bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Regulation of the Arabidopsis High-Affinity Root Iron Uptake System by Local and Long-Distance Signals
Authors: Vert Grégory A. , Briat Jean-François , Curie Catherine
Published: June 2003

Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.
Analysis of Xyloglucan Fucosylation in Arabidopsis
Authors: Perrin Robyn M. , Jia Zhonghua , Wagner Tanya A. , O'Neill Malcolm A. , Sarria Rodrigo , York William S. , Raikhel Natasha V. , Keegstra Kenneth
Published: June 2003

Xyloglucan (XyG) is a load-bearing primary wall component in dicotyledonous and non-graminaceous monocotyledonous plants. XyG fucosyltransferase (FUTase), encoded by the Arabidopsis gene AtFUT1, directs addition of fucose (Fuc) residues to terminal galactose residues on XyG side chains. Reverse transcription-polymerase chain reaction and analysis of promoter-β-glucuronidase transgenic plants indicated highest expression of AtFUT1 in the upper portion of elongating inflorescence stems of Arabidopsis. XyG FUTase activity was highest in Golgi vesicles prepared from growing Arabidopsis tissues and low in those isolated from mature tissues. There was no discernible correlation between the Fuc contents of XyG oligosaccharides derived from different Arabidopsis organs and the level of AtFUT1 expression in the organs. Thus, organ-specific variations in AtFUT1 expression and enzyme activity probably reflect differential rates of cell wall biosynthesis, rather than differences in levels of XyG fucosylation. The effects of manipulating AtFUT1 expression were examined using an Arabidopsis mutant (atfut1) containing a T-DNA insertion in the AtFUT1 locus and transgenic plants with strong constitutive expression of AtFUT1. No Fuc was detected in XyG derived from leaves or roots of atfut1. Plants overexpressing AtFUT1 had higher XyG FUTase activity than wild-type plants, but the XyG oligosaccharides derived from the transgenic and wild-type plants contained comparable amounts of Fuc, indicating that suitable acceptor substrates are limiting. Galactosyl residues had slightly higher levels of O-acetylation in XyG from plants that overexpressed AtFUT1 than in XyG from wild-type plants. O-Acetylation of galactose residues was considerably reduced in Fuc-deficient mutants (atfut1, mur1, and mur2) that synthesize XyG containing little or no Fuc. These results suggest that fucosylated XyG is a suitable substrate for at least one O-acetyltransferase in Arabidopsis.
Seed-Specific Overexpression of an Endogenous Arabidopsis Phytoene Synthase Gene Results in Delayed Germination and Increased Levels of Carotenoids, Chlorophyll, and Abscisic Acid
Authors: Lindgren L. Ove , Stålberg Kjell G. , Höglund Anna-Stina
Published: June 2003

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of β-carotene and a total average amount of β-carotene of approximately 260 μg g–  1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and α-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.
The Identification of Candidate Genes for a Reverse Genetic Analysis of Development and Function in the Arabidopsis Gynoecium
Authors: Scutt Charles P. , Vinauger-Douard Marion , Fourquin Chloé , Ailhas Jérôme , Kuno Norihito , Uchida Kenko , Gaude Thierry , Furuya Masaki , Dumas Christian
Published: June 2003

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.
A Role for the GCC-Box in Jasmonate-Mediated Activation of the PDF1.2 Gene of Arabidopsis
Authors: Brown Rebecca L. , Kazan Kemal , McGrath Ken C. , Maclean Don J. , Manners John M.
Published: June 2003

The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the β-glucoronidase-reporter gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted, we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a 20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box.
Rapid, Noninvasive Screening for Perturbations of Metabolism and Plant Growth Using Chlorophyll Fluorescence Imaging
Authors: Barbagallo Romina P. , Oxborough Kevin , Pallett Kenneth E. , Baker Neil R.
Published: June 2003

