bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrotransposon BARE‐1: expression of encoded proteins and formation of virus‐like particles in barley cells
Authors: Jääskeläinen Marko , Mykkänen Anna‐Helena , Arna Tiina , Vicient Carlos M. , Suoniemi Annu , Kalendar Ruslan , Savilahti Harri , Schulman Alan H.
Published: March 2003

Retrotransposons are ubiquitous and major components of plant genomes, and are characteristically retroviral‐like in their genomic structure and in the major proteins encoded. Nevertheless, few have been directly demonstrated to be transcribed or reverse transcribed. The BARE‐1 retrotransposon family of barley (Hordeum vulgare) is highly prevalent, actively transcribed, and contains well conserved functional regions. Insertion sites for BARE‐1 are highly polymorphic in the barley genome. Here we show that BARE‐1 is translated and the capsid protein (GAG) and integrase (IN) components of the predicted polyprotein are processed into polypeptides of expected size. Some of the GAG sediments as virus‐like particles together with IN and with BARE‐1 cDNA. Reverse transcriptase activity is also present in gradient fractions containing BARE‐1 translation products. Virus‐like particles have also been visualized in fractions containing BARE‐1 components. Thus BARE‐1 components necessary for carrying out the life cycle of an active retrotransposon appear to be present in vivo, and to assemble. This would suggest that post‐translational mechanisms may be at work to prevent rapid genome inflation through unrestricted integration.
Display and isolation of transposon‐flanking sequences starting from genomic DNA or RNA
Authors: Yephremov Alexander , Saedler Heinz
Published: March 2003

Insertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation‐mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon‐flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5′ RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.
Post‐translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14‐3‐3 protein interaction
Authors: Finnemann Jørgen , Schjoerring Jan K.
Published: March 2003

Regulation of the cytosolic isozyme of glutamine synthetase (GS1; EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS1 was the only active GS isozyme in senescing leaves. By use of [γ‐32P]ATP followed by immunodetection, it was shown that GS1 is a phospho‐protein. GS1 is regulated post‐translationally by reversible phosphorylation catalysed by protein kinases and microcystin‐sensitive serine/threonine protein phosphatases. Dephosphorylated GS1 is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS1 changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS1 interacts with 14‐3‐3 proteins as verified by two different methods: a His‐tag 14‐3‐3 protein column affinity method combined with immunodetection, and a far‐Western method with overlay of 14‐3‐3–GFP. The degree of interaction with 14‐3‐3‐proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS1. Thus, the results demonstrate that 14‐3‐3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post‐translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14‐3‐3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.
Gibberellin response mutants identified by luciferase imaging
Authors: Meier Carsten , Bouquin Thomas , Nielsen Mads Eggert , Raventos Dora , Mattsson Ole , Rocher Anne , Schomburg Fritz , Amasino Richard M. , Mundy John
Published: March 2003

The gibberellin (GA) 20‐oxidase encoded by Arabidopsis GA5 catalyzes the synthesis of active GAs. GA5 is a regulatory step in GA biosynthesis as GA5 mRNA levels are negatively regulated by its bioactive GA products. A fusion between the GA5 promoter and the firefly luciferase reporter (GA5‐LUC) was shown to be similarly regulated, indicating GA feedback of GA5 occurs at the transcriptional level. The fidelity of the GA5‐LUC reporter permitted a fusion genetic screen to identify mutants altered in transgene expression. This bioimaging screen identified two types of recessive mutants with increased LUC activity and apparent GA‐related growth phenotypes, a dwarf (lue1) and two late flowering mutants (fpa1‐3 and fpa1‐4). Mutant progeny exhibited altered levels of LUC and of endogenous GA5 and other GA‐regulated mRNAs. SSLP‐based mapping localized lue1 to chromosome I near the ga2 locus, although complementation analyzes showed that lue1 is not allelic to ga2. Mapping and complementation analyzes showed that the late flowering mutants are allelic to fpa1. This provides genetic evidence for crosstalk between the autonomous and gibberellin‐dependent flowering pathways.
Restructuring of wall‐bound xyloglucan by transglycosylation in living plant cells
Authors: Thompson James E. , Fry Stephen C.
Published: March 2003

Xyloglucan endotransglycosylases (XETs) cleave and then re‐join xyloglucan chains and may thus contribute to both wall‐assembly and wall‐loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: ‘integrational’ (in which a newly secreted xyloglucan reacts with a previously wall‐bound one) and ‘restructuring’ (in which one previously wall‐bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in ‘heavy’ or ‘light’ media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml−1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2‐h pulse of l‐[1−3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non‐radioactive walls and chased by light, non‐radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall‐bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall‐bound non‐radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall‐bound [3H]xyloglucan (2−11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall‐bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall‐assembly and ‐loosening.
Coregulation of light‐harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye
Authors: Pursiheimo Saijaliisa , Mulo Paula , Rintamäki Eevi , Aro Eva‐Mari
Published: March 2003

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light‐harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short‐term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.
Molecular cloning and functional identification of (+)‐δ‐cadinene‐8‐hydroxylase, a cytochrome P450 mono‐oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis
Authors: Luo Ping , Wang Yan‐Hong , Wang Guo‐Dong , Essenberg Margaret , Chen Xiao‐Ya
Published: March 2003

In cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)‐δ‐cadinene, forming 8‐hydroxy‐(+)‐δ‐cadinene. This P450 mono‐oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)‐δ‐cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.
Overexpression of RCN1 and RCN2, rice TERMINAL FLOWER 1/CENTRORADIALIS homologs, confers delay of phase transition and altered panicle morphology in rice
Authors: Nakagawa Mayu , Shimamoto Ko , Kyozuka Junko
Published: March 2003

TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)‐like genes play important roles in determining plant architecture, mainly by controlling the timing of phase transition. To investigate the possibility of similar mechanisms operating in the control of inflorescence architecture in rice, we analysed the functions of RCN1 and RCN2, rice TFL1/CEN homologs. Constitutive overexpression of RCN1 or RCN2 in Arabidopsis caused a late‐flowering and highly branching phenotype, indicating that they possess conserved biochemical functions as TFL1. In 35S::RCN1 and 35S::RCN2 transgenic rice plants, the delay of transition to the reproductive phase was observed. The transgenic rice plants exhibited a more branched, denser panicle morphology. Detailed observation of the panicle structure revealed that the phase change from the branch shoot to the floral meristem state was also delayed, leading to the generation of higher‐order panicle branches. These results suggest rice has a pathway that can respond to the overexpressed TFL1/CEN‐like functions, and the molecular mechanisms controlling the phase transition of meristems are conserved between grass and dicot species, at least to some extent.
Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence‐like response and reduced cytokinin levels
Authors: Masferrer Angela , Arró Montserrat , Manzano David , Schaller Hubert , Fernández‐Busquets Xavier , Moncaleán Paloma , Fernández Belén , Cunillera Núria , Boronat Albert , Ferrer Albert
Published: March 2003

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8‐ to 12‐fold as compared to wild‐type plants), the content of sterols and the levels of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence‐like phenotype and grew less vigorously than wild‐type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2‐isopentenyladenine (2‐iP) recovered the wild‐type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin‐type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild‐type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.
Regulation of Arabidopsis thaliana Em genes: role of ABI5
Authors: Carles Cristel , Bies‐Etheve Natacha , Aspart Lorette , Léon‐Kloosterziel Karen M. , Koornneef Maarten , Echeverria Manuel , Delseny Michel
Published: March 2003

In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA showed that the most affected mutant had a weak abi phenotype. Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5–5. In addition to reducing the final level of Em transcripts in the dry seed, the abi5–5 mutation causes a delay in the accumulation of AtEm1 during seed development. An additional characteristic of the abi5–5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol. The abi5–5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene. The wild type and mutant recombinant proteins were produced in E. coli and were assayed for DNA‐binding activity on their target promoters by electrophoretic mobility shift assay (EMSA). The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting. Among the ABRE‐type sequences selected on both Em promoters, the G‐box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.
Multiple plastid signals regulate the expression of the pea plastocyanin gene in pea and transgenic tobacco plants
Authors: Sullivan James A. , Gray John C.
Published: March 2003

The expression of nuclear genes encoding photosynthesis‐related proteins is regulated by signals from plastids. To investigate how the pea PetE gene encoding plastocyanin is regulated by plastid signals, the effects of norflurazon, lincomycin and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), specific inhibitors of plastid‐located processes generating plastid signals, have been examined. RNA‐gel blot analysis of 7‐day‐old pea and tobacco seedlings containing the pea PetE gene showed that treatment with norflurazon and lincomycin, but not DCMU, decreased the accumulation of transcripts of pea PetE and endogenous Lhcb1 genes. Analysis of chimeric PetE gene constructs in tobacco seedlings showed that an intact PetE mRNA 5′ terminus and elements within the PetE coding region were required to confer sensitivity to norflurazon and lincomycin, suggesting post‐transcriptional regulation. Analysis of 4‐week‐old tobacco plants containing chimeric PetE constructs showed that DCMU treatment decreased the accumulation of pea PetE and Lhcb1 transcripts, but had opposite effects on the transcription of the genes in nuclear run‐on assays. DCMU upregulated transcription from the pea PetE promoter whereas transcription of tobacco Lhcb1 genes was decreased. These experiments provide evidence for multiple plastid signals operating at different developmental stages and affecting transcriptional and post‐transcriptional processes regulating expression of the pea PetE gene.
Identification of an essential component of the elicitation active site of the EIX protein elicitor
Authors: Rotblat Barak , Enshell‐Seijffers David , Gershoni Jonathan M. , Schuster Silvia , Avni Adi
Published: March 2003

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen‐related proteins and hypersensitive responses (HR). Pathogen‐derived elicitors trigger these defense responses. The Elicitor Ethylene‐inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX‐specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed β‐strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the β‐1‐4‐endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.
New reference reagent for the quality assurance of anti‐D antibody detection
Authors: Phillips  , Voak  , Downie  , Clark  , Miguta  , Rawlings  , Patel  , Redman
Published: March 2003

A UK BTS‐NIBSC freeze‐dried anti‐D preparation has been prepared and used to monitor the performance of routine antibody detection tests and of the test operators. With the day‐to‐day use of this preparation, adverse changes in test performance and in test operator may be detected and appropriate action taken before the effect becomes significant. Two dilutions of this preparation have been defined, one which should be detected unequivocally in every test; the other, more dilute, may not be detected in every test but is used to monitor changes in performance. Experience with the use of this preparation is reported from three test centres undertaking routine antibody detection tests. By monitoring results over a series of working days, significant differences were noted in operator performance within one test centre, as was a reduced sensitivity of a given test system within one test centre compared with the same system in the other test centres. These differences were detected only by monitoring the results obtained with this preparation.
Evaluation of two new integrated adapters for blood drawing in closed blood bag systems: influence on different molecular coagulation markers
Authors: Siemens  , Klüter  , Brückner  , Kirchner  , Katus  , Wagner
Published: March 2003

We compared a conventional blood donation bag system (A) with two newly developed ones (B, C) with integrated Y‐shaped adapters. The new systems facilitate the withdrawal of blood samples in a closed system under aseptic conditions. The purpose of the study was to determine whether disturbances during blood donation in the different tubings influence the quality of the donated plasma due to activation processes of the coagulation system. In all bag samples from 34 probands for each system, we found higher levels of the global tests (aPTT, TT) compared to intraindividual controls. This was due to a greater amount of anticoagulant in the bag samples. An increase of procoagulant activity was demonstrated for bags A and C. Concerning the fibrinolytic system, values were reduced in all three systems compared to controls. In summary, the quality of the plasma in the three different bag devices was comparable to controls concerning factor activities measured as global tests. The activation of the coagulation system was only slightly higher in the new systems B and C than in the conventional one (A). Therefore, the new closed systems are suitable for blood donation and reveal a high coagulation quality of the plasma.
Fine specificities of murine anti‐Mg monoclonal antibodies
Authors: Jaśkiewicz  , Blanchard  , Rasamoelisolo  , Loirat  , Moulds  , Lisowska
Published: March 2003

The specificities of two murine anti‐Mg monoclonal IgG1 antibodies, 3B10 and 2D5, were determined by pepscan analysis. The peptides which correspond to various fragments of amino‐terminal portions of glycophorin A of group M (GPA‐M), N (GPA‐N) and Mg (GPA‐Mg), and replacement analogues of some of these peptides, were synthesized on plastic pins and tested for binding of the antibodies. Both antibodies bound strongly to the N‐terminal Mg octapeptide 1LSTNEVAM8, but they showed different subspecificities. The essential fragment of the epitope 2D5 are amino acid residues 2STNEV6. Replacement of any of these amino acid residues by Ala, and replacement of Glu5 residue by Gly, abolished or strongly reduced the antibody binding, but replacement of Asn4 by Thr gave only a moderate decrease of peptide activity. In contrast, the Leu1 and Asn4 residues were most essential components of the epitope 3B10, while Ser2, Thr3 and Glu5 seemed to be less important. Our present results and earlier ones on the specificity of human anti‐Mg alloantibodies and monoclonal anti‐M/Mg antibodies showed that antibodies reacting with Mg antigen recognize different fragments and/or different amino acid residues of the amino‐ terminal nonglycosylated domain of GPA‐Mg. The knowledge of fine specificities of antibodies reacting with Mg antigen is interesting in view of the presence of anti‐Mg alloantibodies in 1–2% of human sera.
Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA‐directed probes coupled to electrochemiluminescence
Authors: Chaney  , Rider  , Pamphilon
Published: March 2003

Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin‐ and a ruthenium (ORIGEN®)‐labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin‐coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium‐labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram‐negative and ‐positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL−1. Signals were generated in the range of 1400–80 000 and 3500–500 000 ECL units for unwashed and washed samples, respectively. This is equivalent to 105–108 CFU mL−1. These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.
Prospective audit of cytomegalovirus‐negative blood product utilization in haematology/oncology patients
Authors: Chang  , Hawes  , Hall  , Fuller  , Francombe  , Zuber  , Sher
Published: March 2003

The transfusion management of immunocompromised patients often requires special blood product use such as cytomegalovirus (CMV)‐negative cellular products, which are more costly than standard blood products and occasionally in short supply. We audited the use of CMV‐negative products in haematology/oncology patients to determine the appropriateness of their use. A concurrent‐prospective audit was conducted of all orders for CMV‐negative packed red blood cell (PRBC) and platelet products in 201 haematology/ oncology patients. Once CMV serostatus was determined, orders for inappropriate CMV‐negative units were cancelled, and filled as CMV untested units. During the 21‐month period of this audit, the rates of inappropriate transfusions decreased for PRBC from 73.2% to 14.3% (χ2 = 68.4, P < 0.001) and for platelets from 68.1% to 10.6% (χ2 = 65.6, P < 0.001). The median time to cancellation of inappropriate CMV‐negative orders was 11 days. This audit resulted in estimated cost savings of $16 500 over the 21‐month duration. Inappropriate requests for scarce and expensive blood products are substantially reduced by concurrent‐ prospective auditing of transfusion practice, in a manner that is both simple and cost effective.
Characteristics of hepatitis C‐positive blood donors in Victoria, Australia
Authors: Wong  , Dodd  , Kiely  , Carroll  , Whyte
Published: March 2003

