 is a major downstream effector of Eda and affects Eda-regulated characteristics of tooth morphogenesis, including the number, size and shape of teeth. Fgf20 function is compensated for by other Fgfs, in particular Fgf9 and Fgf4, and is part of an Fgf signaling loop between epithelium and mesenchyme. We showed that removal of Fgf20 in an Eda gain-of-function mouse model results in an Eda loss-of-function phenotype in terms of reduced tooth complexity and third molar appearance. However, the extra anterior molar, a structure lost during rodent evolution 50 million years ago, was stabilized in these mice.     

Evidence for de novo evolution of testis-expressed genes in the Drosophila yakuba/Drosophila erecta clade

The mutational origin and subsequent evolution of de novo genes, which are hypothesized to be genes of recent origin that are not obviously related to ancestral coding sequence, are poorly understood. However, accumulating evidence suggests that such genes may often function in male reproduction. Here we use testis-derived expressed sequence tags (ESTs) from Drosophila yakuba to identify genes that have likely arisen either in D. yakuba or in the D. yakuba/D. erecta ancestor. We found several such genes, which show testis-biased expression and are often X-linked. Comparative data indicate that three of these genes have very short open reading frames, which suggests the possibility that a significant number of testis-biased de novo genes in the D. yakuba/D. erecta clade may be noncoding RNA genes. These data, along with previously published data from D. melanogaster, support the idea that many de novo Drosophila genes function in male reproduction and that a small region of the X chromosome in the melanogaster subgroup may be a hotspot for the evolution of novel testis-biased genes.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.