A rapid, noninvasive technique involving imaging of chlorophyll fluorescence parameters for detecting perturbations of leaf metabolism and growth in seedlings is described. Arabidopsis seedlings were grown in 96-well microtitre plates for 4 d and then treated with eight herbicides with differing modes of action to induce perturbations in a range of different metabolic processes. Imaging of chlorophyll fluorescence emissions from 96 seedlings growing on a microtitre plate enabled images of a number of fluorescence parameters to be rapidly and simultaneously produced for the plants in each well. Herbicideinduced perturbations in metabolism, even in metabolic reactions not directly associated with photosynthetic metabolism, were detected from the changes in the images of fluorescence parameters considerably before any visual effects on seedling growth were observed. Evaluations of seedling growth were made from measurements of the area of chlorophyll fluorescence emission in images of plants growing in the 96-well plates. Decreased seedling growth related directly to herbicideinduced changes in the imaged chlorophyll fluorescence parameters. The applicability of this rapid-screening technique for metabolic perturbations in monocotyledonous species was demonstrated by treating Agrostis tenuis seedlings with Imazapyr, an inhibitor of branched-chain amino acid synthesis.
Refined Annotation of the Arabidopsis Genome by Complete Expressed Sequence Tag Mapping
Authors: Zhu Wei , Schlueter Shannon D. , Brendel Volker
Published: June 2003

Expressed sequence tags (ESTs) currently encompass more entries in the public databases than any other form of sequence data. Thus, EST data sets provide a vast resource for gene identification and expression profiling. We have mapped the complete set of 176,915 publicly available Arabidopsis EST sequences onto the Arabidopsis genome using GeneSeqer, a spliced alignment program incorporating sequence similarity and splice site scoring. About 96% of the available ESTs could be properly aligned with a genomic locus, with the remaining ESTs deriving from organelle genomes and non-Arabidopsis sources or displaying insufficient sequence quality for alignment. The mapping provides verified sets of EST clusters for evaluation of EST clustering programs. Analysis of the spliced alignments suggests corrections to current gene structure annotation and provides examples of alternative and non-canonical pre-mRNA splicing. All results of this study were parsed into a database and are accessible via a flexible Web interface at http://www.plantgdb.org/AtGDB/.
Expression of Antisense Acyl Carrier Protein-4 Reduces Lipid Content in Arabidopsis Leaf Tissue
Authors: Branen Jill K. , Shintani David K. , Engeseth Nicki J.
Published: June 2003

Arabidopsis plants were transformed with acyl carrier protein (ACP)-4 in antisense conformation driven by the cauliflower mosaic virus 35S promoter. It was hypothesized that reduction of ACP4 in leaf tissue would result in a reduction in lipid biosynthesis and, in addition, affect fatty acid composition and leaf physiology. Several transgenic lines have been generated with reduced ACP4 protein in leaf tissue. Dramatic reductions in ACP4 resulted in a reduction of leaf lipid content (22%–60%) based on fresh leaf weight and a bleached appearance and reduced photosynthetic efficiency. In addition, a decrease in 16:3 as a percentage of the total fatty acid composition was noted. There were no changes in leaf lipid class distribution; however, there was a decrease in the relative amount of 16:3 in monogalactosyldiacylglycerol. These results suggest that ACP4 plays a major role in the biosynthesis of fatty acids for chloroplast membrane development. Alterations in the ACP isoform profile of Arabidopsis leaf also appear to alter the flow of fatty acids between the prokaryotic and eukaryotic pathways for assembly of galactolipids. However, it has not yet been determined if the changes in fatty acid composition are due to changes in the profile of ACP isoforms, or if they are actually a reaction to a reduction in fatty acid precursors.
Light Stress-Induced One-Helix Protein of the Chlorophylla/b-Binding Family Associated with Photosystem I
Authors: Andersson Ulrica , Heddad Mounia , Adamska Iwona
Published: June 2003

The superfamily of light-harvesting chlorophyll a/b-binding (Lhc) proteins in higher plants and green algae is composed of more than 20 different antenna proteins associated either with photosystem I (PSI) or photosystem II (PSII). Several distant relatives of this family with conserved chlorophyll-binding residues and proposed photoprotective functions are induced transiently under various stress conditions. Whereas “classical” Lhc proteins contain three-transmembrane α-helices, their distant relatives span the membrane with between one and four transmembrane segments. Here, we report the identification and isolation of a novel member of the Lhc family from Arabidopsis with one predicted transmembrane α-helix closely related to helix I of Lhc protein from PSI (Lhca4) that we named Ohp2 (for a second one-helix protein of Lhc family described from higher plants). We showed that the Ohp2 gene expression is triggered by light stress and that the Ohp2 transcript and protein accumulated in a light intensity-dependent manner. Other stress conditions did not up-regulate the expression of the Ohp2 gene. Localization studies revealed that Ohp2 is associated with PSI under low- or high-light conditions. Because all stress-induced Lhc relatives reported so far were found in PSII, we propose that the accumulation of Ohp2 might represent a novel photoprotective strategy induced within PSI in response to light stress.
Physiological and Molecular Assessment of Altered Expression of Hsc70-1 in Arabidopsis. Evidence for Pleiotropic Consequences
Authors: Sung Dong Yul , Guy Charles L.
Published: June 2003

