Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase

LIM and SH3 domain protein (LASP-1) is a specific focal adhesion protein involved in cell migration. Overlay studies demonstrate that LASP-1 directly binds to the proline-rich domains of zyxin, lipoma preferred partner (LPP), and vasodilator-stimulated phosphoprotein (VASP), with zyxin being the most prominent interacting partner. Despite the LIM/zinc-finger domain, hypothesized to be involved in homodimerization, LASP-1 exists as a monomer. In vitro phosphorylation of recombinant mouse LASP-1 by cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) occurs at serine 61, serine 99, and threonine 156 whereas in intact cells mouse LASP-1 is phosphorylated only at threonine 156. This site is different from the known in vivo phosphorylation sites in human (serine 146) and rabbit (serine 99 and serine 146). Nevertheless, immunofluorescence of LASP-1 in human and mouse mesangial cells revealed no difference in subcellular distribution. Exposure of the cells to forskolin induced a translocation of both, human and mouse LASP-1, from the focal contacts to the cell interior without affecting F-actin structure. Immunoblotting of LASP-1 in various mouse and human tissues detected a similar prominent expression in non-muscle tissue. Altogether, our data suggest so far no functional differences between human and mouse LASP-1.     

Organ-specific mRNA distribution of C-type natriuretic peptide in neonatal and adult mice

C-type natriuretic peptide (CNP) is described as an endothelium-derived vasodilator and a growth inhibitor of vascular smooth muscle cells. In the present study, CNP mRNA was quantified by RNase-protection assay to elucidate organ distribution of CNP in neonatal and adult mice. In adult mice, the highest CNP expressions were detected in uterus and ovary, which exceeded the CNP concentrations of forebrain and brainstem. In contrast, neonatal mice showed highest CNP-mRNA levels in forebrain and brainstem with lower levels in skin, tongue, heart, lung, thymus, skeletal muscle, liver, kidney, stomach, and skull. Thus, CNP-expression pattern diminishes during postnatal development. The observation that the expression level of CNP mRNA is 2.2-fold higher in the adult forebrain compared to the neonatal forebrain allows a comparison between all neonatal and adult organs.     

Cells on a chip--the use of electric properties for highly sensitive monitoring of blood-derived factors involved in angiotensin II type 1 receptor signalling.

BACKGROUND: We developed a highly sensitive cardiomyocyte based screening system for the non-destructive electronic detection of chronotropic drugs and tissue-secreted factors involved in AT1 receptor-mediated cardiovascular diseases. METHODS: For this purpose we cultured spontaneously beating neonatal rat cardiomyocytes on microelectrode arrays (MEAs), and tested the optimised, stable culture parameters for a reproducible real-time recording of alterations in contraction frequency. After the evaluation of culture parameters, computer-based electronic measurement systems were used for counting of contractions by recording of the field potential of cardiomyocytes. RESULTS: Using the biosensor, angiotensin II, the predominant ligand of the AT1 receptor, was detected at very low concentrations of 10(-11) M via altered contractions of cardiomyocytes. Moreover, we demonstrated that cardiomyocyte coupled microarrays allow the detection of blood-derived low concentrated anti-AT1 receptor autoimmune antibodies of pregnant women suffering from preeclampsia. CONCLUSION: This study demonstrates the first well-suited electrophysiological recording of cardiomyocytes on multielectrode arrays as a benefit for functional biomonitoring for the detection of AT1 receptor/ligand interactions and other marker proteins in sera directed to cardiovascular diseases.     

Differences in platelet activation by prolactin and leptin.

Hormones such as prolactin and leptin have recently been recognized as potent platelet aggregation co-activators, and have therefore been postulated as an additional risk factor for both arterial and venous thrombosis. Clinical situations exist that are known to be associated with higher leptin and/or prolactin levels (obesity, pregnancy, prolactinomas and anti-psychotic therapy respectively) and increased venous thrombosis or atherosclerosis risk. Therefore, we compared the impact of both hormones on platelet activation in vitro and in vivo. First, we investigated platelet aggregation and P-selectin expression after stimulation with 1,000 mU/l prolactin or 100 ng/ml leptin in five healthy volunteers in vitro. Prolactin revealed significant higher levels of P-selectin expression and platelet aggregation than leptin in all subjects. We also compared the correlation of prolactin and leptin values with the P-selection expression on platelets. Previously, we detected a significant correlation between prolactin values and ADP-stimulated P-selectin expression on platelets in pregnant women, patients with pituitary tumours, and patients on anti-psychotic therapy. In contrast, leptin did not correlate with P-selectin expression in all subject groups investigated. However, leptin correlated with body mass index in the subjects investigated. Our data indicate that prolactin has a stronger effect on platelet activation as leptin in vitro and in vivo. Moreover, our data suggest that the stronger effect of prolactin on ADP-stimulated platelet aggregation, compared to leptin, depends on higher stimulation of CD62p expression by prolactin.     

Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.     

O-Glycosylation of snails

The glycosylation abilities of snails deserve attention, because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites. In analogy to many other host-pathogen relations, the glycosylation of snail proteins may likewise contribute to these host-parasite interactions. Here we present an overview on the O-glycan structures of 8 different snails (land and water snails, with or without shell): Arion lusitanicus, Achatina fulica, Biomphalaria glabrata, Cepaea hortensis, Clea helena, Helix pomatia, Limax maximus and Planorbarius corneus. The O-glycans were released from the purified snail proteins by β-elimination. Further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and - for the main structures - by gas chromatography/mass spectrometry. Snail O-glycans are built from the four monosaccharide constituents: N-acetylgalactosamine, galactose, mannose and fucose. An additional modification is a methylation of the hexoses. The common trisaccharide core structure was determined in Arion lusitanicus to be N-acetylgalactosamine linked to the protein elongated by two 4-O-methylated galactose residues. Further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present. The typical snail O-glycan structures are different to those so far described. Similar to snail N-glycan structures they display methylated hexose residues.     

Study of the functional interaction between Mcp insulators from the Drosophila bithorax complex: effects of insulator pairing on enhancer-promoter communication

Boundary elements have been found in the Abd-B 3' cis-regulatory region, which is subdivided into a series of iab domains. Previously, a 340-bp insulator-like element, M(340), was identified in one such 755-bp Mcp fragment linked to the PcG-dependent silencer. In this study, we identified a 210-bp core that was sufficient for pairing of sequence-remote Mcp elements. In two-gene transgenic constructs with two Mcp insulators (or their cores) surrounding yellow, the upstream yeast GAL4 sites were able to activate the distal white only if the insulators were in the opposite orientations (head-to-head or tail-to-tail), which is consistent with the looping/bypass model. The same was true for the efficiency of the cognate eye enhancer, while yellow thus isolated in the loop from its enhancers was blocked more strongly. These results indicate that the relative placement and orientation of insulator-like elements can determine proper enhancer-promoter communication.