bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family
Authors: WORMIT Alexandra , TRAUB Michaela , FLÖRCHINGER Martin , NEUHAUS H. Ekkehard , MÖHLMANN Torsten 
Published: September 2004

Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past. This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases. To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides. Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA. Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7). After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent Km values were in the range 3–94 μM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases. Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins. Transport mediated by the AtENT isoforms differed in pH-dependency, e.g. AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent. Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed.
In brain mitochondria the branched-chain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition
Authors: SCHÖNFELD Peter , KAHLERT Stefan , REISER Georg 
Published: September 2004

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) accumulates at high levels throughout the body in the adult form of Refsum disease, a peroxisomal genetic disorder. However, it is still unclear why increased levels of phytanic acid have cytotoxic effects. In the present study, we examined the influence of non-esterified phytanic acid on energy-related functions of mitochondria from adult rat brain. Phytanic acid at low concentrations (5–20 μM, i.e. 5–20 nmol/mg of mitochondrial protein) de-energized mitochondria, as indicated by depolarization, stimulation of non-phosphorylating oxygen uptake and inhibition of the reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. The unbranched homologue palmitic acid exerted much smaller effects. In addition, phytanic acid reduced state 3 respiration, which was partly due to inhibition of the ADP/ATP carrier. Phytanic acid decreased the rate of adenine nucleotide exchange and increased the degree of control, which the ADP/ATP carrier has on state 3 respiration. Important for functional consequences is the finding that mitochondria, which are preloaded with small amounts of Ca2+ (100 nmol/mg of protein), became highly sensitized to rapid permeability transition even when only low concentrations of phytanic acid (below 5 μM) were applied. In conclusion, the incorporation of phytanic acid into the inner mitochondrial membrane increases the membrane H+ conductance and disturbs the protein-linked functions in energy coupling. This is most probably essential for the short-term toxicity of phytanic acid. Thus in neural tissue, which becomes enriched with phytanic acid, the reduction in mitochondrial ATP supply and the facilitation of the opening of the permeability transition pore are two major mechanisms by which the branched-chain fatty acid phytanic acid induces the onset of degenerative processes.
A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2
Authors: MAMPEL Jörg , MAIER Elke , TRALAU Tewes , RUFF Jürgen , BENZ Roland , COOK Alasdair M. 
Published: September 2004

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the inner membrane. tsaT was identified as a 1017-bp ORF (open reading frame) on plasmid pTSA upstream of the TSA-catabolic genes in the tsa operon. Expression of tsaT was regulated by TsaR, the transcriptional activator of the tsa regulon. The presence of tsaT was concomitant with the presence of the tsa operon in different TSA-degrading isolates. tsaT was expressed in Escherichia coli and was detected in the outer membrane. A 22-amino-acid leader peptide was identified. Purified protein reconstituted in lipid bilayer membranes formed anion-selective channels with a single-channel conductance of 3.5 nS in 1 M KCl. Downstream of tsaT, a constitutively expressed 720-bp ORF (tsaS) was identified. tsaS coded for a hydrophobic protein predicted to have six transmembrane helices and which is most likely localized in the cytoplasmic membrane. tsaS is adjacent to tsaT, but showed a different transcriptional profile.
cAMP-response element modulator-τ activates a distinct promoter element for the expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene
Authors: TRAMER Federica , VETERE Amedeo , MARTINELLI Monica , PARONI Federico , MARSICH Eleonora , BOITANI Carla , SANDRI Gabriella , PANFILI Enrico 
Published: September 2004

PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a selenoprotein present in at least three isoforms in testis: cytosolic, mitochondrial and nuclear. All of these derive from the same gene and are structurally related with the exception of the snPHGPx (sperm nucleus-specific form), which differs from the others due to the presence of an arginine-rich N-terminus. It has been demonstrated recently that this N-terminus is encoded by an alternative exon located in the first intron of the PHGPx gene. The expression of snPHGPx has been attributed either to an alternative pre-mRNA splicing or to the presence of a distinct promoter region. Nevertheless, the exact molecular mechanism by which the expression of snPHGPx occurs has not been demonstrated so far. Preliminary sequence analysis of the region located upstream of the alternative exon revealed some potential DNA-binding sites, one of which is specific to the binding of CREM (cAMP-response element modulator) transcription factors. By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-τ protein can specifically bind to this element. Furthermore, we cloned a 1059 bp comprising the intron and the alternative exon for snPHGPx in the pCAT®3 reporter vector. By transient transfection experiments, we demonstrated that the expression of the transcription factor CREM-τ can induce the activation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments performed on highly purified rat spermatid cells. On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-τ, which acts as a cis-acting element localized in the first intron of the PHGPx gene.
Diminished L-arginine bioavailability in hypertension
Authors: MOSS Monique B. , BRUNINI Tatiana M. C. , SOARES de MOURA Roberto , NOVAES MALAGRIS Lúcia E. , ROBERTS Norman B. , ELLORY J. Clive , MANN Giovanni E. , MENDES RIBEIRO Antônio C. 
Published: September 2004

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175±19 μmol/l) compared with control subjects (137±8 μmol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60±7 μmol·l−1·cells−1·h−1; n=16) compared with controls (90±17 μmol·l−1·cells−1·h−1; n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86±17 pmol·109 cells−1·min−1 in controls compared with 36±9 pmol·109 cells−1·min−1 in hypertensive patients (n=10; P&lt;0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12±0.02 pmol/108 cells; n=8) compared with controls (0.22±0.01 pmol/108 cells; n=8; P&lt;0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.
Tumour cell growth in culture: dependence on arginine
Authors: CASO Giuseppe , McNURLAN Margaret A. , McMILLAN Nelson D. , EREMIN Oleg , GARLICK Peter J. 
Published: September 2004

The amino acid arginine has been shown to affect the growth of several tumours, although the mechanisms of its action are not clear. In the present study, using a human breast tumour cell line (MCF-7), we investigated the arginine requirements of tumour cells for optimal protein synthesis and growth, and the metabolic pathway responsible for the arginine-dependent growth. The results showed that MCF-7 cells are highly dependent on arginine for growth and that the requirement for arginine is much higher than for an indispensable amino acid, leucine, indicating that arginine is needed for pathways other than protein synthesis. In arginine-free cultures, growth could be completely restored by the urea cycle intermediate citrulline. However, arginine could not be replaced by the urea cycle intermediate and the direct precursor for polyamine synthesis, ornithine, or by the polyamine putrescine, suggesting that the high dependence on arginine is not due to a requirement for polyamine synthesis. Moreover, inhibition of NOS [NO (nitric oxide) synthase] did not affect cell protein synthesis and growth, and the arginine analogue and substrate for NOS, homoarginine, could not replace arginine, implying that the conversion of arginine into NO is not involved in the growth-promoting effects of arginine. The major determinant for the high dependence of MCF-7 cells for arginine was found to be the irreversible conversion of this amino acid into ornithine by the intracellular enzyme arginase. The conversion into ornithine caused a progressive depletion of arginine from the culture medium, which ultimately inhibited cell protein synthesis and halted growth. Intracellular arginase activity may be the major factor determining the requirement for arginine of all cells in culture.
Weight loss leads to reductions in inflammatory biomarkers after a very-low-carbohydrate diet and a low-fat diet in overweight men
Authors: SHARMAN Matthew J. , VOLEK Jeff S. 
Published: September 2004