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44°C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.
Identification and Expression Analysis of a Gene Encoding a Bacterial-Type Phosphoenolpyruvate Carboxylase from Arabidopsis and Rice
Authors: Sánchez Rosario , Cejudo Francisco Javier
Published: June 2003

Phosphoenolpyruvate carboxylase (PEPC) is distributed in plants and bacteria but is not found in fungi and animal cells. Important motifs for enzyme activity and structure are conserved in plant and bacterial PEPCs, with the exception of a phosphorylation domain present at the N terminus of all plant PEPCs reported so far, which is absent in the bacterial enzymes. Here, we describe a gene from Arabidopsis, stated as Atppc4, encoding a PEPC, which shows more similarity to Escherichia coli than to plant PEPCs. Interestingly, this enzyme lacks the phosphorylation domain, hence indicating that it is a bacterial-type PEPC. Three additional PEPC genes are present in Arabidopsis, stated as Atppc1, Atppc2, and Atppc3, encoding typical plant-type enzymes. As most plant PEPC genes, Atppc1, Atppc2, and Atppc3 are formed by 10 exons interrupted by nine introns. In contrast, Atppc4 gene has an unusual structure formed by 20 exons. A bacterial-type PEPC gene was also identified in rice (Oryza sativa), stated as Osppc-b, therefore showing the presence of this type of PEPC in monocots. The phylogenetic analysis suggests that both plant-type and bacterial-type PEPCs diverged early during the evolution of plants from a common ancestor, probably the PEPC from γ-proteobacteria. The diversity of plant-type PEPCs in C3, C4, and Crassulacean acid metabolism plants is indicative of the evolutionary success of the regulation by phosphorylation of this enzyme. Although at a low level, the bacterial-type PEPC genes are expressed in Arabidopsis and rice.
Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps
Authors: Groover Andrew T. , Fontana Joseph R. , Arroyo Juana M. , Yordan Cristina , McCombie W. Richard , Martienssen Robert A.
Published: June 2003

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a “secretion trap” screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a β-glucuronidase reporter enzyme that is inhibited by N-linked glycosylation specific to the secretory pathway. Treatment of seedlings with tunicamycin inhibits glycosylation, resulting in increased activity of secreted β-glucuronidase fusions that result from gene trap integration downstream of exons encoding signal peptides. In the 2,059 gene trap lines that we screened, 32 secretion trap expression patterns were identified in a wide variety of tissues including embryos, meristems, and the developing vasculature. Genes disrupted by the secretion traps encode putative extracellular signaling proteins, membrane transport proteins, and novel secreted proteins of unknown function missed by conventional mutagenesis and gene prediction. Secretion traps provide a unique reagent for gene expression studies and can guide the genetic combination of loss of function alleles in related genes.
Changes in Gene Expression in Arabidopsis Shoots during Phosphate Starvation and the Potential for Developing Smart Plants
Authors: Hammond John P. , Bennett Malcolm J. , Bowen Helen C. , Broadley Martin R. , Eastwood Dan C. , May Sean T. , Rahn Clive , Swarup Ranjan , Woolaway Kathryn E. , White Philip J.
Published: June 2003

Our aim was to generate and prove the concept of “smart” plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as β-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these “late” genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.
Analysis of the Molecular Basis of Flowering Time Variation in Arabidopsis Accessions
Authors: Gazzani Silvia , Gendall Anthony R. , Lister Clare , Dean Caroline
Published: June 2003

Allelic variation at the FRI (FRIGIDA) and FLC (FLOWERING LOCUS C) loci are major determinants of flowering time in Arabidopsis accessions. Dominant alleles of FRI confer a vernalization requirement causing plants to overwinter vegetatively. Many early flowering accessions carry loss-of-function fri alleles containing one of two deletions. However, some accessions categorized as early flowering types do not carry these deletion alleles. We have analyzed the molecular basis of earliness in five of these accessions: Cvi, Shakhdara, Wil-2, Kondara, and Kz-9. The Cvi FRI allele carries a number of nucleotide differences, one of which causes an in-frame stop codon in the first exon. The other four accessions contain nucleotide differences that only result in amino acid substitutions. Preliminary genetic analysis was consistent with Cvi carrying a nonfunctional FRI allele; Wil-2 carrying either a defective FRI or a dominant suppressor of FRI function; and Shakhdara, Kondara, and Kz-9 carrying a functional FRI allele with earliness being caused by allelic variation at other loci including FLC. Allelic variation at FLC was also investigated in a range of accessions. A novel nonautonomous Mutator-like transposon was found in the weak FLC allele in Landsberg erecta, positioned in the first intron, a region required for normal FLC regulation. This transposon was not present in FLC alleles of most other accessions including Shakhdara, Kondara, or Kz-9. Thus, variation in Arabidopsis flowering time has arisen through the generation of nonfunctional or weak FRI and FLC alleles.
Microarray Analysis of the Nitrate Response in Arabidopsis Roots and Shoots Reveals over 1,000 Rapidly Responding Genes and New Linkages to Glucose, Trehalose-6-Phosphate, Iron, and Sulfate Metabolism
Authors: Wang Rongchen , Okamoto Mamoru , Xing Xiujuan , Crawford Nigel M.
Published: June 2003

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 μm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.
Gene Expression Phenotypes of Arabidopsis Associated with Sensitivity to Low Temperatures
Authors: Provart Nicholas J. , Gil Pedro , Chen Wenqiong , Han Bin , Chang Hur-Song , Wang Xun , Zhu Tong
Published: June 2003

Chilling is a common abiotic stress that leads to economic losses in agriculture. By comparing the transcriptome of Arabidopsis under normal (22°C) and chilling (13°C) conditions, we have surveyed the molecular responses of a chilling-resistant plant to acclimate to a moderate reduction in temperature. The mRNA accumulation of approximately 20% of the approximately 8,000 genes analyzed was affected by chilling. In particular, a highly significant number of genes involved in protein biosynthesis displayed an increase in transcript abundance. We have analyzed the molecular phenotypes of 12 chilling-sensitive mutants exposed to 13°C before any visible phenotype could be detected. The number and pattern of expression of chilling-responsive genes in the mutants were consistent with their final degree of chilling injury. The mRNA accumulation profiles for the chilling-lethal mutants chs1, chs2, and chs3 were highly similar and included extensive chilling-induced and mutant-specific alterations in gene expression. The expression pattern of the mutants upon chilling suggests that the normal function of the mutated loci prevents a damaging widespread effect of chilling on transcriptional regulation. In addition, we have identified 634 chilling-responsive genes with aberrant expression in all of the chilling-lethal mutants. This reference gene list, including genes related to lipid metabolism, chloroplast function, carbohydrate metabolism and free radical detoxification, represents a potential source for genes with a critical role in plant acclimation to suboptimal temperatures. The comparison of transcriptome profiles after transfer of Arabidopsis plants from 22°C to 13°C versus transfer to 4°C suggests that quantitative and temporal differences exist between these molecular responses.
The SAC Domain-Containing Protein Gene Family in Arabidopsis
Authors: Zhong Ruiqin , Ye Zheng-Hua
Published: June 2003

The SAC domain was first identified in the yeast (Saccharomyces cerevisiae) Sac1p phosphoinositide phosphatase protein and subsequently found in a number of proteins from yeast and animals. The SAC domain is approximately 400 amino acids in length and is characterized by seven conserved motifs. The SAC domains of several proteins have been recently demonstrated to possess phosphoinositide phosphatase activities. Sac1p has been shown to regulate the levels of various phosphoinositides in the phosphoinositide pool and affect diverse cellular functions such as actin cytoskeleton organization, Golgi function, and maintenance of vacuole morphology. The Arabidopsis genome contains a total of nine genes encoding SAC domain-containing proteins (AtSACs). The SAC domains of the AtSACs possess the conserved amino acid motifs that are believed to be important for the phosphoinositide phosphatase activities of yeast and animal SAC domain proteins. AtSACs can be divided into three subgroups based on their sequence similarities, hydropathy profiles, and phylogenetic relationship. Gene expression analysis demonstrated that the AtSAC genes exhibited differential expression patterns in different organs and, in particular, the AtSAC6 gene was predominantly expressed in flowers. Moreover, the expression of the AtSAC6 gene was highly induced by salinity. These results provide a foundation for future studies on the elucidation of the cellular functions of SAC domain-containing proteins in Arabidopsis.
Systemic Gene Expression in Arabidopsis during an Incompatible Interaction with Alternaria brassicicola
Authors: Schenk Peer M. , Kazan Kemal , Manners John M. , Anderson Jonathan P. , Simpson Robert S. , Wilson Iain W. , Somerville Shauna C. , Maclean Don J.
Published: June 2003

