bluuebunny/crossref_metadata_2025_split huggingface dataset:
Seed‐specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons
Authors: Rosche Elke , Blackmore Daniel , Tegeder Mechthild , Richardson Terese , Schroeder Hart , Higgins Thomas J. V. , Frommer Wolf B. , Offler Christina E. , Patrick John W. 
Published: March 2003

SummaryDuring the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [14C]sucrose influx into cotyledons above wild‐type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2‐fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.
Mitochondrial oxidative burst involved in apoptotic response in oats
Authors: Yao Nan , Tada Yasuomi , Sakamoto Masaru , Nakayashiki Hitoshi , Park Pyoyun , Tosa Yukio , Mayama Shigeyuki 
Published: March 2003

SummaryApoptotic cell response in oats is induced by victorin, a host‐selective toxin secreted by Cochliobolus victoriae and thought to exert toxicity by inhibiting mitochondrial glycine decarboxylase (GDC) in Pc‐2/Vb oats. We examined the role of mitochondria, especially the organelle‐derived production of reactive oxygen species (ROS), in the induction of apoptotic cell death. Cytofluorimetric analysis showed that victorin caused mitochondrial ΔΨm breakdown and mitochondrial oxidative burst. Ultrastructural analysis using a cytochemical assay based on the reaction of H2O2 with CeCl3 detected H2O2 eruption at permeability transition pore‐like sites on the mitochondrial membrane in oat cells treated with victorin. ROS generation preceded the apoptotic cell responses seen in chromatin condensation and DNA laddering. Both aminoacetonitrile (a specific GDC inhibitor) and antimycin A (a mitochondrial complex III inhibitor) also induced mitochondrial H2O2 eruption, and led to the apoptotic response in oat cells. ROS scavengers such as N‐acetyl‐l‐cysteine and catalase suppressed the mitochondrial oxidative burst and delayed chromatin condensation and DNA laddering in the victorin‐ or antimycin A‐treated leaves. These findings indicate possible involvement of mitochondria, especially mitochondrial‐derived ROS generation, as an important regulator in controlling apoptotic cell death in oats.
Constructing plant radiation hybrid panels
Authors: Wardrop Julie , Snape John , Powell Wayne , Machray Gordon C. 
Published: March 2003

SummaryRadiation hybrid (RH) mapping, a somatic cell genetic technique, has been developed in animal systems as a general approach for the construction of long‐range physical maps of chromosomes. This statistical method relies on X‐ray induced breakage of chromosomes to determine the physical distance between markers, as well as their order on the chromosome. The method can be applied to single chromosomes or across the whole genome. The generation of plant (barley) radiation hybrids and their culture in vitro is described here. PCR‐based marker systems are used to verify hybrid status and to demonstrate genome coverage. RH panels of the type generated can be used for physical mapping, map‐based cloning, or sequence contig assembly. RH resources will greatly aid the physical characterisation of crop plants with large genomes.
A novel interaction partner for the C‐terminus of Arabidopsis thaliana plasma membrane H+‐ATPase (AHA1 isoform): site and mechanism of action on H+‐ATPase activity differ from those of 14‐3‐3 proteins#
Authors: Morandini Piero , Valera Marco , Albumi Cristina , Bonza Maria Cristina , Giacometti Sonia , Ravera Giuseppe , Murgia Irene , Soave Carlo , De Michelis Maria Ida 
Published: March 2003

SummaryUsing the two‐hybrid technique we identified a novel protein whose N‐terminal 88 amino acids (aa) interact with the C‐terminal regulatory domain of the plasma membrane (PM) H+‐ATPase from Arabidopsis thaliana (aa 847–949 of isoform AHA1). The corresponding gene has been named Ppi1 for Protonpumpinteractor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C‐terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50–70% at the aa level over stretches of 200–600 aa) to Ppi1. The PPI1 N‐terminus, expressed in bacteria as a fusion protein with either GST or a His‐tag, binds the PM H+‐ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+‐ATPase activity. The effect of the His‐tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C‐terminal 10 kDa fragment. The His‐tagged peptide binds also the trypsinised H+‐ATPase. Altogether these results indicate that PPI1 N‐terminus is able to modulate the PM H+‐ATPase activity by binding to a site different from the 14‐3‐3 binding site and is located upstream of the trypsin cleavage site.
An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants
Authors: Rademacher Thomas , Häusler Rainer E. , Hirsch Heinz‐Josef , Zhang Li , Lipka Volker , Weier Dagmar , Kreuzaler Fritz , Peterhänsel Christoph 
Published: March 2003

