Demonstration of PGl2 production by isolated perfused rat lungs with platelet aggregation test

Platelet aggregation test was used for PGl₂ measurements. The use of 6 % CO₂ in air stabilized human platelet rich plasma (PRP) so that it could be used for up to 7 hours in these measurements. The PGl₂ caused inhibition of aggregation in response to ADP was concentration dependent in the range of 0.5 ng/ml to 50 ng/ml. Isolated perfused rat lungs released spontaneously 190 ng/min PGl₂ and about 3 % of infused arachidonic acid potassium salt (equivalent to 25 μg/min arachidonic acid) was converted to PGl₂.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

Expression of the gene for mitoribosomal protein S12 is controlled in human cells at the levels of transcription, RNA splicing, and translation.

The human gene RPMS12 encodes a protein similar to bacterial ribosomal protein S12 and is proposed to represent the human mitochondrial orthologue. RPMS12 reporter gene expression in cultured human cells supports the idea that the gene product is mitochondrial and is localized to the inner membrane. Human cells contain at least four structurally distinct RPMS12 mRNAs that differ in their 5'-untranslated region (5'-UTR) as a result of alternate splicing and of 5' end heterogeneity. All of them encode the same polypeptide. The full 5'-UTR contains two types of sequence element implicated elsewhere in translational regulation as follows: a short upstream open reading frame and an oligopyrimidine tract similar to that found at the 5' end of mRNAs encoding other growth-regulated proteins, including those of cytosolic ribosomes. The fully spliced (short) mRNA is the predominant form in all cell types studied and is translationally down-regulated in cultured cells in response to serum starvation, even though it lacks both of the putative translational regulatory elements. By contrast, other splice variants containing one or both of these elements are not translationally regulated by growth status but are translated poorly in both growing and non-growing cells. Reporter analysis identified a 26-nucleotide tract of the 5'-UTR of the short mRNA that is essential for translational down-regulation in growth-inhibited cells. Such experiments also confirmed that the 5'-UTR of the longer mRNA variants contains negative regulatory elements for translation. Tissue representation of RPMS12 mRNA is highly variable, following a typical mitochondrial pattern, but the relative levels of the different splice variants are similar in different tissues. These findings indicate a complex, multilevel regulation of RPMS12 gene expression in response to signals mediating growth, tissue specialization, and probably metabolic needs.     

Gene dosage and selective expression modify phenotype in a Drosophila model of human mitochondrial disease

Human mitochondrial disease manifests with a wide range of clinical phenotypes of varying severity. To create a model for these disorders, we have manipulated the Drosophila gene technical knockout, encoding mitoribosomal protein S12. Various permutations of endogenous and transgenic alleles create a range of phenotypes, varying from larval developmental arrest through to mild neurological defects in the adult, and also mimic threshold effects associated with human mtDNA disease. Nuclear genetic background influences mutant phenotype by a compensatory mechanism affecting mitochondrial RNA levels. Selective expression of the wild-type allele indicates critical times and cell-types in development, in which mitochondrial protein synthesis deficiency leads to specific phenotypic outcomes.     

Heterodimerization of yLAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y⁺LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y⁺LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y⁺LAT-1 and 4F2hc in HEK293 cells expressing y⁺LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y⁺LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y⁺LAT-1 to the plasma membrane.     

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.     

No effects of short-term GSM mobile phone radiation on cerebral blood flow measured using positron emission tomography

The present study investigated the effects of 902.4 MHz Global System for Mobile Communications (GSM) mobile phone radiation on cerebral blood flow using positron emission tomography (PET) with the ¹⁵O‐water tracer. Fifteen young, healthy, right‐handed male subjects were exposed to phone radiation from three different locations (left ear, right ear, forehead) and to sham exposure to test for possible exposure effects on brain regions close to the exposure source. Whole‐brain [¹⁵O]H₂O–PET images were acquired 12 times, 3 for each condition, in a counterbalanced order. Subjects were exposed for 5 min in each scan while performing a simple visual vigilance task. Temperature was also measured in the head region (forehead, eyes, cheeks, ear canals) during exposure. The exposure induced a slight temperature rise in the ear canals but did not affect brain hemodynamics and task performance. The results provided no evidence for acute effects of short‐term mobile phone radiation on cerebral blood flow. Bioelectromagnetics 33:247–256, 2012. © 2011 Wiley Periodicals, Inc.     

