A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin

In the T(1;2)dor ^var7 multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)_n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon. Received: 3 October 1998 / Accepted: 3 December 1998     

Revising the selfish DNA hypothesis: new evidence on accumulation of transposable elements in heterochromatin

The bulk of the eukaryotic genome is composed of families of repetitive sequences that are genetically silent and exhibit various types of instability. Transposable elements (TEs) are particularly common in heterochromatic regions of the genome - a location where TEs might do less damage to their host. Recent advances suggest that the relationship between TEs and heterochromatin might not be quite so straightforward.     

Automated multi-well device to measure transepithelial electrical resistances under physiological conditions

Measurement of transendothelial or transepithelial electrical resistances (TERs) is a straightforward in situ experimental approach to monitor the expression or modulation of barrier-forming cell-to-cell contacts (tight junctions) in cultured cells grown on porous filters. Although widely accepted, there is currently no device available to automatically measure the time course of TERs under ordinary cell culture conditions (37 degrees C, 5% or 10% CO2). This paper describes a development from our laboratory that is capable of following in parallel the TERs of several filter-grown cell layers with time and in an entirely computer-controlled fashion. The cell cultures can be followed even in long-term experiments without any manual assistance or opening of the incubator Besides reading TER values, this approach also returns the electrical capacitance of the cell layers, which is indicative of the expression of microvilli and other membrane extrusions. The device is based on reading the frequencydependent impedance of the cell layer, followed by equivalent circuit modeling to extract the cell-related parameters. It is compatible with several multi-well formats (up to 96 wells) and controlled by custom-designed software that reads, analyzes, and presents the data.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.     

Synthesis of benzoylpyrimidines as antagonists of the corticotropin-releasing factor-1 receptor

A series of benzoylpyrimidines derived from the anilinepyrimidine CRF(1) antagonists were synthesized. Several synthetic routes were developed to explore the SAR of this series of compounds. Compounds such as 8d (K(i) = 15 nM) exhibited high binding affinities at the human CRF(1) receptor.     

Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.     

Remodeling of the adventitia during coronary arteriogenesis

We studied the role of the adventitia in adaptive arteriogenesis during the phase of active growth of coronary collateral vessels (CV) induced by chronic occlusion of the left circumflex coronary artery in canine hearts. We used electron microscopy and immunoconfocal (IF) labeling for bFGF, matrix metalloproteinase (MMP)-2, MMP-9, tissue-type plasminogen activator (tPA), its inhibitor (PAI-1), fibronectin (FN), and Ki-67. Proliferation of smooth muscle cells and adventitial fibroblasts was evident. Quantitative IF showed that adventitial MMP-2, MMP-9, and FN were 9.2-, 7.5-, and 8.6-fold, bFGF was 5.1-fold, and PAI-1 was 3.4-fold higher in CV than in normal vessels (NV). The number of fibroblasts was 5-fold elevated in CV, but the elastic fiber content was 25-fold greater in NV than in CV. Perivascular myocyte damage and induction of endothelial nitric oxide synthase in peri-CV capillaries indicate expansion of CV. It was concluded that adventitial activation is associated with the development of CV through cell proliferation, production of growth factors, and induction of extracellular proteolysis thereby contributing to remodeling during adaptive arteriogenesis.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.