SummaryPhosphoenolpyruvate carboxylase (PEPC) plays a central role in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3plants. Furthermore, in both C4 and CAM plants photosynthetic isoforms are pivotal for the fixation of atmospheric CO2. Potato PEPC was mutated either by modifications of the N‐terminal phosphorylation site or by an exchange of an internal cDNA segment for the homologous sequence of PEPC from the C4plantFlaveria trinervia. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition and an increased affinity for PEP. These effects were enhanced by a combination of both mutated sequences and pulse labelling with14CO2in vivorevealed clearly increased fixation into malate for this genotype. Activity levels correlated well with protein levels of the mutated PEPC. Constitutive overexpression of PEPC carrying both N‐terminal and internal modifications strongly diminished plant growth and tuber yield. Metabolite analysis showed that carbon flow was re‐directed from soluble sugars and starch to organic acids (malate) and amino acids, which increased four‐fold compared with the wild type. The effects on leaf metabolism indicate that the engineered enzyme provides an optimised starting point for the installation of a C4‐like photosynthetic pathway in C3plants.
Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds
Authors: Duval Frédéric D. , Renard Michelle , Jaquinod Michel , Biou Valérie , Montrichard Françoise , Macherel David 
Published: March 2003

SummaryImplication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N‐terminal α‐helix. CaM was shown to be encoded by a small multigenic family, and full‐length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5′ and 3′ untranslated regions. Expression studies, performed by semiquantitative RT–PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep‐tagged proteins (CaM1‐ST, CaM2‐ST and CaM3‐ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1‐ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2‐ST and CaM3‐ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp‐3, an idealised helical CaM‐binding peptide. However, the three proteins activated in the same way the CaM‐dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.
Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid
Authors: Köhler Barbara , Blatt Michael R. 
Published: March 2003

SummaryProtein phosphorylation and cytosolic‐free [Ca2+] ([Ca2+]i) contribute to signalling cascades evoked by the water‐stress hormone abscisic acid (ABA) that lead to stomatal closure in higher‐plant leaves. ABA activates an inward‐rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage‐sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de‐)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2, 1.3 ± 0.7 mm) but not the non‐hydrolyzable analog ATPγS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A‐type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage‐sensitivities of the Ca2+ current and [Ca2+]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane‐delimited interaction between 1/2 A‐type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de‐)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+]i increases in the guard cells.
The cellulose‐deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II, an enzyme processing N‐glycans during ER quality control
Authors: Burn Joanne E. , Hurley Ursula A. , Birch Rosemary J. , Arioli Tony , Cork Ann , Williamson Richard E. 
Published: March 2003

Summary rsw3 is a temperature‐sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose. The rsw3 gene was identified by a map‐based strategy, and shows high similarity to the catalytic α‐subunits of glucosidase II from mouse, yeast and potato. These enzymes process N‐linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding. Putative β‐subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER‐retention signal) with β‐subunits in mammals and yeast. The genes encoding the putative α‐ and β‐subunits are single copy and, like the rsw3 phenotype, widely expressed. rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems. Most features of the rsw3 phenotype are shared with other cellulose‐deficient mutants, but some – notably, production of multiple rosettes and a lack of secreted seed mucilage – are not and may reflect glucosidase II affecting processes other than cellulose synthesis. The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature. This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.
Serine/threonine kinase activity in the putative histidine kinase‐like ethylene receptor NTHK1 from tobacco
Authors: Xie Can , Zhang Jin‐Song , Zhou Hua‐Lin , Li Jian , Zhang Zhi‐Gang , Wang Dao‐Wen , Chen Shou‐Yi 
Published: March 2003

