bluuebunny/crossref_metadata_2025_split huggingface dataset:
A CELD‐fusion method for rapid determination of the DNA‐binding sequence specificity of novel plant DNA‐binding proteins
Authors: Xue Gang‐Ping 
Published: February 2005

SummaryThe current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA‐binding sites, in conjunction with genome‐wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA‐binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA‐binding protein (DBP) to 6×His‐tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP‐DNA complex in the selection of binding sites from a pool of biotinylated random‐sequence oligonucleotides and as a reporter for measurement of DNA‐binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP‐DNA complexes and assays of DNA‐binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high‐throughput capacity of CELD‐based DNA‐binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP‐selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high‐throughput analysis on the DNA‐binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA‐binding domains.
K+ currents through SV‐type vacuolar channels are sensitive to elevated luminal sodium levels
Authors: Ivashikina Natalya , Hedrich Rainer 
Published: February 2005

SummaryNon‐selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch‐clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150‐fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient‐driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.
Reduced neuronal expression of synaptic transmission modulator HNK‐1/neural cell adhesion molecule as a potential consequence of amyloid β‐mediated oxidative stress: a proteomic approach
Authors: Thomas Stefani N. , Soreghan Brian A. , Nistor Mihaela , Sarsoza Floyd , Head Elizabeth , Yang Austin J. 
Published: February 2005

AbstractOxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid β (Aβ) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Aβ‐mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two‐dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Aβ‐induced oxidative stress in cultured primary cortical mouse neurons. The Golgi‐resident enzyme glucuronyltransferase (GlcAT‐P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK‐1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Aβ, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK‐1 expression. Moreover, in vivo, HNK‐1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Aβ‐mediated oxidation of GlcAT‐P is impairment of its enzymatic function, thereby disrupting HNK‐1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK‐1 modulates synaptic plasticity and is critically involved in memory consolidation.
Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol‐3′‐kinase pathway
Authors: Madziar Beata , Lopez‐Coviella Ignacio , Zemelko Victoria , Berse Brygida 
Published: February 2005

AbstractNerve growth factor (NGF) exerts anti‐apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen‐activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up‐regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF‐induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter‐luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF‐induced cholinergic differentiation.
Proteasome inhibition protects HT22 neuronal cells from oxidative glutamate toxicity
Authors: Van Leyen Klaus , Siddiq Ambreena , Ratan Rajiv R. , Lo Eng H. 
Published: February 2005

AbstractOxidative stress caused by glutathione depletion after prolonged exposure to extracellular glutamate leads to a form of neuronal cell death that exhibits morphologically mixed features of both apoptosis and necrosis. However, specific downstream executioners involved in this form of cell death have yet to be identified. We report here that glutamate exposure does not activate caspase‐3 in the HT22 neuronal cell line. Furthermore, no cytoprotection was achieved with either the pan‐caspase inhibitor Z‐VAD‐fmk or the caspase‐3‐specific inhibitor DEVD‐CHO. In contrast, inhibition of the proteasome by lactacystin protected both HT22 cells and rat primary neuronal cells against cell lysis. In parallel, oxidatively altered and ubiquitinated proteins accumulated in the mitochondrial fraction of cells after proteasome inhibition. These findings suggest that caspases can be decoupled from oxidative stress under some conditions, and implicate the ubiquitin/proteasome pathway in neuronal cell death caused by oxidative glutamate toxicity.
A role for caveolin‐1 in post‐injury reactive neuronal plasticity
Authors: Gaudreault Sophie B. , Blain Jean‐François , Gratton Jean‐Philippe , Poirier Judes 
Published: February 2005

AbstractRemodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin‐1 is a cholesterol‐binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up‐regulation of caveolin‐1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin‐1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin‐1‐overexpressing cells revealed increases in synaptophysin and GAP‐43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin‐1‐overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin‐1‐overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin‐1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin‐1 plays an active role in the regulation of injury‐induced synaptic and terminal remodeling in the adult CNS.
Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease
Authors: Clayton Rebecca , Clark John B. , Sharpe Martyn 
Published: February 2005

AbstractApoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all‐or‐none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for ‘buffering’ cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non‐synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage‐dependent anion channel rather than non‐specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.
A gene‐targeted mouse model for chorea‐acanthocytosis
Authors: Tomemori Yuko , Ichiba Mio , Kusumoto Akira , Mizuno Emiko , Sato Daisuke , Muroya Shinji , Nakamura Masayuki , Kawaguchi Hiroaki , Yoshida Hiroki , Ueno Shu‐ichi , Nakao Kazuki , Nakamura Kenji , Aiba Atsu , Katsuki Motoya , Sano Akira 
Published: February 2005

