bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene‐associated CpG islands in plants as revealed by analyses of genomic sequences
Authors: Ashikawa Ikuo
Published: March 2003

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG‐rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG‐rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG‐rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5′‐end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5′‐end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue‐specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5′‐end of the genes.
Light regulation of circadian clock‐controlled gene expression in rice
Authors: Sugiyama Nobuko , Izawa Takeshi , Oikawa Tetsuo , Shimamoto Ko
Published: March 2003

Using transgenic rice seedlings expressing a firefly luciferase (luc) gene under the control of a rice CAB (chlorophyll a/b binding protein) promoter, we demonstrated how light affects circadian clocks in the transcription of the CAB gene. Rhythmic luc expression was observed for more than 5 d under constant light and dark (DD) conditions after light/dark entrainment. After a light pulse was applied at different time points in DD various temporal patterns of CAB gene expression were individually observed. We first examined two distinct properties related to the entrainment mechanism of the circadian clock: fluence‐rate dependence of free‐running periods (FRPs) and phase resetting by a light pulse. Although fluence‐rate dependent shortening of FRP was demonstrated, the FRP in DD was almost equal to that in constant light of a middle fluence‐rate, indicating that this fluence‐rate dependence may not fully describe the entrainment of the circadian clock in rice. Typical phase responses of the circadian clock by a single light pulse were also observed at the transcriptional level in rice seedlings. Thus, the phase resettings upon the light/dark transitions of daily cycles may be sufficient to explain the entrainment mechanisms of rice. We have further demonstrated that, in addition to having a gating effect to acute response, a light pulse can activate the circadian clock‐controlled CAB1R gene expression at the first circadian peak in a phase‐dependent manner. This suggests that light activates circadian clock activity in the diurnal CAB gene expression under daily light/dark cycles.
Trans‐dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses
Authors: Pontier Dominique , Miao Zhong‐He , Lam Eric
Published: March 2003

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic‐leucine‐zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans‐dominant mutant that specifically eliminates DNA‐binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA‐binding activities is correlated with suppression of two xenobiotic‐responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis‐related (PR) genes by SA. In addition, these TGA‐suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.
Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance
Authors: Ton Jurriaan , De Vos Martin , Robben Charlotte , Buchala Anthony , Métraux Jean‐Pierre , Van Loon L. C. , Pieterse Corné M. J.
Published: March 2003

In Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers jasmonate (JA)‐ and ethylene (ET)‐dependent induced systemic resistance (ISR) that is effective against different pathogens. Arabidopsis genotypes unable to express rhizobacteria‐mediated ISR against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) exhibit enhanced disease susceptibility towards this pathogen. To identify novel components controlling induced resistance, we tested 11 Arabidopsis mutants with enhanced disease susceptibility (eds) to pathogenic P. syringae bacteria for WCS417r‐mediated ISR and pathogen‐induced systemic acquired resistance (SAR). Mutants eds4‐1, eds8‐1 and eds10‐1 failed to develop WCS417r‐mediated ISR, while mutants eds5‐1 and eds12‐1 failed to express pathogen‐induced SAR. Whereas eds5‐1 is known to be blocked in salicylic acid (SA) biosynthesis, analysis of eds12‐1 revealed that its impaired SAR response is caused by reduced sensitivity to this molecule. Analysis of the ISR‐impaired eds mutants revealed that they are non‐responsive to induction of resistance by methyl jasmonate (MeJA) (eds4‐1, eds8‐1 and eds10‐1), or the ET precursor 1‐aminocyclopropane‐1‐carboxylate (ACC) (eds4‐1 and eds10‐1). Moreover, eds4‐1 and eds8‐1 showed reduced expression of the plant defensin gene PDF1.2 after MeJA and ACC treatment, which was associated with reduced sensitivity to either ET (eds4‐1) or MeJA (eds8‐1). Although blocked in WCS417r‐, MeJA‐ and ACC‐induced ISR, eds10‐1 behaved normally for several other responses to MeJA or ACC. The results indicate that EDS12 is required for SAR and acts downstream of SA, whereas EDS4, EDS8 and EDS10 are required for ISR acting either in JA signalling (EDS8), ET signalling (EDS4), or downstream JA and ET signalling (EDS10) in the ISR pathway.
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