Biomine: predicting links between biological entities using network models of heterogeneous databases

ABSTRACT: BACKGROUND: Biological databases contain large amounts of data concerning the functions and associationsof genes and proteins. Integration of data from several such databases into a single repositorycan aid the discovery of previously unknown connections spanning multiple types ofrelationships and databases. RESULTS: Biomine is a system that integrates cross-references from several biological databases into agraph model with multiple types of edges, such as protein interactions, gene-diseaseassociations and gene ontology annotations. Edges are weighted based on their type,reliability, and informativeness. We present Biomine and evaluate its performance in linkprediction, where the goal is to predict pairs of nodes that will be connected in the future,based on current data. In particular, we formulate protein interaction prediction and diseasegene prioritization tasks as instances of link prediction. The predictions are based on aproximity measure computed on the integrated graph. We consider and experiment withseveral such measures, and perform a parameter optimization procedure where different edgetypes are weighted to optimize link prediction accuracy. We also propose a novel method fordisease-gene prioritization, defined as finding a subset of candidate genes that cluster togetherin the graph. We experimentally evaluate Biomine by predicting future annotations in thesource databases and prioritizing lists of putative disease genes. CONCLUSIONS: The experimental results show that Biomine has strong potential for predicting links when aset of selected candidate links is available. The predictions obtained using the entire Biominedataset are shown to clearly outperform ones obtained using any single source of data alone,when different types of links are suitably weighted. In the gene prioritization task, anestablished reference set of disease-associated genes is useful, but the results show that underfavorable conditions, Biomine can also perform well when no such information is available.The Biomine system is a proof of concept. Its current version contains 1.1 million entities and8.1 million relations between them, with focus on human genetics. Some of its functionalitiesare available in a public query interface at http://biomine.cs.helsinki.fi, allowing searching forand visualizing connections between given biological entities.     

Setup and dosimetry for exposing anaesthetised pigs in vivo to 900 MHz GSM mobile phone fields

The aim of this study was a dosimetrical analysis of the setup used in the exposure of the heads of domestic pigs to GSM-modulated radio frequency electromagnetic fields (RF-EMF) at 900 MHz. The heads of pigs were irradiated with a half wave dipole using three different exposure routines; short bursts of 1-3 s at two different exposure levels and a continuous 10-min exposure. The electroencephalogram (EEG) was registered continuously during the exposures to search for RF-EMF originated changes. The dosimetry was based on simulations with the anatomical heterogeneous numerical model of the pig head. The simulation results were validated by experimental measurements with the exposure dipole and a homogeneous liquid phantom resembling the pig head. The specific absorption rate (SAR), defined as a maximum average over 10 g tissue mass (SAR(10g)), was 7.3 W/kg for the first set of short bursts and 31 W/kg for the second set of short bursts. The SAR(10g) in the continuous 10-min exposure was 31 W/kg. The estimated uncertainty for the dosimetry was +/-25% (K = 2).     

Lipid Metabolism of Caddisfly Larvae at Low pH

The influence of low pH (5.0 and 4.0) on lipid metabolism of caddisfly larvae Hydropsyche contubernalis L. (Trichoptera) was studied in 48 h toxicity experiments. The results were correlated with lipid composition of caddisfly larvae directly isolated from natural water. Phospholipids, cholesterol, mono-, di-, triacylglycerols, and fatty acids were detected by thin-layer and liquid chromatography. Minimal environmental changes were shown to initiate the biochemical adaptation mechanisms strengthening the cellular membranes through their condensation due to additional phospholipid and cholesterol synthesis. In the natural medium the adaptation processes are more active than in the artificial medium. More serious changes, such as pH decrease to 4.0, suppress the adaptation processes in the first medium and terminate them in the second one.