bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tonoplast vesicles of Beta vulgaris storage root show functional aquaporins regulated by protons
Authors: Sutka Moira , Alleva Karina , Parisi Mario , Amodeo Gabriela 
Published: October 2005

Background information. Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re‐evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation.Results. An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped‐flow technique. The preparation showed high water permeability (485 μm·s−1), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl2 (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western‐blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition.Conclusions. Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl2, not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell‐to‐cell pathway.
Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage
Authors: Wu Xiaoming , Yang Zhengguan , Liu Yiyong , Zou Yue 
Published: October 2005

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.
Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence
Authors: Solomaha Elena , Palfrey H. Clive 
Published: October 2005

The effects of guanine nucleotides on the intrinsic and extrinsic fluorescence properties of dynamin were assessed. The intrinsic Trp (tryptophan) fluorescence spectra of purified recombinant dynamin-1 and -2 were very similar, with a maximum at 332 nm. Collisional quenching by KI was weak (∼30%), suggesting that the majority of Trp residues are buried. Binding of guanine nucleotides decreased intrinsic fluorescence by 15–20%. Titration of the effects showed that GTP and GDP bound to a single class of non-interacting sites in dynamin tetramers with apparent dissociation constants (Kd) values of 5.4 and 7.4 μM (dynamin-1) and 13.2 and 7.1 μM (dynamin-2) respectively. Similar dissociation constant values for both nucleotides were obtained by titrating the quenching of IAEDANS [N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine]-labelled dynamin-2. Despite the similar binding affinities, GTP and GDP result in different conformations of the protein, as revealed by sensitivity to proteinase K fragmentation. Dynamins contain five Trp residues, of which four are in the PH domain (pleckstrin homology domain) and one is in the C-terminal PRD (proline/arginine-rich domain). Guanine nucleotides quenched fluorescence emission from a truncated (ΔPRD) mutant dynamin-1 to the same extent as in the full-length protein, suggesting conformational coupling between the G (groove)-domain and the PH domain. Efficient resonance energy transfer from PH domain Trp residues to bound mant-GTP [where mant stands for 2′-(3′)-O-(N-methylanthraniloyl)] suggests that the G-domain and PH domain are in close proximity (5–6 nm). Promotion of dynamin-2 oligomerization, by reduction in ionic strength or increasing protein concentration, had little effect on intrinsic dynamin fluorescence. However, fluorescence emission from IAEDANS·dynamin-2 showed a significant spectral shift on oligomerization. In addition, energy transfer was observed when oligomerization was promoted in mixtures of IAEDANS·dynamin-2 and 4-(4-dimethylaminophenylazo)benzoic acid-coupled dynamin-2, an effect that was counteracted by GTP but not GDP.
Molecular cloning and characterization of PLC-η2
Authors: Zhou Yixing , Wing Michele R. , Sondek John , Harden T. Kendall 
Published: October 2005

PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-η2, was cloned and characterized. Most of the coding sequence of PLC-η2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5′-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-η2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-η2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-η2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-η2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-η2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-η2, each containing possible alternatively spliced first exons. Co-expression of PLC-η2 with Gβ1γ2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-η2 may in part function downstream of G-protein-coupled receptors.
Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-κB in A2A adenosine receptor induction
Authors: Murphree Lauren J. , Sullivan Gail W. , Marshall Melissa A. , Linden Joel 
Published: October 2005

The A2A adenosine receptor (A2AAR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (&lt;2-fold) increase in the expression of A2AAR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMΦ (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a &gt;100-fold increase in A2AAR mRNA. LPS-induced increases in mRNA for A2AAR and TNFα (tumour necrosis factor α) are reduced by 90% in IPMΦ pretreated with the NF-κB (nuclear factor κB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 μM}. In Wehi-3 cells exposed to LPS, A2AAR and A2BAR transcripts are elevated by 290- and 10-fold respectively, the A1AR transcript is unchanged and the A3AR transcript is decreased by 67%. The induction of A2AAR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A2AAR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors·cell−1·min−1 to a Bmax of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A2A agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A2A agonist, ATL146e, to reduce TNFα release from IPMΦ was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A2AAR, is part of a delayed feedback mechanism initiated through NF-κB to terminate the activation of human and mouse macrophages.
N-Acetylcysteine ameliorates the late phase of liver ischaemia/reperfusion injury in the rabbit with hepatic steatosis
Authors: Fusai Giuseppe , Glantzounis George K. , Hafez Tarek , Yang Wenxuan , Quaglia Alberto , Sheth Hemant , Kanoria Sanjeev , Parkes Harry , Seifalian Alexander , Davidson Brian R. 
Published: October 2005