In recent years, it has become apparent that low-grade vascular inflammation plays a key role in all stages of the pathogenesis of atherosclerosis. Weight loss has been shown to improve blood inflammatory markers; however, it is unknown if weight-loss diets varying in macronutrient composition differentially affect inflammatory responses. The primary purpose of the present study was to compare a very-low-carbohydrate diet and a low-fat weight-loss diet on inflammatory biomarkers in overweight men. In a randomized cross-over design, 15 overweight men (body fat, &gt;25%; body mass index, 34 kg/m2) consumed two experimental weight-loss diets for two consecutive 6-week periods: a very-low-carbohydrate diet (&lt;10% energy via carbohydrate) and a low-fat diet (&lt;30% energy via fat). Both the low-fat and the very-low-carbohydrate diets resulted in significant decreases in absolute concentrations of hsTNF-α (high-sensitivity tumour necrosis factor-α), hsIL-6 (high-sensitivity interleukin-6), hsCRP (high-sensitivity C-reactive protein) and sICAM-1 (soluble intercellular cell-adhesion molecule-1). There was no significant change in absolute sP-selectin (soluble P-selectin) concentrations after either diet. Normalized inflammatory values represented as the delta change per 1 kg reduction in body mass showed a significant difference between the two diets only for sP-selectin (P&lt;0.05). In summary, energy-restricted low-fat and very-low-carbohydrate diets both significantly decreased several biomarkers of inflammation. These data suggest that, in the short-term, weight loss is primarily the driving force underlying the reductions in most of the inflammatory biomarkers.
Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification
Authors: Yanai Itai , Benjamin Hila , Shmoish Michael , Chalifa-Caspi Vered , Shklar Maxim , Ophir Ron , Bar-Even Arren , Horn-Saban Shirley , Safran Marilyn , Domany Eytan , Lancet Doron , Shmueli Orit 
Published: September 2004

Abstract
Motivation: Genes are often characterized dichotomously as either housekeeping or single-tissue specific. We conjectured that crucial functional information resides in genes with midrange profiles of expression.
Results: To obtain such novel information genome-wide, we have determined the mRNA expression levels for one of the largest hitherto analyzed set of 62 839 probesets in 12 representative normal human tissues. Indeed, when using a newly defined graded tissue specificity index τ, valued between 0 for housekeeping genes and 1 for tissue-specific genes, genes with midrange profiles having 0.15 &lt; τ &lt; 0.85 were found to constitute &gt;50% of all expression patterns. We developed a binary classification, indicating for every gene the IB tissues in which it is overly expressed, and the 12 − IB tissues in which it shows low expression. The 85 dominant midrange patterns with IB = 2–11 were found to be bimodally distributed, and to contribute most significantly to the definition of tissue specification dendrograms. Our analyses provide a novel route to infer expression profiles for presumed ancestral nodes in the tissue dendrogram. Such definition has uncovered an unsuspected correlation, whereby de novo enhancement and diminution of gene expression go hand in hand. These findings highlight the importance of gene suppression events, with implications to the course of tissue specification in ontogeny and phylogeny.
Availability: All data and analyses are publically available at the GeneNote website, http://genecards.weizmann.ac.il/genenote/ and, GEO accession GSE803.
Contact: doron.lancet@weizmann.ac.il
Supplementary information: Four tables available at the above site.
APART: Automated Preprocessing for NMR Assignments with Reduced Tedium
Authors: Pawley Norma H. , Gans Jason D. , Michalczyk Ryszard 
Published: September 2004

Abstract
Motivation: High-throughput NMR structure determination is a goal that will require progress on many fronts, one of which is rapid resonance assignment. An important rate-limiting step in the resonance assignment process is accurate identification of resonance peaks in the NMR spectra. Peak-picking schemes range from incomplete (which lose essential assignment connectivities) to noisy (which obscure true connectivities with many false ones). We introduce an automated preassignment process that removes false peaks from noisy peak lists by requiring consensus between multiple NMR experiments and exploiting a priori information about NMR spectra. This process is designed to accept multiple input formats and generate multiple output formats, in an effort to be compatible with a variety of user preferences.
Results: Automated preprocessing with APART rapidly identifies and removes false peaks from initial peak lists, reduces the burden of manual data entry, and documents and standardizes the peak filtering process. Successful preprocessing is demonstrated by the increased number of correct assignments obtained when data are submitted to an automated assignment program.
Availability: APART is available from http://sir.lanl.gov/NMR/APART.htm
Contact: npawley@lanl.gov; rmichalczyk@lanl.gov
Supplementary information: Manual pages with installation instructions, procedures and screen shots can also be found at http://sir.lanl.gov/NMR/APART_Manual1.pdf
A key role for ALD1 in activation of local and systemic defenses in Arabidopsis
Authors: Song Jong Tae , Lu Hua , McDowell John M. , Greenberg Jean T. 
Published: September 2004

SummaryThe Arabidopsis thaliana agd2‐like defense response protein1 (ald1) mutant was previously found to be hypersusceptible to the virulent bacterial pathogen Pseudomonas syringae and had reduced accumulation of the defense signal salicylic acid (SA). ALD1 was shown to possess aminotransferase activity in vitro, suggesting it generates an amino acid‐derived defense signal. We now find ALD1 to be a key defense component that acts in multiple contexts and partially requires the PHYTOALEXIN DEFICIENT4 (PAD4) defense regulatory gene for its expression in response to infection. ald1 plants have increased susceptibility to avirulent P. syringae strains, are unable to activate systemic acquired resistance and are compromised for resistance to the oomycete pathogen Peronospora parasitica in mutants with constitutively active defenses. ALD1 and PAD4 can act additively to control SA, PATHOGENESIS RELATED GENE1 (PR1) transcript and camalexin (an antimicrobial metabolite) accumulation as well as disease resistance. Finally, ALD1 and PAD4 can mutually affect each other's expression in a constitutive defense mutant, suggesting that these two genes can act in a signal amplification loop.
Expression of RPS4 in tobacco induces an AvrRps4‐independent HR that requires EDS1, SGT1 and HSP90
Authors: Zhang Yan , Dorey Stephan , Swiderski Michal , Jones Jonathan D. G. 
Published: September 2004

SummaryThe ArabidopsisRPS4 gene belongs to the Toll/interleukin‐1 receptor/nucleotide‐binding site/leucine‐rich repeat (TIR‐NB‐LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4‐independent hypersensitive response (HR). The same phenotype is seen after expression of a full‐length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P‐loop motif of the NB domain abolish the HR. Using virus‐induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.
The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins
Authors: Mori Takashi , Maruyama Nobuyuki , Nishizawa Keito , Higasa Takahiko , Yagasaki Kazuhiro , Ishimoto Masao , Utsumi Shigeru 
Published: September 2004

SummaryGlycinin (11S) and β‐conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi‐derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER‐derived protein bodies (PBs) with a diameter of 0.3–0.5 μm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.
Novel cell wall architecture of isoxaben‐habituated Arabidopsis suspension‐cultured cells: global transcript profiling and cellular analysis
Authors: Manfield Iain W. , Orfila Caroline , McCartney Lesley , Harholt Jesper , Bernal Adriana J. , Scheller Henrik Vibe , Gilmartin Philip M. , Mikkelsen Jørn D. , Paul Knox J. , Willats William G. T. 
Published: September 2004

SummaryThe herbicide isoxaben is a highly specific and potent inhibitor of cellulose synthesis in plants. Nevertheless, suspension‐cultured cells can be habituated to grow in high concentrations of isoxaben, and apparently compensate for the disruption of cellulose synthesis by the modulation of other cell wall components. We have habituated Arabidopsis cells to isoxaben and characterized the cellular and genetic consequences. Near whole‐genome transcript profiling implicated novel genes in cell wall assembly and extended our understanding of the activity of known cell wall‐related genes including glycosyltransferases involved in cellulose and pectin biosynthesis. Habituation does not appear to be mediated by stress response processes, nor by functional redundancy within the cellulose synthase (AtCesA) family. Uniquely, amongst the cellulose synthase superfamily, AtCslD5 was highly upregulated and may play a role in the biosynthesis of the novel walls of habituated cells. In silico analysis of differentially expressed genes with unknown functions identified a putative glycosyltransferase and collagen‐like putative cell wall protein.