Identification of cardiac malformations in mice lacking <Emphasis Type="Italic">Ptdsr</Emphasis>using a novel high-throughput magnetic resonance imaging technique

Background  

Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.     

Solution stability and degradation pathway of deoxyribonucleoside phosphoramidites in acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O-cyanoethyl-N,N-diisopropyl-phosphoramidites of 5'-O-dimethoxytrityl (DMT) protected deoxyribonucleosides (dG(ib), dA(bz), dC(bz), T) were analyzed by HPLC-MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile-induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid-catalyzed hydrolysis could also be reduced by addition of small amounts of base.     

Vascular NAD(P)H oxidases: specific features,expression, and regulation

The importance of reactive oxygen species (ROS) in vascular physiology and pathology is becoming increasingly evident. All cell types in the vascular wall produce ROS derived from superoxide-generating protein complexes similar to the leukocyte NADPH oxidase. Specific features of the vascular enzymes include constitutive and inducible activities, substrate specificity, and intracellular superoxide production. Most phagocyte enzyme subunits are found in vascular cells, including the catalytic gp91phox (aka, nox2), which was the earliest member of the newly discovered nox family. However, smooth muscle frequently expresses nox1 rather than gp91phox, and nox4 is additionally present in all cell types. In cell culture, agonists increase ROS production by activating multiple signals, including protein kinase C and Rac, and by upregulating oxidase subunits. The oxidases are also upregulated in vascular disease and are involved in the development of atherosclerosis and a significant part of angiotensin II-induced hypertension, possibly via nox1 and nox4. Likewise, enhanced vascular oxidase activity is associated with diabetes. Therefore, members of this enzyme family appear to be important in vascular biology and disease and constitute promising targets for future therapeutic interventions.     

Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.     

Comparison of prostaglandin F2alpha,bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.