A roof plate-dependent enhancer controls the expression of Homeodomain only protein in the developing cerebral cortex

The smallest known homeodomain protein, Homeodomain only protein (Hop), was identified and described here as a temporally and spatially restricted gene in the neurogenic regions of the developing murine CNS including the cerebral cortex. Furthermore, an evolutionarily conserved 418 base pair upstream cis-regulatory DNA sequence was found to confine the Hop expression to the CNS of transgenic mice, but not to the heart which is the second major Hop expressing organ Chen, F., Kook, H., Milewski, R., Gitler, A.D., Lu, M.M., Li, J., Nazarian, R., Schnepp, R., Jen, K., Biben, C., Runke, G., Mackay, J.P., Novotny, J., Schwartz, R.J., Harvey, R.P., Mullins, M.C., Epstein, J.A., 2002. Hop is an unusual homeobox gene that modulates cardiac development. Cell 110, 713-723; Shin, C.H., Liu, Z.P., Passier, R., Zhang, C.L., Wang, D.Z., Harris, T.M., Yamagishi, H., Richardson, J.A., Childs, G., Olson, E.N., 2002. Modulation of cardiac growth and development by HOP, an unusual homeodomain protein. Cell 110, 725-735. The forebrain enhancer activity was successfully reproduced in vitro utilizing a combination of the electroporation and the organotypic brain culture method. Using this approach, the minimal requirement for the forebrain-specific enhancer sequence was delineated down to 200 base pairs. We further demonstrate that the Hop enhancer activity is inducible ectopically in a transgenic tissue by wild-type roof plate transplantation in vitro. Thus Hop is regulated in the forebrain by a so far unidentified paracrine signaling factor from the roof plate. Furthermore, the identified enhancer sequence provides an important tool for the targeted expression of transgenes in the medial cortex and the cortical hem.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

Enzyme-independent nitric oxide formation during UVA challenge of human skin: characterization, molecular sources, and mechanisms

Many of the local UV-induced responses including erythema and edema formation, inflammation, premature aging, and immune suppression can be influenced by nitric oxide synthase (NOS)-produced NO which is known to play a pivotal role in cutaneous physiology. Besides NOS-mediated NO production, UV radiation might trigger an enzyme-independent NO formation in human skin by a mechanism comprising the decomposition of photo-reactive nitrogen oxides. Therefore, we have examined the chemical-storage forms of potential NO-generating agents, the mechanisms and kinetics of their decomposition, and their biological relevance. In normal human skin specimens we find nitrite and S-nitrosothiols (RSNO) at concentrations 25- or 360-fold higher than those found in plasma of healthy volunteers. UVA irradiation of human skin leads to high-output formation of bioactive NO due to photo-decomposition of RSNO and nitrite which represents the primary basis for NO formation during UVA exposure. Interestingly, reduced thiols strongly augment photo-decomposition of nitrite and are essential for maximal NO release. The enzyme-independent NO formation found in human skin opens a completely new field in cutaneous physiology and will extend our understanding of mechanisms contributing to skin aging, inflammation, and cancerogenesis.     

C-terminus of p47(phox) is required for interaction with leukocyte type rat 12-lipoxygenase

In yeast two-hybrid system, rat 12-lipoxygenase (12-LO) bound to complete (390 amino acids) or the N-terminus truncated form of human p47 (phox), but not to the C-terminus truncated form (residues 1-286). When glutathione S-transferase fused human p47(phox) was added to an in vitro 12-LO enzyme activity assay, formation of 12-hydroperoxyeicosatetraenoic acid was reduced significantly compared to the C-terminus truncated form. These results indicate that C-terminus of p47(phox) is important for its interaction to rat 12-LO.     

Implication of CcpN in the regulation of a novel untranslated RNA (SR1) in Bacillus subtilis

Antisense-RNAs have been investigated in detail over the past 20 years as the principal regulators in accessory DNA elements such as plasmids, phages and transposons. However, only a few examples of chromosomally encoded bacterial antisense RNAs were known. Meanwhile, approximately 70 small non-coding RNAs from the Escherichia coli genome have been found, the functions of the majority of which remain to be elucidated. Only one systematic search has been performed for Gram-positive bacteria, so far. Here, we report the identification of a novel small (205 nt) non-translated RNA--SR1--encoded in the Bacillus subtilis genome. SR1 was predicted by a computational approach and verified by Northern blotting. Knockout or overexpression of SR1 did not affect growth. SR1 was derepressed under conditions of gluconeogenesis, but repressed under glycolytic conditions. Two regulatory levels could be identified, one involving CcpA, the second, more important, involving the recently identified regulator CcpN.     

Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in vivo and in vitro

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5-1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.     

Isomeric thiazole derivatives as ligands for the neuropeptide Y5 receptor

Sets of isomeric thiazole derivatives 1 and 2 have been synthesised in a parallel iterative solution-phase synthesis approach guided by the SAR analysis derived from biological results and computer-aided design and analysis. This synergistic and streamlined working procedure led to highly active isomeric NPY5 receptor ligands. However, a 10-fold difference at least in their respective binding affinities was consistently found for all isomeric pairs 1 and 2. The analysis of conformational differences due to heteroatom interactions in 1 and 2 revealed a favourable C=O...S interaction in 1, whereas thiazoles 2 showed a repulsive C=O...N interaction.     

Redox-responsive and calcium-dependent switching of glycosyldisulfide interactions with Concanavalin A

Glycosyldisulfides can interact efficiently with carbohydrate-binding entities. This has been shown for a range of thiosaccharide dimers when tested for their effects against the lectin Concanavalin A using a modified quartz crystal microbalance-technique. Contrary to the thiosaccharide monomers, showing no significant binding up to 10 mM, several of the dimers showed IC(50)-values in the low millimolar range. Three of the glycosyldisulfides tested also displayed very high positive apparent cooperativity effects that were found to be both calcium-dependent and redox-responsive.     

Müllerian mimicry: an examination of Fisher's theory of gradual evolutionary change

In 1927, Fisher suggested that Müllerian mimicry evolution could be gradual and driven by predator generalization. A competing possibility is the so-called two-step hypothesis, entailing that Müllerian mimicry evolves through major mutational leaps of a less-protected species towards a better-protected, which sets the stage for coevolutionary fine-tuning of mimicry. At present, this hypothesis seems to be more widely accepted than Fisher's suggestion. We conducted individual-based simulations of communities with predators and two prey types to assess the possibility of Fisher's process leading to a common prey appearance. We found that Fisher's process worked for initially relatively similar appearances. Moreover, by introducing a predator spectrum consisting of several predator types with different ranges of generalization, we found that gradual evolution towards mimicry occurred also for large initial differences in prey appearance. We suggest that Fisher's process together with a predator spectrum is a realistic alternative to the two-step hypothesis and, furthermore, it has fewer problems with purifying selection. We also examined the factors influencing gradual evolution towards mimicry and found that not only the relative benefits from mimicry but also the mutational schemes of the prey types matter.