Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria.

Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus 8 and F. succinogenes S85.     

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3 end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5 flanking region of gene Vst1 contains a TATAA box at nucleotide –48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

雌激素神经保护作用机制:线粒体功能的调节

大量研究表明雌激素具有神经保护作用,但其机制尚不清楚。近年来研究提示,雌激素的神经保护作用与线粒体有着密切联系。线粒体是细胞内能量和活性氧自由基(ROS)的主要来源,对细胞内信号转导、细胞存活与死亡调节等具有十分重要的影响。在生理和病理条件下,雌激素可多方面调节线粒体功能,包括影响ATP与ROS的生成、稳定线粒体膜电位、维护细胞内钙稳态,以及调节线粒体基因和蛋白表达等。本文主要从线粒体角度综述了雌激素神经保护作用及其机制。     

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Nonlinear regulation of unitary synaptic signals by CaV(2.3) voltage-sensitive calcium channels located in dendritic spines

The roles of voltage-sensitive sodium (Na) and calcium (Ca) channels located on dendrites and spines in regulating synaptic signals are largely unknown. Here we use 2-photon glutamate uncaging to stimulate individual spines while monitoring uncaging-evoked excitatory postsynaptic potentials (uEPSPs) and Ca transients. We find that, in CA1 pyramidal neurons in acute mouse hippocampal slices, CaV(2.3) voltage-sensitive Ca channels (VSCCs) are found selectively on spines and act locally to dampen uncaging-evoked Ca transients and somatic potentials. These effects are mediated by a regulatory loop that requires opening of CaV(2.3) channels, voltage-gated Na channels, small conductance Ca-activated potassium (SK) channels, and NMDA receptors. Ca influx through CaV(2.3) VSCCs selectively activates SK channels, revealing the presence of functional Ca microdomains within the spine. Our results suggest that synaptic strength can be modulated by mechanisms that regulate voltage-gated conductances within the spine but do not alter the properties or numbers of synaptic glutamate receptors.     

Deletion of psbJ leads to accumulation of Psb27–Psb28 photosystem II complexes in Thermosynechococcus elongatus

The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27–Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (< 10 kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using ¹⁵N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50 μmol photons m^? 2 s^? 1). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

First Report of Peronospora belbahrii on Sweet Basil in Baja California Sur,México

In Baja California Sur, Mexico, a foliar disease occurred on sweet basil which seriously affected its quality and yield. The most common symptoms were yellowing and necrosis on leaves, caused by a downy mycelium growth on the lower leaf surface. Symptomatic leaves from two sampling sites were collected for morphological studies and molecular analysis of pathogen DNA. Based on morphological characteristics (sporangiophore size of 240–530 × 7–11 μm, branches of 5–8 order and a sporangia size of 27–31 × 21–25 μm) and molecular analysis (the GenBank blast of the PCR assays showed unique rDNA sequence data with 99% similarity to P. belbahrii), the pathogen was identified as Peronospora belbahrii, the causal agent of basil downy mildew. This is the first report of P. belbahrii affecting sweet basil in Mexico.