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of “high alert,” otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the β-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.
Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice
Authors: Baxter Ivan , Tchieu Jason , Sussman Michael R. , Boutry Marc , Palmgren Michael G. , Gribskov Michael , Harper Jeffrey F. , Axelsen Kristian B.
Published: June 2003

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2  +-ATPases (endoplasmic reticulum-type Ca2  +-ATPase and autoinhibited Ca2  +-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions.
‘Accommodating of Thame Selfis to Heir the Worde’: Preaching, Pews and Reformed Worship in Scotland, 1560–1638
Authors: Spicer Andrew
Published: June 2003

The liturgical implications of the teachings of Jean Calvin had a dramatic impact on the internal arrangement and furnishing of churches. Notably the pulpit replaced the altar as the focal point in religious worship. The congregation were expected to be attentive listeners to the Word of God delivered by the minister from the pulpit. This article looks at the impact that these teachings had in Scotland and in particular considers the liturgical and theological importance of establishing a reverent and attentive audience from the beginning of the Reformation. The provision of seating was an important part of these measures, with the Kirk being keen to ensure that the minister could be seen and heard by the congregation and vice versa. This article also shows that seating came to provide the means by which certain groups or individuals demonstrated their importance and status in the local community and was the cause of tension and dispute within the Kirk.
Good Pastors or Careless Shepherds? Parish Ministers and the English Reformation
Authors: Carlson Eric Josef
Published: June 2003

The post‐Reformation English parish clergy were not pastoral failures or ‘careless shepherds’ who were forced by parishioners to change their doctrine and tactics. From the beginning, the reformed church taught an ideal of ministry that was extremely sensitive to its audience. This ideal emphasized preaching a balance of repentance and mercy and rejected divisive behaviour such as attacking individual sinners from the pulpit. Moreover, preaching was not the only activity required of godly ministers. They were also expected to be ‘good pastors’ and act as peacemakers and sources of hospitality. Divisive and unpopular ministers existed, but they can be explained by incompetence and failure to follow the norms set out by the church. This article argues that the changes in the quality of parish ministry which took place during the late sixteenth and early seventeenth centuries were affected more by the church's attempt to spread the word about its own expectations than pressure from the laity.
Tolerance and Intolerance, 1976–1998
Authors: Mondak Jeffery J. , Sanders Mitchell S.
Published: June 2003

Early students of political tolerance projected a rapid rise in levels of tolerance, but subsequent research has failed to offer conclusive evidence regarding whether tolerance has, in fact, increased. The General Social Survey (GSS) included the same 15 dichotomous tolerance items in the period 1976–1998, seemingly permitting assessment of trends in tolerance through examination of a standard 0–15 scale. Unfortunately, the validity of these data is uncertain because we cannot rule out the possibility that changes in affect toward the five GSS target groups, not true changes in tolerance, drive movement in the longitudinal series. To address this problem, we reexamine the GSS data from the perspective of past discussions of the meaning of tolerance and intolerance. We argue that that the GSS scale captures two aspects of tolerance: whether respondents are tolerant or intolerant; and, among the intolerant, the breadth and depth of their intolerance. We further argue that the first of these dimensions can be measured validly using the GSS data. Our analyses reveal that, at most, tolerance has increased only marginally in the period 1976–1998.
Dynamics of Viral and Proviral Loads of FelineImmunodeficiency Virus within the Feline Central Nervous System duringthe Acute Phase following IntravenousInfection
Authors: Ryan G. , Klein D. , Knapp E. , Hosie M. J. , Grimes T. , Mabruk M. J. E. M. F. , Jarrett O. , Callanan J. J.
Published: June 2003