SummaryA histidine kinase‐based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase‐like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP‐binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1‐KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase‐type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.
Expression profiling of the host response to bacterial infection: the transition from basal to induced defence responses in RPM1‐mediated resistance
Authors: Torres Marta de , Sanchez Pedro , Fernandez‐Delmond Isabelle , Grant Murray 
Published: March 2003

SummaryChanges in transcription in leaves of Arabidopsis thaliana were characterised following challenge with strains of Pseudomonas syringae pv. tomato DC3000 allowing differentiation of basal resistance (hrpA mutants), gene‐specific resistance (RPM1‐specified interactions) and susceptibility (wild‐type pathogen). In planta avirulence gene induction, changes in host [Ca2+]cyt and leaf collapse were used to delineate the transition from infection to induced resistance. The plant responds rapidly, dynamically and discriminately to infection by phytopathogenic bacteria. Within the first 2 h host transcriptional changes are common to all challenges indicating that Type III effector function does not contribute to early events in host transcriptome re‐programming. The timing of induction for specific transcripts was reproducible, hierarchical and modulated at least in part through EDS1 function. R gene‐specific transcripts were not observed until 3 h after inoculation. Intriguingly, the R gene‐specific response proteins are expected to localise to diverse cellular addresses indicative of a global impact on cellular homeostasis. The altered transcriptional response rapidly manifests into initial symptoms of leaf collapse within 2 h, although establishment of the full macroscopic HR occurs significantly later.
Current practice in neonatal exchange transfusions: a retrospective audit based at one transfusion centre
Authors: Stern  , Cockburn  , De Silva  
Published: March 2003

As a result of the widespread use of phototherapy, the need for neonatal exchange transfusion has declined markedly over the last 25 years. However, in spite of this, we have discovered a marked disparity in the number of requests for units of blood suitable for exchange transfusion made by neonatal units of similar size. A retrospective audit was therefore performed to establish current practice. A questionnaire was sent to consultant neonatologists and an audit trail of all units of blood issued from our Transfusion Centre for exchange transfusion over an 18‐month period was followed.A significant finding was the large amount of over‐ordering of blood by neonatologists, resulting in units produced expensively to high specification being given to other patients. Only 42% of the blood issued for exchange transfusions was actually used for that purpose. Also, published guidelines are not being adhered to, particularly with respect to the specification of the blood products being used. Better communication between neonatologists, hospital haematologists and transfusion medicine specialists would help to ensure that infants requiring exchange transfusion are given the best product and that valuable resources are not wasted.
A first approach to compare efficacy and safety of lepirudin and danaparoid in patients with heparin‐induced thrombocytopenia (HIT)
Authors: Farner B. , Eichler P. , Kroll H. , Urban G. , Greinacher A. 
Published: March 2003

B ackground HIT is a hyper–coagulable syndrome requiring further anticoagulation.O bjective To compare the outcome of HIT patients treated with danaparoid or lepirudin regarding the single or combined endpoints of new thromboembolic complications (TECs), amputations and/or death, and major bleedings.M ethod Cohort study comparing HIT patients treated during the same time period with danaparoid or lepirudin.P atients The efficacy of lepirudin was investigated in two prospective trials. HIT patients, who were identified in the same two laboratories during the same time period, but who were not enrolled into these studies but treated with danaparoid, were assessed retrospectively according to a standardized questionnaire.R esults 126 danaparoid (50 male, 70 female) and 175 lepirudin treated patients (73 male, 102 female) fulfilled the same inclusion and exclusion criteria. In a time‐to‐event‐analysis the cumulative risk of combined endpoint was higher in HIT patients without thromboembolic complications at study entry treated with danaparoid as compared to lepirudin (P = 0·02), whereas HIT patients with thrombosis at study entry had a similar outcome in both treatments groups (P = 0·91). Major bleedings occurred in 2·5% [95% CI 0·5–7·0%] of danaparoid treated patients as compared to 10·4% [95% CI 6·3–15·9%] of lepirudin treated patients until day 42 (P = 0·009).C onclusion The efficacies of therapeutic dosages of danaparoid or lepirudin do not differ largely, but the risk of bleedings seems to be higher in lepirudin treated patients. However the prophylactic dosage of danaparoid (750 antifactor Xa units b.i.d. or t.i.d. s.c.) approved in European Union for HIT without TEC's seems to be suboptimal.
Effectiveness of chemoprophylaxis and other determinants of malaria in travellers to Kenya
Authors: Muehlberger  , Muehlberger  , Jelinek  , Schlipkoeter  , V. Sonnenburg  , Nothdurft  
Published: March 2003

Summary
objective To investigate the effectiveness of chemoprophylaxis and the determinants of malaria importation from Kenya. method In a population‐based case‐control study, 51 travellers from Bavaria diagnosed with falciparum malaria imported from Kenya (cases) and a sample of 383 healthy Bavarian travellers returning from Kenya (controls) were interviewed. Data were analysed by multiple logistic regression. results Mefloquine (OR = 0.055; 95% Cl 0.019‐0.16) and chloroquine combined with proguanil (OR = 0.128; 95% CI 0.039‐0.419) were highly protective against P. falciparum malaria, whereas other drugs were ineffective (OR = 1.225; 95% CI 0.536‐2.803). Ineffective prophylaxis (10.4%) and non‐prophylaxis (11.2%) were the main reasons for malaria importation. Travelling alone or with friends, male sex, and travel duration over 4 weeks could be identified as additional risk factors. The main reason for inadequate chemoprophylaxis was inappropriate medical advice (87.5%). Prophylaxis refusal occurred frequently despite correct advice (58.1%). Diagnosis was often delayed unnecessarily (27.5%). conclusion Malaria importation from Kenya could be reduced substantially (34%) by eliminating inappropriate medical advice.
Diagnostic PCR with Leishmania donovani specificity using sequences from the variable region of kinetoplast minicircle DNA
Authors: Singh Neeloo , Curran Martin D. , Rastogil Anil K. , Middleton Derek , Sundar Shyam 
Published: March 2003

Summary Kala azar or visceral leishmaniasis, a parasitic disease caused by Leishmania donovani, is presently causing an epidemic in the eastern region of India. Diagnosis of kala azar is often complicated. We developed a pair of oligonucleotides suitable as primers from the variable region of a predominant sequence class of minicircles of L. donovani. These primers were used in a nonisotopic polymerase chain reaction and found to be highly specific for the parasites of L. donovani complex. Using these primers, amplification of L. donovani kinetoplast DNA minicircle from the peripheral blood of kala azar patients results in a product of 204 bp. The patient group was comprised of individuals from a highly endemic region of India. We feel that PCR could assess the efficacy of new leishmanicidal drugs under investigation in these patients. PCR could also predict response to therapy which would be useful for both clinical and research applications.
The epidemiology of a recent focus of mixed Schistosoma haematobium and Schistosoma mansoni infections around the ‘Lac de Guiers’ in the Senegal River Basin, Senegal
Authors: De Clercq D. , Vercruysse J. , Picquet M. , Shaw D. J. , Diop M. , Ly A. , Gryseels B. 
Published: March 2003

Summary A village with mixed Schistosoma mansoni and S. haematobium infections (probably in a early endemic phase) was identified around the Lac de Guiers in the Senegal River Basin. In documenting the epidemiology of both schistosomes, we focused on prevalence and intensity of infection, transmission patterns and the impact of treatment. S. mansoni prevalences (near 100%) and egg counts (overall geometric mean eggs per gram of faeces (epg) of 589 were high in all age groups, with 35% of individuals excreting > 1000 epg, and showing a slow decline in egg output only after the age of 30 years. The overall prevalence (28%) and egg counts (2% > 50 eggs/10 ml) of S. haematobium were low, with mean counts of 6.3 eggs/10 ml. Maximal mean S. mansoni egg counts were found in 5–9 year‐old boys and in 15–19 year‐old girls; S. haematobium maximal counts in 1–4 year‐old boys and in girls aged 5–9. Extremely high Biomphalaria pfeifferi infection ratios were recorded over the whole year. Following a single treatment, re‐infection was rapid with prevalences and mean egg counts of both Schistosoma species reaching pretreatment levels within 7 months.