Steatotic livers are highly susceptible to I/R (ischaemia/reperfusion) injury and, therefore, the aim of the present study was to evaluate the in vivo effect of NAC (N-acetylcysteine) on hepatic function in the early and initial late phase of warm liver I/R injury in steatotic rabbits. Twelve New Zealand White rabbits were fed a high-cholesterol (2%) diet. The control group (n=6) underwent lobar liver ischaemia for 1 h, followed by 6 h of reperfusion. In the treated group receiving NAC (n=6), an intravenous infusion of NAC was administered prior to and during the 6 h reperfusion period. Systemic and hepatic haemodynamics were monitored continuously. ALT (alanine aminotransferase) activity and bile production were measured. NMR spectroscopy was used to analyse bile composition. Oxidation of DHR (dihydrorhodamine) to RH (rhodamine) was used as a marker of production of reactive oxygen and nitrogen species. Moderate centrilobular hepatic steatosis was demonstrated by histology. The results showed that NAC administration significantly improved portal flow, hepatic microcirculation, bile composition and bile flow after 5 h of reperfusion. NAC administration was also associated with less hepatocellular injury, as indicated by ALT serum activity, and decreased the oxidation of DHR to RH. In conclusion, NAC administration decreased the extent of I/R injury in the steatotic liver, particularly during the late phase of reperfusion.
Proteomic approach to identify changes in protein expression modified by 17β-oestradiol in bovine vascular smooth muscle cells
Authors: Molero Laura , García-Méndez Antonio , Alonso-Orgaz Sergio , Carrasco Carolina , Macaya Carlos , López Farré Antonio J. 
Published: October 2005

The aim of the present study was to use proteomics to analyse modifications in the level of expression of different proteins in BVSMCs (bovine vascular smooth muscle cells) incubated in the absence and presence of 17β-oestradiol. By using two-dimensional electrophoresis with a pH range of 4–7, we identified several areas on the gels in which the level of expression of proteins were different between control BVSMCs and cells incubated for 24 h with 17β-oestradiol. Changes in several isoforms of α-enolase, HSP60 (heat-shock protein 60), vimentin and PDI (protein disulphide-isomerase) were observed in BVSMCs. The expression of α-enolase isoform 1 was enhanced after 17β-oestradiol treatment. The expression of HSP60 isoform 3, vimentin isoforms 2 and 3 and caldesmon was reduced by 17β-oestradiol. Finally, the expression of PDI isoforms was reduced by 17β-oestradiol. In summary, 17β-oestradiol modified the expression of isoforms of proteins associated with smooth muscle cell proliferation (α-enolase, vimentin and HSP-60), cell contraction (vimentin and caldesmon) and cell redox modulation (PDI). These findings confirm that 17β-oestradiol may modulate a wide range of signalling pathways in vascular smooth muscle cells.
L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic β-cell line and protects from pro-inflammatory cytokine-induced apoptosis
Authors: Cunningham Grainne A. , Mcclenaghan Neville H. , Flatt Peter R. , Newsholme Philip 
Published: October 2005

Acute effects of nutrient stimuli on pancreatic β-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic β-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (NG-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] was additionally assessed by flow cytometry. Culture for 24 h with 10 mM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD11 cells. Sixty-six genes were up-regulated &gt;1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD11 cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in β-cell function in vivo.
Temporal Mitochondrial DNA Variation in the Basque Country: Influence of Post‐Neolithic Events
Authors: Alzualde A. , Izagirre N. , Alonso S. , Alonso A. , De La Rúa C. 
Published: October 2005

SummaryThe Basque population has been considered an outlier in a large number of genetic studies, due to its hypothesized antiquity and greater genetic isolation. The present paper deals with an analysis of the mtDNA variability of the historical population of Aldaieta (VI–VII c. AD; Basque Country) which, together with genetic data existing for other prehistoric populations of the Basque Country (4,500–5,000 YBP), permits an appraisal of the hypotheses proposed for the origin of the genetic differentiation of the Basque population. Given that this is an aDNA study, application has been made both of standard precautions, to avoid contamination, and of authentication criteria (analysis of duplicates, replication in an independent laboratory, quantification of target DNA, sequencing and cloning of PCR products). The variability of the mtDNA haplogroups of the historical population of Aldaieta falls within the range of the present‐day populations of Europe's Atlantic fringe, whereas the prehistoric populations of the Basque Country display clear differentiation in relation to all others. Consequently, we suggest that between 5,000–1,500 YBP approximately, there may have been gene flow amongst the western European populations that homogenised mtDNA lineages.
Validation of the Spanish version of the physical activity questionnaire used in the Nurses' Health Study and the Health Professionals' Follow-up Study
Authors: Martínez-González Miguel Angel , López-Fontana Constanza , Varo José Javier , Sánchez-Villegas Almudena , Martinez J Alfredo 
Published: October 2005