Animal models of human immunodeficiency virus 1, such as feline immunodeficiency virus (FIV), provide the opportunities to dissect the mechanisms of early interactions of the virus with the central nervous system (CNS). The aims of the present study were to evaluate viral loads within CNS, cerebrospinal fluid (CSF), ocular fluid, and the plasma of cats in the first 23 weeks after intravenous inoculation with FIVGL8. Proviral loads were also determined within peripheral blood mononuclear cells (PBMCs) and brain tissue. In this acute phase of infection, virus entered the brain in the majority of animals. Virus distribution was initially in a random fashion, with more diffuse brain involvement as infection progressed. Virus in the CSF was predictive of brain parenchymal infection. While the peak of virus production in blood coincided with proliferation within brain, more sustained production appeared to continue in brain tissue. In contrast, proviral loads in the brain decreased to undetectable levels in the presence of a strengthening PBMC load. A final observation in this study was that there was no direct correlation between viral loads in regions of brain or ocular tissue and the presence of histopathology.
Mutational Analysis of the HLA Class II Interaction with Epstein-Barr Virus Glycoprotein 42
Authors: McShane Marisa P. , Mullen Maureen M. , Haan Keith M. , Jardetzky Theodore S. , Longnecker Richard
Published: June 2003

Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.
Ovarian Sex Cord–Stromal Tumors in Children and Adolescents
Authors: Schneider D.T. , Calaminus G. , Wessalowksi R. , Pathmanathan R. , Selle B. , Sternschulte W. , Harms D. , Göbel U.
Published: June 2003

Purpose: To develop diagnostic standards and a risk-adapted therapeutic strategy for ovarian sex cord–stromal tumors (OSCST).

Patients and Methods: Fifty-four patients were prospectively enrolled as follow-up patients onto the German Maligne Keimzelltumoren protocols. Surgical protocols and histopathology were reviewed centrally (53 patients with complete data). Surgery included ovariectomy in 18 patients, salpingo-ovariectomy in 34 patients, and hysterectomy in one patient. Patients with stage IA tumors were followed-up at regular intervals, whereas nine patients with stage IC and six patients with stage II to III tumors were treated with cisplatin-based chemotherapy.

Results: International Federation of Gynecology and Obstetrics stage was IA in 27 patients, IC in 21 patients, II in three patients, and III in three patients. After a median follow-up of 59 months (range, 6 to 193 months), event-free survival ± SD was 0.86 ± 0.05 (47 of 54 patients) and overall survival was 0.89 ± 0.05 (49 of 54 patients). Prognosis correlated with stage (event-free survival ± SD: IA, 1.0 [27 of 27 patients]; IC, 0.76 ± 0.09 [16 of 21 patients]; and II/III, 0.67 ± 0.19 [four of six patients]; P = .02). Ten of 15 patients treated with chemotherapy, including four of six stage II to III patients, are alive after a median follow-up of 33 months.

Conclusion: On the basis of a standardized clinical and histopathologic assessment, risk-adapted therapeutic strategies for OSCST can be evaluated. Considering our experience, we would recommend that stage IA tumors be followed up at regular intervals, whereas we would recommend cisplatin-based chemotherapy in stage IC tumors with preoperative rupture or malignant ascites, especially those with high mitotic activity. Finally, cisplatin-based chemotherapy also seems to be effective in advanced-stage tumors.
Current Developments in Cancer Vaccines and Cellular Immunotherapy
Authors: Ribas Antoni , Butterfield Lisa H. , Glaspy John A. , Economou James S.
Published: June 2003

This article reviews the immunologic basis of clinical trials that test means of tumor antigen recognition and immune activation, with the goal to provide the clinician with a mechanistic understanding of ongoing cancer vaccine and cellular immunotherapy clinical trials. Multiple novel immunotherapy strategies have reached the stage of testing in clinical trials that were accelerated by recent advances in the characterization of tumor antigens and by a more precise knowledge of the regulation of cell-mediated immune responses. The key steps in the generation of an immune response to cancer cells include loading of tumor antigens onto antigen-presenting cells in vitro or in vivo, presenting antigen in the appropriate immune stimulatory environment, activating cytotoxic lymphocytes, and blocking autoregulatory control mechanisms. This knowledge has opened the door to antigen-specific immunization for cancer using tumor-derived proteins or RNA, or synthetically generated peptide epitopes, RNA, or DNA. The critical step of antigen presentation has been facilitated by the coadministration of powerful immunologic adjuvants, the provision of costimulatory molecules and immune stimulatory cytokines, and the ability to culture dendritic cells. Advances in the understanding of the nature of tumor antigens and their optimal presentation, and in the regulatory mechanisms that govern the immune system, have provided multiple novel immunotherapy intervention strategies that are being tested in clinical trials.
Randomized Trial of 2 Versus 5 Years of Adjuvant Tamoxifen for Women Aged 50 Years or Older With Early Breast Cancer: Italian Interdisciplinary Group for Cancer Evaluation Study of Adjuvant Treatment in Breast Cancer 01
Authors: Sacco M. , Valentini M. , Belfiglio M. , Pellegrini F. , De Berardis G. , Franciosi M. , Nicolucci A.
Published: June 2003