AbstractChorea‐acanthocytosis (CHAC) is a hereditary neurodegenerative disorder with autosomal recessive transmission, in which selective degeneration of striatum has been reported in brain pathology. Clinically, CHAC shows Huntington's disease‐like neuropsychiatric symptoms and red blood cell acanthocytosis. Recently, we identified the gene, CHAC, encoding a novel protein, chorein, in which a deletion mutation was found in Japanese families with CHAC. In the present study, we have identified the mouse CHAC cDNA sequence and the exon–intron structures of the gene and produced a CHAC model mouse introducing no. 60–61 exon deletion corresponding to a human disease mutation by a gene‐targeting technique. The mice began to show acanthocytosis and motor disturbance in old age. In behavioral observations, locomotor activity was significantly decreased and the contact time at social interaction test was decreased significantly in the model mice. In the brain pathology, many apoptotic cells were observed in the striatum of the mutant mice. In neurochemical determinations, the dopamine metabolite, homovanillic acid, concentration decreased significantly in the portion including the midbrain of the mutant mice. These findings are consistent with the human results reported elsewhere and indicate that the CHAC model mice showed a mild phenotype with late adult onset. The CHAC model mouse therefore provides a good model system to study the human disease.
Identification of autotaxin as a neurite retraction‐inducing factor of PC12 cells in cerebrospinal fluid and its possible sources
Authors: Sato Koichi , Malchinkhuu Enkhzol , Muraki Takeshi , Ishikawa Koichi , Hayashi Kensuke , Tosaka Masahiko , Mochiduki Akikazu , Inoue Kinji , Tomura Hideaki , Mogi Chihiro , Nochi Hiromi , Tamoto Koichi , Okajima Fumikazu 
Published: February 2005

AbstractCerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.
Mutational studies using a cation‐conducting GABAA receptor reveal the selectivity determinants of the Cys‐loop family of ligand‐gated ion channels
Authors: Jensen Marianne L. , Pedersen Lone N. , Timmermann Daniel B. , Schousboe Arne , Ahring Philip K. 
Published: February 2005

AbstractThe present study evaluates how four key amino acid residue positions (− 4′ to − 1′) within the M1–M2 linker of the GABAA receptor β subunit influences ion selectivity of a cation‐conducting GABA receptor. Cation selectivity was found to be highly dependent on the side‐chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the − 1′ position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a −1′ Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1–M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three‐dimensional position of the −1′ Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction.
Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity
Authors: Chai Yunrong , Winans Stephen C. 
Published: February 2005

ABSTRACTTraR ofAgrobacterium tumefaciensis a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations oftraRand identified two constitutive alleles. Surprisingly, neither contained a point mutation within thetraRgene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycosideN-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library oftraRalleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed inEscherichia coliand showed detectabletrabox binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases.
Identification and Characterization of a Novel Allele of
Escherichia coli dnaB
Helicase That Compromises the Stability of Plasmid P1
Authors: Slavcev Roderick A. , Funnell Barbara E. 
Published: February 2005

ABSTRACT

Bacteriophage P1 lysogenizes
Escherichia coli
cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for
E. coli
chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the
dnaB
helicase gene (
dnaB277
) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host;
dnaB277
cells were not viable at temperatures above 34°C. Shifting
dnaB277
cells to 42°C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The
dnaB277
allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.
Weighted Genome Trees: Refinements and Applications
Authors: Gophna Uri , Doolittle W. Ford , Charlebois Robert L. 
Published: February 2005

ABSTRACTThere are many ways to group completed genome sequences in hierarchical patterns (trees) reflecting relationships between their genes. Such groupings help us organize biological information and bear crucially on underlying processes of genome and organismal evolution. Genome trees make use of all comparable genes but can variously weight the contributions of these genes according to similarity, congruent patterns of similarity, or prevalence among genomes. Here we explore such possible weighting strategies, in an analysis of 142 prokaryotic and 5 eukaryotic genomes. We demonstrate that alternate weighting strategies have different advantages, and we propose that each may have its specific uses in systematic or evolutionary biology. Comparisons of results obtained with different methods can provide further clues to major events and processes in genome evolution.
Reconstitution of O-Specific Lipopolysaccharide Expression in
Burkholderia cenocepacia
Strain J2315, Which Is Associated with Transmissible Infections in Patients with Cystic Fibrosis
Authors: Ortega Ximena , Hunt Tracey A. , Loutet Slade , Vinion-Dubiel Arlene D. , Datta Anup , Choudhury Biswa , Goldberg Joanna B. , Carlson Russell , Valvano Miguel A. 
Published: February 2005

ABSTRACT

Burkholderia cenocepacia
is an opportunistic bacterium that infects patients with cystic fibrosis.
B. cenocepacia
strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS
402
interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two
N
-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.