During 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

Apoptotic 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

Radiation 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

Using 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

Phosphoenolpyruvate 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

Implication 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

Protein 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

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

A 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

Changes 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.
Fatal reaction to transfusion of red‐cell concentrate contaminated with Serratia liquefaciens
Authors: Boulton  , Chapman  , Walsh
Published: March 2003

A 60‐year‐old woman undergoing surgery died from endotoxic shock and DIC after receiving a 19‐day‐old unit of optimal additive red‐cell concentrate found contaminated with Serratia liquefaciens. No source of contamination could be found. This normally free‐living organism is usually of low pathogenicity. It is a very unusual contaminant of stored donated blood, although it appears to be on the increase. When transfused, blood contaminated with S. liquefaciens always causes severe morbidity and is associated with a high death rate. This is the fifth report in the English literature.
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.
Complement activation during plasma production depends on the apheresis technique
Authors: Sonntag  , Emeis  , Vornwald  , Strauss  , Maier
Published: March 2003

Elevated anaphylatoxin concentrations have been found in fresh frozen plasma packs produced by apheresis. The aim of this study was to investigate anaphylatoxin generation during apheresis production in relation to two frequently used techniques: employing either centrifugation alone or centrifugation with simultaneous filtration. The concentrations of C3a, C5a and sC5b‐9 were measured in 50 plasmas after apheresis and before freezing generated by combined centrifugation and filtration and in 50 plasmas generated solely by centrifugation and in the corresponding 100 donors before apheresis. The median C3a concentration increased during apheresis by centrifugation alone from 62 (donor) to 380 μg L−1 (FFP pack) and during apheresis by combined centrifugation and filtration from 70 to 992 μg L−1. The median C5a concentration increased during apheresis by centrifugation alone from 0.38 to 0.83 μg L−1 and during apheresis by combined centrifugation and filtration from 0.29 to 4.9 μg L−1. The soluble terminal complement complex increased only by combined centrifugation and filtration apheresis from 162 to 426 μg L−1. Complement activation during apheresis by combined centrifugation and filtration is more pronounced than that obtained by the centrifugation method alone. Changes in the construction or the materials used in the membrane‐type separation units may reveal possibilities of prevention.
Flow cytometric monitoring of red blood cell chimerism after bone marrow transplantation
Authors: David  , Bernard  , Navenot  , Muller  , Blanchard
Published: March 2003

Chimerism after bone marrow transplantation (BMT) was investigated by flow cytometry analysis of red blood cells (RBCs) and of reticulocytes using a series of selected monoclonal or polyclonal antibodies directed against ABO, Rhesus, Kell, Duffy or MNSs antigens. The method allows the routine detection of less than 0.1% of positive cells in artificial mixed field populations. Blood samples from 135 patients undergoing BMT were investigated around days 15, 30, 45, 60, 90, 180, and then every 6 months after transplantation. Characteristic patterns showing expression of donor red blood cell antigens (expansion markers) and concomitant decrease of recipient specific antigens (depletion markers) within days 16–20 were observed for 125 successfully engrafted patients. Distinct patterns were obtained in 10 patients. A delay in engraftment was evidenced in four patients by the absence of chimerism during the first 6 months without any sign of relapse. Re‐appearance of recipient RBCs and reticulocytes was observed in five patients; it was consistent with relapse that was later confirmed by clinical, haematological and cytogenetic studies. Finally, a stable and partial chimerism with 20% of RBCs expressing a marker from the recipient was observed in one patient without any sign of relapse. The reported investigation demonstrated that flow cytometry of RBCs and reticulocytes represents a powerful method to efficiently monitor bone marrow transplanted patients on a long‐term basis.
Haemodynamic response following a 10% topload infusion of HemolinkTM in conscious, anaesthetized and treated spontaneously hypertensive rats
Authors: Ning  , Wong  , Christoff  , Carmichael  , Biro
Published: March 2003

HemolinkTM (HLK), a haemoglobin‐based oxygen carrier (HBOC), is currently undergoing Phase II/III clinical trials in surgical patients. It causes some blood pressure rise in animal and human tests. This study was designed to investigate the systemic haemodynamic response to HemolinkTM in spontaneously hypertensive rats (SHR rats). Conscious or anaesthetized SHR rats and control Wistar Kyoto rats (WKY rats) received either HemolinkTM or homologous plasma as a 10% topload infusion. Some awake animals were pretreated with nifedipine and followed by HLK infusion. In the conscious animal study, HLK induced a greater pressure rise and less bradycardia in SHR rats than in WKY rats. In the anaesthetized animal experiment, HLK‐induced pressure rise and bradycardia were similar in both strains and less pronounced than in the conscious animals. In the nifedipine pretreated SHR rats, HLK‐induced pressure rise was significantly smaller than that observed in nontreated SHR rats and was not different from that of nontreated WKY rats. The HLK‐induced bradycardia was significantly smaller in nifedipine‐treated animals than in the nontreated SHR or WKY rats. This study suggests that the pressor effect of HemolinkTM can be attenuated in hypertensive animals with general anaesthesia or treatment with antihypertensive agents.
Transfusion of platelet concentrates from pooled buffy‐coats: comparison of bedside vs. prestorage leukofiltration
Authors: Müller‐Steinhardt  , Schlenke  , Wagner  , Klüter
Published: March 2003

Prestorage leucocyte filtration of platelet concentrates (PC) has been considered to be superior to bedside filtration with regard to the incidence of nonhaemolytic transfusion reactions (NHTR) or HLA‐alloimmunization. It is currently a matter of debate whether prestorage leucocyte filtration has an impact on the storability of platelets and on the transfusion results of PC. In a clinical retrospective study we investigated the transfusion results of PC from pooled buffy‐coats (PC‐BC) in haematological patients without known refractoriness to platelets, and compared bedside filtration (n = 228/36 patients) vs. prestorage filtration (n = 271/25 patients). Leukocyte and platelet content of the PC, duration of storage, platelet count of the patient before transfusion and 20 h after transfusion were determined and platelet increment and corrected count increment (CCI) 20 h after transfusion were calculated. The mean leucocyte content of the bedside filtered PC was 66 ± 50 × 106 and < 0.1 × 106 for the prestorage filtered PC (P < 0.001). Mean platelet content of the PC (2.6 × 1011 vs. 2.7 × 1011) and the duration of PC storage (2.7 vs. 2.6 days) were almost identical in both groups. The platelet increment after 20 h (14.6 × 109 L−1 vs. 14.9 × 109 L−1) was equal for both groups, but CCI values were significantly higher for the bedside filtered PC (14.1 ± 9.5 vs. 11.4 ± 6.8) (P = 0.008). Correlation with the storage time revealed that CCI levels were higher for bedside filtered PC after short‐term storage (< 36 h) (P = 0.014), but declined more rapidly compared with prestorage filtered PC. A patient‐based analysis including fewer cases revealed superior but nonsignificant results for bedside filtration. In conclusion, bedside filtered PC‐BC resulted in better CCI results after short‐term storage, but values equalized with prestorage filtered PC‐BC after longer storage intervals. Prestorage‐leucocyte filtration did not improve platelet recovery in vivo, but CCI values decreased only moderately throughout storage. Both preparations showed excellent transfusion results even after a 5‐day storage interval.
Alloimmunization in Chinese with warm autoimmune haemolytic anaemia — incidence and characteristics
Authors: So  , Wong  , Yu  , Kwan  , Lee
Published: March 2003

We perform a retrospective study to determine the incidence and characteristics of alloimmunization in Chinese patients with warm autoimmune haemolytic anaemia. Among 67 patients studied, clinically significant alloantibodies were found in only eight patients, giving a rate of alloimmunization of 11.3%. The latter contrasts with the much higher rate reported in the Western population. This probably reflects the genetic homogeneity in Chinese with respect to the red cell phenotype. The alloimmunization rate, however, is still significant and therefore comprehensive pretransfusion testing is required for Chinese patients with warm autoantibodies so as to safeguard patient's safety.
Induction of a secondary human HPA‐5B alloimmune response in SCID mice engrafted with human lymphocytes and antigen‐pulsed autologous dendritic cells
Authors: Hughes D. , Jackson D. , Kumpel B. , Garner S. , Ouwehand W. H.
Published: March 2003