AbstractObjectiveThe objective of this analysis was to test the validity of the estimates of energy expenditure and sedentary lifestyle obtained through a self-administered questionnaire of physical activity for Spanish-speaking people adapted from US questionnaires (Nurses' Health Study and Health Professionals' Follow-up Study) using a triaxial accelerometer (RT3 Triaxial Research Tracker) as the reference.Design and setting: Validation study, calculating the non-parametric correlation coefficients between the level of physical activity and sedentary lifestyle collected by the self-administered questionnaire and the triaxial accelerometer measurements. Percentage of misclassification and kappa coefficients were also calculated.SubjectsThe study population consisted of a sample of 40 obese women who were participants of the SUN (Seguimiento Universidad de Navarra) project (a prospective cohort study among Spanish university alumni). They were selected because of their peculiar metabolic characteristics, in the search for a sub-optimal scenario for validity.ResultsPhysical activity during leisure time (estimated as MET-h week-1) derived from the self-administered questionnaire moderately correlated with kcal day-1 assessed through the accelerometer (Spearman's ρ = 0.507, 95% confidence interval (CI) 0.232, 0.707). The Spearman correlation between the ratio of sedentary lifestyle to physical activity obtained through the questionnaire and the direct estimation (RT3) was -0.578 (95% CI -0.754, -0.325). The kappa index was 0.25 (P = 0.002) when assessing the cross-classification into quintiles and 0.41 for the dichotomous estimation of a sedentary lifestyle. Only 2.5% of participants were misclassified by the questionnaire more than two quintiles apart from the estimates of the RT3.ConclusionsThe moderate values obtained for correlation in a sub-optimal scenario for validity and the low percentage of extreme misclassification suggest the validity of the questionnaire to assess physical activity in Spanish-speaking women aged 20–50 years.
Large-scale prokaryotic gene prediction and comparison to genome annotation
Authors: Nielsen Pernille , Krogh Anders 
Published: October 2005

Abstract
Motivation: Prokaryotic genomes are sequenced and annotated at an increasing rate. The methods of annotation vary between sequencing groups. It makes genome comparison difficult and may lead to propagation of errors when questionable assignments are adapted from one genome to another. Genome comparison either on a large or small scale would be facilitated by using a single standard for annotation, which incorporates a transparency of why an open reading frame (ORF) is considered to be a gene.
Results: A total of 143 prokaryotic genomes were scored with an updated version of the prokaryotic genefinder EasyGene. Comparison of the GenBank and RefSeq annotations with the EasyGene predictions reveals that in some genomes up to ∼60% of the genes may have been annotated with a wrong start codon, especially in the GC-rich genomes. The fractional difference between annotated and predicted confirms that too many short genes are annotated in numerous organisms. Furthermore, genes might be missing in the annotation of some of the genomes. We predict 41 of 143 genomes to be over-annotated by &gt;5%, meaning that too many ORFs are annotated as genes. We also predict that 12 of 143 genomes are under-annotated. These results are based on the difference between the number of annotated genes not found by EasyGene and the number of predicted genes that are not annotated in GenBank.
We argue that the average performance of our standardized and fully automated method is slightly better than the annotation.
Availability: The EasyGene 1.2 predictions and statistics can be accessed at
Contact: pern@binf.ku.dk
GSMA: software implementation of the genome search meta-analysis method
Authors: Pardi Fabio , Levinson Douglas F. , Lewis Cathryn M. 
Published: October 2005

Abstract
Meta-analysis can be used to pool results of genome-wide linkage scans. This is of great value in complex diseases, where replication of linked regions occurs infrequently. The genome search meta-analysis (GSMA) method is widely used for this analysis, and a computer program is now available to implement the GSMA.
Availability:
Contact: Cathryn.lewis@genetics.kcl.ac.uk
ISER: selection of differentially expressed genes from DNA array data by non-linear data transformations and local fitting
Authors: Drummond R. D. , Pinheiro A. , Rocha C. S. , Menossi M. 
Published: October 2005

AbstractSummary: This report describes an algorithm (intensity-dependent selection of expression ratios or ISER) developed to analyse DNA array data by optimizing the selection of genes with the most significant variations in expression amongst two RNA samples. The algorithm is designed for use when little or no replication of array hybridizations is available.Availability: ISER is written in R language, and its code and on-line version are freely available atContact: menossi@unicamp.brSupplementary information: