bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in GA 20‐oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants
Authors: Carrera Esther , Bou Jordi , García‐Martínez José Luis , Prat Salomé
Published: March 2003

Gene StGA20ox1 encoding potato GA 20‐oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over‐expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20‐oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.
Probing expansin action using cellulose/hemicellulose composites
Authors: Whitney Sarah E. C. , Gidley Mike J. , McQueen‐Mason Simon J.
Published: March 2003

Cellulose‐based composite materials containing xyloglucans or mannan‐based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical α‐expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose‐only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose‐only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1‐4) β‐glucan to (1‐4) β‐glucan hydrogen bonded contacts can also serve as substrates.
Interactions within a network of phytochrome, cryptochrome and UV‐B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue
Authors: Wade Helena K. , Bibikova Tatiana N. , Valentine William J. , Jenkins Gareth I.
Published: March 2003

The Arabidopsis gene encoding the key flavonoid biosynthesis enzyme chalcone synthase (CHS) is regulated by several environmental and endogenous stimuli. Here we dissect the network of light signalling pathways that control CHS expression in mature leaves using cryptochrome (cry) and phytochrome (phy) deficient mutants. The UV‐A/blue light induction of CHS is mediated principally by cry1, but neither cry1 nor cry2 is involved in UV‐B induction or in the UV‐A and blue light signalling pathways that interact synergistically with the UV‐B pathway to enhance CHS expression. Moreover, these synergistic responses do not require phyA or phyB. Phytochrome is a positive regulator of the cry1 inductive pathway, mediating distinct potentiation and coaction effects. A red light pretreatment enhances subsequent cry1‐mediated CHS induction. This potentiation is unaltered in phyA and phyB mutants but much reduced in a phyA phyB double mutant, indicating that it requires principally phyA or phyB. In contrast, the cry1‐mediated induction of CHS, without pretreatment, is much reduced in phyB but not phyA mutants, indicating coaction between cry1 and phyB. Further experiments with phy‐deficient mutants demonstrate that phyB is a negative regulator of the UV‐B inductive pathway. We further show that phyB acts upstream of the points of interaction of the UV‐A and blue synergism pathways with the UV‐B pathway. We propose that phyB functions to balance flux through the cry1 and UV‐B signalling pathways.
Analysis of the SINE S1 Pol III promoter from Brassica; impact of methylation and influence of external sequences
Authors: Arnaud Philippe , Yukawa Yasushi , Lavie Laurence , Pélissier Thierry , Sugiura Masahiro , Deragon Jean‐Marc
Published: March 2003

Transcription is an important control point in the transposable element mobilization process. To better understand the regulation of the plant SINE (Short Interspersed Elements) S1, its promoter sequence was studied using an in vitro pol III transcription system derived from tobacco cells. We show that the internal S1 promoter can be functional although upstream external sequences were found to enhance this basal level of transcription. For one putative ‘master’ locus (na7), three CAA triplets (in positions –12, –7 and –2) and two overlapping TATA motifs (in positions –54 to –43) were important to stimulate transcription. For this locus, two transcription initiation regions were characterized, one centered on position + 1 (first nucleotide of the S1 element) and one centered on position − 19 independently of the internal motifs. The CAA triplets only influence transcription in + 1 and work in association with the internal motifs. We show that methylation can inhibit transcription at the na7 locus. We also observe that S1 RNA is cleaved in a smaller Poly (A) minus product by a process analogous to the maturation of mammalian SINEs.
Generation and properties of ascorbic acid‐deficient transgenic tobacco cells expressing antisense RNA for L‐galactono‐1,4‐lactone dehydrogenase
Authors: Tabata Kazufumi , Ôba Kazuko , Suzuki Kanichi , Esaka Muneharu
Published: March 2003