Severe combined immunodeficient (SCID) mice possess a mutation which impairs the recombination of antigen receptor genes, rendering them severely deficient in B and T lymphocytes. Such mice support reconstitution with human lymphocytes. We have reconstituted 14 SCID mice with peripheral blood lymphocytes (PBLs) from a female donor who was HPA‐5b alloimmunized in pregnancy over 15 years ago. Nine mice received PHA stimulated PBLs and were challenged with HPA‐5b on days 0 and 7. Five mice (3 with PHA stimulated PBLs and 2 with PBLs not stimulated with PHA) received in addition autologous, monocyte‐derived dendritic cells pulsed with HPA‐5b. Sera were screened for human anti‐HPA‐5b IgG/M by the MAIPA method at 7 day intervals after reconstitution. Anti‐HPA‐5b was only detected (at 14 and 21 days, respectively) in two of the three mice reconstituted with pulsed APCs and PHA stimulated PBLs. We have demonstrated that SCID mice support a secondary HPA‐5b alloimmune response when reconstituted with immune donor lymphocytes, and the use of antigen‐pulsed dendritic cells. We are now cloning the rearranged V gene repertoire of these mice for phage display to obtain a monoclonal HPA‐5b antibody.
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.
Glanzmann thrombasthenia and isoimmunization
Authors: Kaplan C. , Bianchi F. , Jallu V.
Published: March 2003

Glanzmann thrombasthenia is a rare autosomal recessive inherited bleeding disorder characterized by the inability of the platelets to aggregate. The abnormality results from a quantitative or qualitative defect in the platelet membrane glycoprotein (GP) IIb‐IIIa. A potential consequence of platelet membrane GP deficiency is isoantibody (isoAb) production following platelet transfusion or during pregnancy, when the patient has contact with normal platelets. IsoAb have been reported in Glanzmann's thrombasthenia patients lacking GPIIb‐IIIa (GTI), which recognize distinct epitopes on the GPIIb‐IIIa complex. These antibodies (Ab) are of clinical significance as they can lead to platelet refractoriness by destruction of the transfused platelets and thus to therapeutical difficulties. The development of antigen specific assays (as the MAIPA technique) has been extremely useful, allowing the binding of the antibody to its epitope in a conformational state. One of the major disadvantages of this technique is the existence of false negative results due to a competition between the human Ab and monoclonal antibodies (MoAbs) for the same or closely related epitopes. Negative or discordant results for identification or characterization of Ab are a matter of concern during investigation for isoimmunization. In an attempt to demonstrate the importance of the choice of MoAbs for accurate biological diagnosis and to show the serological heterogeneity among isoAb, we screened serum from GTI patients by the MAIPA technique with a panel of MoAbs. These studies were particularly informative as we found striking differences in the binding of the human Ab to their target antigen depending on the MoAbs present in the reaction. Furthermore the choice of MoAbs can be crucial for detecting a specific Ab in case of low reactivity or low titre. Further investigations are needed to clarify whether this heterogeneity plays a role in clinical conditions.
Effectiveness of washed platelet concentrate and red cell transfusions for a patient with anhaptoglobinemia with antihaptoglobin antibody
Authors: Nishiki S. , Hino M. , Kumura T. , Hashimoto S. , Ohta K. , Yamane T. , Takubo T. , Tatsumi N. , Kitagawa S. , Kamitani T. , Watanabe Y. , Shimada E. , Juji T. , Iida S.
Published: March 2003

Summary A 71‐year‐old Japanese male with myelodysplastic syndrome progressing to overt leukaemia and hepatocellular carcinoma developed dyspnea and urticaria immediately after infusion of platelet concentrate (PC). He exhibited an identical reaction following blood transfusion. Serum haptoglobin was undetectable. The patient was determined to be homozygous for Hpdel by polymerase chain reaction (PCR). Antibody to haptoglobin was detected by enzyme‐linked immunosorbent assay (ELISA) and Western blot analysis. No antibodies against human leucocyte antigen (HLA) or platelet‐specific antigens were detected. Washed PC and washed red blood cells were effective in preventing the transfusion‐related anaphylactoid reactions.
A new murine monoclonal antibody against Kx protein
Authors: Hattab C. , Blanchard D. , Gane P. , Verkarre V. , Petit‐Leroux Y. , Loirat M.‐J. , Cartron J.‐P. , Bertrand O.
Published: March 2003

Summary. Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross‐reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross‐reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell–Kx complex from RBC membrane extract by immunochromatography.
Transfusion medicine in India: a survey of current practice
Authors: Bray T. J. , Salil P. , Weiss H. A. , Porter J. D. H.
Published: March 2003

Summary. The new Indian National Blood Policy intends to improve the provision of easily accessible safe blood and blood components, available according to need. There is a requirement for information on patterns of blood transfusion in India, to help understand the potential for changes in transfusion practice and the organization of blood services, and to help ensure optimal use of this valuable and limited human resource. This study involved a questionnaire survey of blood transfusion practice with reference to Indian National AIDS Control Organisation clinical guidelines at selected blood centres in four study areas (Delhi, Bangalore, Nasik District and Imphal). Information on 1062 transfusion episodes was analysed. Adult recipients accounted for 87% of transfusions, and amongst the age group of 25–34, 73% of transfusions were to women. Anaemia was listed as a reason for 60% of transfusions, surgery for 42%, acute haemorrhage for 26% and pregnancy for 16%. Seventy‐four per cent of adult transfusions were inappropriate when assessed against criteria derived from government transfusion guidelines. Possible common proximate causes for inappropriate transfusions include unnecessary transfusion for iron‐deficiency anaemia and transfusion as a first choice for volume replacement. Options to promote good transfusion practice in India should be appraised.
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.
Outbreak of Crimean‐Congo haemorrhagic fever in Quetta, Pakistan: contact tracing and risk assessment
Authors: Altaf Arshad , Luby Stephen , Jamil Agha , Najam Ahmed , Aamir Zaidi , Khan Javed , Mirza Sheper , McCormick Joseph , Fisher‐Hoch Susan
Published: March 2003

Summary In December 1994 in a private hospital in Quetta, Pakistan, 3 health‐workers contracted Crimean‐Congo haemorrhagic fever (CCHF) after surgery on a bleeding patient who later died. We conducted a retrospective study to determine transmission risks among contacts. Fifty contacts gave blood for antibody tests and answered questions about exposure. Two of four people exposed percutaneously and one of five with cutaneous exposure contracted CCHF. The person with cutaneous exposure was a surgeon who tore his glove during surgery and noted blood on his hand but no cut. There were no anti‐CCHF antibodies or CCHF cases among persons whose skin came into contact with body fluids other than blood (0/4), who had skin‐to‐skin contact (0/16) with patients or were physically close to them (0/21). Three index case relatives reported that although 10 family members had cutaneous exposure, none developed CCHF. The family refused blood tests. CCHF transmission in resource‐constrained settings can be limited by focusing on avoiding health worker contact with blood.
Towards a kala azar risk map for Sudan: mapping the potential distribution of Phlebotomus orientalis using digital data of environmental variables
Authors: Thomson M. C. , Elnaiern D. A. , Ashford R. W. , Connor S. J.
Published: March 2003

Summary The need to define the geographical distribution of Phlebotomus orientalis results from its importance as the dominant vector of kala azar (visceral leishmaniasis) in Sudan. Recent epidemics of this disease in southern and eastern Sudan caused an estimated 100000 deaths and have renewed the impetus for defining the ecological boundaries of the vector. This information is an essential prerequisite to the production of a risk map for kala azar. This study uses data on the presence and absence of P. orientalis from 44 collecting sites across the central belt of Sudan. A logistic regression model was used to estimate the probability of the presence of P. orientalis at each collecting site as a function of climatic and environmental variables (rainfall; temperature; altitude; soil type and the satellite‐derived environmental proxies – Normalized Difference Vegetation Index and Land Surface Temperature). The logistic regression model indicates mean annual maximum daily temperature and soil type as the most important ecological determinants of P. orientalis distribution. An initial risk map was created in a raster‐based geographical information system which delineates the area where P. orientalis may occur. This map was then refined using a mask layer indicating the known rainfall‐based boundaries of the distribution of Acacia‐Balanites woodland – a woodland type known to be associated with the distribution of this vector. The predictive performance of the risk map is discussed.
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.