Purpose: To compare 2 with 5 years of adjuvant tamoxifen therapy in the treatment of early breast cancer.

Patients and Methods: Women with breast carcinoma T1–3, N0–3, M0, who were between 50 and 70 years of age, were eligible irrespective of menopausal status, tumor grade, or estrogen receptor (ER) status. Patients who were event-free after 2 years of tamoxifen therapy were randomly assigned to stop or continue tamoxifen therapy for an additional 3 years. The primary end point was length of disease-free survival (DFS). Secondary end points included overall survival (OS) and toxicity.

Results: From 1989 through 1996, 1,901 patients were randomly assigned either to stop treatment (n = 958) or to receive tamoxifen for 3 additional years (n = 943). The median duration of postrandomization follow-up was 52 months. We found no statistically significant differences between the 5-year arm and the 2-year arm in terms of DFS (hazard ratio [HR], 0.91; 95% confidence interval [CI], 0.76 to 1.08) and OS (HR, 1.16; 95% CI, 0.92 to 1.46). In ER-positive patients, a statistically significant prolongation of DFS related to longer treatment duration was observed (HR, 0.74; 95% CI, 0.59 to 0.93), whereas no difference in OS could be detected (HR, 0.98; 95% CI, 0.72 to 1.32). No differences in terms of endometrial cancers, cardiac or cerebrovascular events, or fractures were detected, whereas a doubling in the risk of thromboembolic events was found in the 5-year arm.

Conclusion: Our results confirm previous research that shows that 5 years of tamoxifen decreases recurrence compared to 2 years in patients with ER-positive tumors.
Radiation Therapy Plus Tamoxifen Versus Tamoxifen Alone After Breast-Conserving Surgery in Postmenopausal Women With Stage I Breast Cancer: A Decision Analysis
Authors: Punglia Rinaa S. , Kuntz Karen M. , Lee Jason H. , Recht Abram
Published: June 2003

Purpose: To compare outcomes for hypothetical cohorts of postmenopausal patients with estrogen receptor–positive tumors that are ≤ 2 cm in size, with pathologically uninvolved axillary nodes, treated with radiation therapy plus tamoxifen versus tamoxifen alone after breast-conserving surgery.

Methods: A Markov model was used to simulate patients’ clinical course and estimate overall survival, recurrence-free survival, time with an intact breast, and death from breast cancer. Probabilities were derived from randomized trials and retrospective studies. Analyses were performed separately by age of diagnosis in 5-year increments from 50 to 80 years. Sensitivity analyses tested the stability of radiation benefit.

Results: The modeled recurrence-free survival benefit of giving radiation therapy was 3.35 years for women who were 50 years of age at diagnosis, versus 0.61 years for women who were 80 years of age. In the 50-year-old cohort, radiation therapy resulted in additional 0.60 years survival, compared with 0.04 years among 80-year-olds. A 50-year-old woman who received radiation therapy plus tamoxifen was less likely to die from breast cancer than if she received tamoxifen alone (2.43% v 5.29%; relative-risk reduction, 54%). An 80-year-old woman had a 1.17% chance of dying from breast cancer if she received radiation therapy plus tamoxifen, versus 2.02% with tamoxifen alone (relative-risk reduction, 42%). Sensitivity analyses showed that the magnitude of benefit was strongly influenced by including unequal rates of developing distant disease after breast recurrence between the treatment arms and varying rates of local recurrence.

Conclusion: The absolute and relative benefits of radiation therapy and individual patient preferences for different health states should be considered when selecting treatment.