In higher plants, the terminal step of l‐ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L‐galactono‐1,4‐lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA‐deficient transgenic tobacco BY‐2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY‐2 cells using PCR. Two transgenic cell‐lines, AS1–1 and AS2–2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild‐type BY‐2 cells. In synchronous cultures, division of AS1–1 and AS2–2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild‐type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild‐type cells. The calli of AS1–1 and AS2–2 appeared to be sticky and soft. Back extrusion method also showed that AsA‐deficient BY‐2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1–1 and AS2–2 cells were abnormally slender, suggesting a potential for a significant and a uni‐axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.
Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor
Authors: Karsies Aletta , Hohn Thomas , Leclerc Denis
Published: March 2003

Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N‐terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N‐terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.
Analysis of heat‐shock transcription factor–DNA binding in Arabidopsis suspension cultures by UV laser crosslinking
Authors: Zhang Lemin , Eggers‐Schumacher Gabriele , Schöffl Fritz , Prändl Ralf
Published: March 2003

Crosslinking techniques are important in examining protein–DNA interactions in living cells. Formaldehyde and UV light emitted by conventional lamps are the most commonly used crosslinking agents. The crosslinking step is followed by immunoprecipitation of specific protein–DNA adducts, and by analysis and quantification of the co‐immunoprecipitated DNA. There are a few reported cases of fruitful in vivo protein–DNA crosslinking experiments, but not in plants. In this report, we analyse the binding of heat‐shock transcription factor (HSF) to heat‐shock promoters in intact Arabidopsis cells. Efficient protein–DNA crosslinking by irradiation of Arabidopsis suspension culture tissue was achieved using UV laser pulses. In addition, methods for immunoprecipitation and detection of the co‐immunoprecipitated DNA are reported. Our results suggest that Arabidopsis HSFs immunoreactive for HSF1 antibodies bind constitutively to the HSP18.2 gene.
BZI‐1 specifically heterodimerises with the tobacco bZIP transcription factors BZI‐2, BZI‐3/TBZF and BZI‐4, and is functionally involved in flower development
Authors: Strathmann Anne , Kuhlmann Markus , Heinekamp Thorsten , Dröge‐Laser Wolfgang
Published: March 2003

The tobacco (Nicotiana tabacum) protein BZI‐1 is closely related to the plant bZIP transcription factors CPRF2, G/HBF‐1 and OHP1. Using the C‐terminal part of BZI‐1, which includes the bZIP domain, as a bait in a yeast two‐hybrid screen, three BZI‐1 interacting bZIP transcription factors, referred to as BZI‐2, BZI‐3/TBZF and BZI‐4, were isolated. The observed interactions are due to the leucine zipper dimerisation domain and have been found to be specific, in so far as other bZIP transcription factors do not interact with BZI‐1. The formation of heterodimers is favoured to homodimerisation. Furthermore, physical protein–protein interaction was confirmed by in vitro binding studies. Expression analysis reveals that BZI‐2 mRNA is predominantly located in the stems and parts of the flower. BZI‐3/TBZF expression has been observed predominantly in flowers and, to a lesser extent, in the vegetative parts of the plant. In particular, BZI‐4 is transcribed specifically in the stamen, the petals and the pistils of the tobacco flower. Since BZI‐1 is expressed ubiquitously in tobacco plants, co‐localisation with BZI‐2, BZI‐3/TBZF or BZI‐4 might influence BZI‐1 heterodimerisation and consequently target gene selection. Analysis of transgenic plants displaying changes in BZI‐1 protein level revealed that BZI‐1 regulates BZI‐4 expression. Moreover, a reduction in functional BZI‐1 protein resulted in flowers having a reduced size; in particular, the stamen and the petals are affected. Consequently, BZI‐1 homo‐ or heterodimers are involved in the control of the size of the organs of flowers. Based on these data, we discuss a model postulating BZI transcription factor heterodimerisation as a mechanism determining target gene selection and regulating processes involved in plant development.
Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes fromChlamydomonas reinhardtii
Authors: Hippler Michael , Klein Jens , Fink Andreas , Allinger Tanja , Hoerth Patric
Published: March 2003

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild‐type and mutant strains ofChlamydomonas reinhardtii.Thylakoid membrane proteins were separated by high‐resolution two‐dimensional gel electrophoresis (2‐DE) and analysed by immuno‐blotting and mass spectrometry for the presence of membrane‐spanning proteins. Our data show that light‐harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2‐DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2‐DE revealed the presence of 18 LHCI protein spots. The use of two peptide‐specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post‐translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2‐DE. Comparison between 2‐DE maps from thylakoid membrane proteins isolated from a PSI‐deficient (Δycf4)andacrd1mutant, which is conditionally reduced in PSI and LHCI under copper‐deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2‐DE, and (ii) that high‐resolution 2‐DE facilitates the comparative analysis of membrane protein complexes in wild‐type and mutants cells.
Hydroxyl radical‐induced cell‐wall loosening in vitro and in vivo: implications for the control of elongation growth
Authors: Schopfer Peter
Published: March 2003

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell‐wall polysaccharides in a site‐specific reaction. I investigated the hypothesis that cell‐wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load‐bearing cell‐wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre‐loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin‐induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2–), an OH precursor, in the growth‐controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall‐loosening factor acting in auxin‐mediated elongation growth of plant species with widely differing cell‐wall polysaccharide compositions.
Ribozyme termination of RNA transcripts down‐regulate seed fatty acid genes in transgenic soybean
Authors: Buhr Tony , Sato Shirley , Ebrahim Farida , Xing Aiqiu , Zhou You , Mathiesen Michelle , Schweiger Bruce , Kinney Anthony , Staswick Paul ,   Tom Clemente
Published: March 2003

We investigated whether termination of transcripts with a self‐cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease specific plant gene expression. Nuclear retention was first monitored in tobacco using the β‐glucuronidase gene terminated with either the 35S CaMV 3′ untranslated sequence (UTR) or a cis‐acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme‐terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme‐terminated transcripts were subsequently tested as a gene down‐regulation strategy in soybean. The embryo‐specific Δ‐12 fatty acid desaturase FAD2‐1 gene was targeted because its down‐regulation elevates oleic acid content of seed storage lipids. Both ribozyme‐terminated antisense and standard antisense constructs were capable of gene down‐regulation, producing over 57% oleic acid compared with less than 18% in wild‐type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene down‐regulation and the simultaneous down‐regulation of two embryo‐specific genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2‐1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2‐1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2‐1 and the FatB gene encoding a palmitoyl‐thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to specifically down‐regulate endogenous gene expression in soybean.
The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid and ethylene signalling in cell death and defence against pathogens
Authors: Devadas Sendil K. , Enyedi Alexander , Raina Ramesh
Published: March 2003

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response‐like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA‐ and ET/JA‐responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA‐depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel‐blot analysis of RNA isolated separately from the lesion+ and the lesion– leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA‐responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA‐depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration‐dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR‐1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.
Downregulation of a chloroplast‐targeted β‐amylase leads to a starch‐excess phenotype in leaves
Authors: Scheidig Andreas , Fröhlich Anja , Schulze Silke , Lloyd James R. , Kossmann Jens
Published: March 2003

A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast‐targeted β‐amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional β‐amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the β‐amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques. Analysis of plants reduced in the presence of this β‐amylase isoform showed that their leaves had a starch‐excess phenotype, indicating a defect in starch degradation. In addition, it was shown that the antisense plants degraded only 8–30% of their total starch, in comparison with 50% in the wild type, over the dark period. This is the first time that a physiological role for a β‐amylase in plants has been demonstrated.
Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells
Authors: Pay Aniko , Resch Katja , Frohnmeyer Hanns , Fejes Erzsebet , Nagy Ferenc , Nick Peter
Published: March 2003

In animals and yeast, the small GTP‐binding protein Ran has multiple functions – it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re‐assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP‐like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double‐immunofluorescence shows that RanGAPs are co‐localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co‐assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.
The plant disease resistance gene Asc‐1 prevents disruption of sphingolipid metabolism during AAL‐toxin‐induced programmed cell death
Authors: Spassieva Stefka D. , Markham Jonathan E. , Hille Jacques
Published: March 2003

The nectrotrophic fungus Alternaria alternata f.sp. lycopersici infects tomato plants of the genotype asc/asc by utilizing a host‐selective toxin, AAL‐toxin, that kills the host cells by inducing programmed cell death. Asc‐1 is homologous to genes found in most eukaryotes from yeast to humans, suggesting a conserved function. A yeast strain with deletions in the homologous genes LAG1 and LAC1 was functionally complemented by Asc‐1, indicating that Asc‐1 functions in an analogous manner to the yeast homologues. Examination of the yeast sphingolipids, which are almost absent in the lag1Δlac1Δ mutant, showed that Asc‐1 was able to restore the synthesis of sphingolipids. We therefore examined the biosynthesis of sphingolipids in tomato by labeling leaf discs with l‐[3‐3H]serine. In the absence of AAL‐toxin, there was no detectable difference in sphingolipid labeling between leaf discs from Asc/Asc or asc/asc leaves. In the presence of pathologically significant concentrations of AAL‐toxin however, asc/asc leaf discs showed severely reduced labeling of sphingolipids and increased label in dihydrosphingosine (DHS) and 3‐ketodihydrosphingosine (3‐KDHS). Leaf discs from Asc/Asc leaves responded to AAL‐toxin treatment by incorporating label into different sphingolipid species. The effects of AAL‐toxin on asc/asc leaflets could be partially blocked by the simultaneous application of AAL‐toxin and myriocin. Leaf discs simultaneously treated with AAL‐toxin and myriocin showed no incorporation of label into sphingolipids or long‐chain bases as expected. These results indicate that the presence of Asc‐1 is able to relieve an AAL‐toxin‐induced block on sphingolipid synthesis that would otherwise lead to programmed cell death.
SVISS – a novel transient gene silencing system for gene function discovery and validation in tobacco plants
Authors: Gosselé Véronique , Faché Ina , Meulewaeter Frank , Cornelissen Marc , Metzlaff Michael
Published: March 2003

We developed a novel, two‐component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV‐U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus‐induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well‐characterized bridging species for the Solanaceae plant family.
Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids
Authors: Ceci Luigi R. , Volpicella Mariateresa , Rahbé Yvan , Gallerani Raffaele , Beekwilder Jules , Jongsma Maarten A.
Published: March 2003

The mustard trypsin inhibitor, MTI‐2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI‐2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 × 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI‐2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild‐type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI‐2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 µg ml−1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild‐type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.
The frequency of human platelet antigen (HPA) genotypes in the Polish population
Authors: Drzewek  , Brojer  , Z˙upańska
Published: March 2003

The human platelet antigens (HPA1–5) in the Polish population were investigated using the PCR with sequence specific primers (SSP). The HPA gene frequency was: 1a — 0.874, 1b — 0.126; 2a — 0.898, 2b — 0.102; 3a — 0.592, 3b — 0.408; 4a — 1.00, 4b — 0.00; 5a — 0.937, 5b — 0.063. The HPA2 and HPA5 differed from those observed in some other European populations — German and both German and Austrian, respectively. The HPA5 alleles frequency was most similar to those observed in the Finnish population. The modification of SSP described allowed the genotyping of HPA1–4 under the same PCR conditions.
Monitoring the clearance of fetal RhD‐positive red cells in FMH following RhD immunoglobulin administration
Authors: Lubenko  , Williams  , Johnson  , Pluck  , Armstrong  , MacLennan
Published: March 2003

Anti‐RhD immunoglobulin was administered to RhD‐negative women based on estimates of fetal bleed size obtained using a direct immunofluorescence flow cytometric technique employing a FITC‐conjugated monoclonal human anti‐D (BRAD 3). The effectiveness of the dose administered was assessed by (i) measuring the fraction of RhD‐positive fetal cells in the maternal circulation at d0, and between d2 and d10 post RhD Ig administration, (ii) quantifying the amount of anti‐D detectable in maternal plasma following RhD Ig injection in the perinatal period and (iii) assessing maternal serum for the presence of immune anti‐D in follow‐up samples taken 3 months to 3 years after delivery. Fifty‐four women were assessed, 29 having fetal bleeds in excess of 4 mL. Follow‐up samples were received from 20/29 mothers after RhD Ig administration; 43–99% and 69–99% of fetal cells had been cleared by d2/3 and d5/6, respectively, in 14/20 mothers, whereas less than 50% had been cleared in the remaining mothers. Long‐term follow‐up samples were obtained from eight of the 29 mothers (four with bleeds ≥20 mL, two with bleeds >95 mL): none had detectable anti‐D in the serum 4 months to 3 years after delivery despite the persistence of up to 36% fetal RhD‐positive cells in the maternal circulation six days after delivery.
A randomized clinical trial comparing intravenous immunoglobulin with high‐dose intravenous methylprednisolone, with or without subsequent oral prednisone administration, in adults with previously untreated severe autoimmune thrombocytopenic purpura
Authors: Godeau B. B. , Chevret S. , Varet B. , Zini J. M. , Bassompierre F. , Bierling P.
Published: March 2003

To compare the response to intravenous immunoglobulin (IVIg) with the response to high‐dose methylprednisolone (HDMP) in previously untreated adults with severe AITP and to determine if oral prednisone (PRDN) is required after IVIg or HDMP infusions.

M ethods 

116 adults, previously untreated, with platelet count ≤ 20 × 109/L were assigned by simultaneous double randomization to receive either IVIg (0·7 g/kg body weight per day) or HDMP (15 mg/kg body weight per day, total daily dose less than 1 g) from day 1 to day 3 (randomization A), and then to receive either PRDN, 1 mg/kg body weight per day, or a placebo (randomization B), from day 4 to day 21 in blinded manner.

R esults 

Between day 1 and day 21, the number of days on which platelet counts were above 50 × 109/L and above 20 × 109/L was significantly higher among the 56 patients receiving IVIg than among the 60 patients receiving HDMP (P = 0·008 and P = 0·013, respectively), and the percentage of patients who had a platelet count exceeding 50 × 109/L on day 5 was also higher in the IVIg group. During the second treatment period, PRDN was more effective than the placebo on all short‐term end points. Patients who received IVIg followed by PRDN had a significantly larger number of days with platelet counts > 50 × 109/L and > 20 × 109/L between day 1 and day 21 (P = 0·008 and P = 0·005, respectively) than patients treated with HDMP followed by PRDN. As regards the long‐term response, the percentage of remitters was 40% overall and was not influenced by the treatment allocation in either randomization period (A or B).

C onclusion 

IVIg followed by PRDN appears to be the more effective treatment in adults with severe AITP.
Vigam‐S, a solvent/detergent‐treated intravenous immunoglobulin, in idiopathic thrombocytopenic purpura
Authors: Newland A. C. , Burton I. , Cavenagh J. D. , Copplestone A. , Dolan G. , Houghton J. , Reilly T.
Published: March 2003

. The efficacy of Vigam‐S, a highly purified intravenous immunoglobulin, was investigated by an open, noncomparative study in 20 adults with chronic idiopathic thrombocytopenic purpura (ITP). Fifteen patients responded to the initial 3‐day infusion of 0·4 g kg −1 day−1 by exhibiting an incremental increase in platelet count of ≥ 30 × 109 L−1, in eight of whom platelet count normalized (> 150 × 109 L−1). The peak platelet count for responders on day 4 was 163 × 109 L−1 (baseline = 18 × 109 L−1). No benefit was derived from an extra 2 days infusion in nonresponders. Further treatment (either a single 0·8 g kg−1 dose or another 3‐day infusion) given to responders when platelet counts fell below 30 × 109 L−1 was effective on eight of 14 occasions. Increases in total serum IgG concentration (to a mean peak of 25·3 g L−1) were not correlated with platelet response. There was no evidence of seroconversion to virus markers, or of alteration in renal function, following Vigam‐S infusion. Most adverse events were mild and transient; however, three patients had severe headache and vomiting (possible aseptic meningitis syndrome) and one had marked superficial thrombophlebitis. Therefore Vigam‐S provides effective and well tolerated therapy in the management of adults with ITP although individual patient response remains difficult to predict.
Expression of CD2 and activation markers on blood T‐helper cell subsets in patients with transfusional iron overload
Authors: Jensen P.‐D. , Heickendorff L. , Carlson I. , Jensen F. T. , Christensen T. , Ellegaard J.
Published: March 2003

. The aim of the present study was to investigate the relationship between different measures of iron status, and the expression of CD2, and the activation markers CD25, CD71, CD45RO, HLADR CD38 within the Th‐cell subset in patients with progressive transfusional iron overload. We estimated the expression of the activation surface markers on the Th cells of peripheral blood by flow cytometry from 22 multiply transfused patients. The number of CD2 binding sites (BS) on Th cells was significantly higher in the patients (82 917 ± 30 801) than in age‐matched normal controls (41 145 ± 6989, P < 0·0001). When investigating whether this difference could be due to the iron overload we found the number of CD2 BS closely related to the iron saturation of serum transferrin (TfS) (R2 = 0·78, P < 0·001). The relationship to the serum ferritin concentration and to the number of blood units given was weaker, but also significant (R2 = 0·22, P < 0·027, respectively, R2 = 0·21, P < 0·032). Also the fraction of mature memory Th cells which express CD45RO at a high level was directly related to the TfS (R2 = 0·57, P < 0·0001), while the expression of CD38 within the Th cell fraction was inversely related to the TfS (R2 = − 0·43, P = 0·009). The expression of HLA‐DR (but not of CD25 and CD71) was also directly related to the TfS (R2 = 0·29, P = 0·01). Our results show a clear, statistical relationship between the iron status and the expression of surface markers within Th cells in multiply transfused patients.
Perceptions and motivations of Canadian autologous blood donors
Authors: Moltzan C. , Proulx N. , Bormanis J. , Lander N. , DeGroot H. , Rock G.
Published: March 2003

. Preoperative autologous blood donation in Canada has increased in the last decade due to concerns about allogeneic blood safety. As economic policies necessitate the validation of autologous blood donation, it is important to assess potential changes. This study examines the motivations and perceptions of patients participating in a Canadian autologous blood donation programme.

The study was conducted at the Ottawa Hospital, Civic Campus. Questionnaires were developed and then administered to 100 consecutive adult autologous blood donors. The questionnaires assessed patient demographics, motivation for participation and perceptions about blood safety.

Autologous donation was presented to 82% of patients as an option by their physicians. However, 59% of all patients felt that they were motivated to donate by their own fears. Patients (87%) also felt that directed blood donation should be offered due to their perception that directed donation is safer. Three‐quarters of patients would be willing to pay for autologous donation.

Patients are concerned about the safety of allogeneic blood and they are motivated to seek other alternatives despite the fact that allogeneic blood is very safe. These perceptions and motivations must be taken into account as health care policy changes are considered.
Autologous placental blood transfusion after a planned neonatal pacemaker implantation
Authors: Domanović D. , Završnik T. , Vesel S.
Published: March 2003

. A fetal third‐degree atrio‐ventricular block was diagnosed in a 22‐year‐old woman at 24 weeks of gestation. During clinical follow‐up a mild to moderate tricuspid regurgitation was detected at 35 weeks and maternal connective tissue diseases were excluded. Early postnatal cardiac pacing therapy was planned and autologous placental blood transfusion was proposed for the treatment of probable blood loss due to pacemaker implantation. A male infant was delivered at 38 weeks vaginaly and 87 mL of placental blood was collected from the undelivered placenta. The placental blood was negative for viral markers and syphilis. Subsequent tests for bacteriological cultures were also negative. Within 6 h of delivery, the baby underwent cardiac pacemaker implantation and received 45 mL of autologous placental blood. Autologous placental blood transfusion was successfully used for the treatment of predicted blood loss after a planned neonatal surgical procedure.
In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression
Authors: Chambers D. R. , Procter J. , Muratova O. , Byrne K. , Keister D. , Shanks D. , Magill A. , Stroncek D.
Published: March 2003

Summary  Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/collections before and after exposure to P. falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite, and blinded serologic studies were completed. CD35 (complement receptor 1), CD55 (decay‐accelerating factor), CD59 (membrane inhibitor of reactive lysis) and CD47 (integrin‐associated protein) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa‐stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty‐three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.
A prospective study of blood transfusion history and liver cancer in a high‐endemic area of Japan
Authors: Fujino Y. , Mizoue T. , Tokui N. , Yoshimura T.
Published: March 2003

SummaryBackground: The high prevalence of liver cancer in Japan may be owing to infection with hepatitis C virus (HCV), for which the major transmission route is blood transfusion. However, among the general population, there have been few follow‐up studies of people with a history of transfusion. The purpose of this study was to examine whether such a history correlates with the development of liver cancer among the general population of this high‐endemic area.

Study design and methods: The baseline survey was conducted between 1986 and 1991 among the population (15 597 people, aged 30–79) of Fukuoka prefecture, Japan. The participants were assessed for their transfusion history and other covariates using a baseline questionnaire. The relative risk (RR) of blood transfusion for liver cancer was estimated using the Cox proportional hazard model.

Results: The RRs of transfusion for subsequent liver cancer were 1·86 (95% confidence interval (CI) = 1·05–3·29) in men and 4·20 (95% CI = 1·83–9·61) in women, after adjusting for covariates. Transfusion was associated with an increased risk of liver cancer, mainly in the group without hepatitis and cirrhosis (RR = 1·81, 95% CI = 0·84–3·91 – men; RR = 4·39, 95% CI = 1·37–14·02 – women).

Conclusion: A history of blood transfusion carried a significant risk of developing liver cancer in the study region. The people with a history of transfusion should be monitored more aggressively for viral infections and liver disease, as they may not report the infection or they may be unaware that they are infected.
Use of hospital data for Safe Motherhood programmes in South Kalimantan, Indonesia
Authors: Ronsmans Carine , Achadi Endang , Sutratikto Gunawan , Zazri Ali , McDermott Jeanne
Published: March 2003

Summary The evaluation of Safe Motherhood programmes has been hampered by difficulties in measuring the preferred outcomes of maternal mortality and morbidity. The need for adequate indicators has led researchers and programme managers alike to resort to indicators of utilization and quality of health services. In this study we assess the magnitude of four indicators of use of essential obstetric care (EOC) and one indicator of quality of care in health facilities in three districts in South Kalimantan, Indonesia. The general picture which emerges for South Kalimantan is that the use of obstetric services is low. Even in the more urban district of Banjar where facility‐based coverage is highest, fewer than 14% of all deliveries take place in an EOC facility, 2% of expected births are admitted to such a facility with a major obstetric intervention (MOI), and 1% of expected births have an MOI for an absolute maternal indication. The use of facility‐based EOC is consistently lower in Barito Kuala compared to the other districts, and the differences persist regardless of the indicators used. In this setting with low utilization rates, general rates of utilization of EOC facilities seem to be as satisfactory an indicator of relative access to EOC as more elaborate indicators specifying the reasons for admission. The inequalities in access to care revealed by the various indicators of use of EOC services may prove to be a more powerful stimulus for change than the widely reported and highly inaccurate accounts of the high levels of maternal mortality.
Short communication: Wuchereria bancrofti antigenaemia in Sri Lanka
Authors: Itoh M. , Weerasooriya M. V. , Gunawardena N. K. , Mudalige M. P. S. , Samarawickrema W. A. , Kimura E.
Published: March 2003

The prevalence of Wuchereria bancrofti antigenaemia determined in 353 subjects in Matara, Sri Lanka by Og4C3 ELISA was 20.7%. Positive rates obtained with the same subjects by 1 ml Nuclepore filtration and 60 Âµl thick blood smear were 11.3% and 7.9%, respectively. Antigen levels were positively associated with microfilaria counts. Two‐thirds of antigen‐positive and microfilaria‐negative (Ag+/Mf‐) individuals were > 25‐year‐old, but younger age groups (≤ 25‐year‐old) tended to have proportionally more Ag+/Mf‐ cases. Possible origins of the Ag+/Mf‐ status are discussed.
Morbidity assessment in urinary schistosomiasis infection through ultrasonography and measurement of eosinophil cationic protein (ECP) in urine
Authors: Leutscher Peter D. C. , Reimert Claus M. , Vennervald Birgitte J. , Ravaoalimalala Voahangy E. , Ramarokoto Charles E. , Serieye Jean , Raobelison Ando , Rasendramino Mamy , Christensen Niels O. , Esterre Philippe
Published: March 2003

Summary In a Schistosoma haematobium‐endemic village in western Madagascar we evaluated ultrasonography and Eosinophil Cationic Protein (ECP) in urine as means to detect the associated urinary tract pathology. 192 individuals were matched according to age and sex, and grouped into infected persons with bladder and, if present, kidney pathology (n= 96); infected persons without pathology (n= 48) and noninfected persons without pathology (n= 48). The median urinary egg count was significantly higher in individuals with ultrasonographically detectable urinary tract pathology (115 eggs/10 ml urine) than in infected persons without (45 eggs/10 ml of urine). At 136 ng/ml, the median ECP level was significantly higher in the 144 infected individuals than in the 48 noninfected persons (0.35 ng/ml). Egg excretion correlated positively with ECP level. The median ECP level was significantly higher in the group with ultrasonographically detectable urinary tract pathology than in the group without (183 ng/ml vs. 67 ng/ml). The results suggest that minor degrees of pathology, particularly at an early stage of infection with S. haematobium, might be overlooked by ultrasonography despite the presence of marked inflammation, as indicated by markedly increased urinary ECP levels in infected individuals without ultrasonographically detectable urinary tract pathology. ECP may therefore provide important information on the evolution of S. haematobium‐associated urinary tract morbidity.
Genotypic patterns of multiple isolates of M. tuberculosis from tuberculous HIV patients
Authors: Lourenço M. C. S. , Grinsztejn B. , Fandinho‐Montes F. C. O. , da Silva M. G. , Saad M. H. F. , Fonseca L. S.
Published: March 2003

Summary We investigated whether the recurrence of tuberculosis in HIV‐infected patients is due to an exogenous reinfection or relapses after antituberculosis chemotherapy. We reviewed clinical information on 32 patients at a Rio de Janeiro hospital from whom multiple Mycobacterium tuberculosis isolates were taken. All isolates were analysed by DRE‐PCR fingerprinting technique, and those with identical DRE‐PCR patterns were analysed by the RFLP method. Twenty patients had M. tuberculosis simultaneously isolated from different organs. These patients and nine others with sequential positive cultures after 2 months of therapy showed stable DRE‐PCR and RFLP patterns. One patient's isolate became resistant to isoniazid, but the molecular pattern remained unchanged despite the development of drug resistance. In three patients, the DRE‐PCR patterns of the isolates changed dramatically. Clinical and microbiological evidence was consistent with active tuberculosis caused by a new strain of M. tuberculosis. The exogenous reinfection of the three patients was not due to an outbreak, but the isolates from each patient showed unique patterns.