Data from 3156 voluntary blood donors in Victoria, Australia, who were repeatedly reactive on anti‐HCV screening test were analysed. It showed that between 1990 and 1997, 0.97% of routine blood donors were anti‐HCV repeat reactive. Sixty‐four (64) donors tested positive for HCV RNA. They were more likely to be male, had a mean age of 32.7 ± 10.9 years and a mean serum ALT level of 70.9 ± 46.5 iu L−1: the latter value being significantly different from donors with biological false reactivity (P < 0.0001). Among HCV antibody‐positive donors there was a significantly increased rate of prior injecting drug use (34 vs. 1%), transfusion (25 vs. 10%) and tattooing (31 vs. 7%) than among donors with biological false reactivity (all P < 0.0001). The association of these risk factors is stronger with positivity for both immunoblot and HCV RNA than immunoblot alone. The data suggest that efforts should be made to increase the efficacy of donor questioning about prior drug use.
In vitro properties of red cells prepared from half‐strength citrate CPD/RAS‐2 (Erythro‐Sol) donations in PL‐146 plastic
Authors: Hornsey  , MacDonald  , Drummond  , Bethel  , Walker  , Prowse
Published: March 2003

Whole blood donations were collected into 0.5CPD anticoagulant in PL‐146 plastic. This was shown to improve the stability of plasma FVIII levels when compared with CPD. RAS‐2 was used as additive and this improved the in vitro properties of the red cells, such that post processing 2,3‐DPG levels were maintained for 21 days and ATP levels were maintained for 28 days. Whether or not such improvements in red cell properties yield a benefit in clinical use remains to be established.
The ‘transfusion trigger’ in three large West of Scotland hospitals: medical staff questionnaire survey
Authors: Garrioch  , Sandbach  , McIlveney
Published: March 2003

Efficient use of blood is desirable due to increased demand for blood, shortfall in donations, and the risk of mismatch transfusion accidents and transfusion‐transmitted infections. We surveyed transfusion practice in three hospitals. Low haemoglobin was a feature in 91% of transfusion requests and is the commonest ‘trigger’ for transfusion. Forty‐eight per cent of transfusions were initiated with the aim of restoring haemoglobin to 10 g dL−1. Seventy‐three per cent of transfusions were given when the patient's haemoglobin was between 7 and 10 g dL−1. These figures suggest liberal blood use when compared with international practice. Major healthcare resource and patient safety implications result from this. Evidence‐based guidelines issued at both local and national levels would be helpful.
Antigen capture ELISA for platelet antibody detection: choice of conjugate influences assay result
Authors: Lubenko  , Savage
Published: March 2003

The performance of an anti‐IgG/A/M and two anti‐IgG alkaline phosphatase (AP) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) contained in 11 sera. Three anti‐HPA‐1a and one anti‐HPA‐3a were not detected by the anti‐IgG/A/M conjugate, but both anti‐IgG conjugates detected all HPA antibodies as a consequence of increased sensitivity in detecting specific antibody binding. This increase varied from 10% to 500%, depending on the serum being tested. The increased sensitivity in some instances was also accompanied by an apparent increase in nonspecific binding, as measured by activity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti‐IgG conjugates would appear to be preferable for detecting platelet‐reactive antibodies in many clinical situations; however, the choice of anti‐IgG conjugate should be made judiciously (and appropriately validated), in order to avoid false positive results arising from increased nonspecific binding, which may otherwise be erroneously attributed to the presence of auto‐antibodies in the serum under investigation.
HLA class II Ab in TRALI donors
Authors: Lubenko A. , Rodi K. , Savage J. , Hilton D.
Published: March 2003

TRALI (Transfusion Related Acute Lung Injury) occurs in 11–16 patients and is associated with 3–4 deaths per year in the UK. It is caused by HLA (e.g. anti‐HLA‐A2), neutrophil (e.g. anti‐NA2) and leucocyte‐panreactive (e.g. anti‐5b) Ab. Recently an IvIg preparation enriched for HLA class I and II Ab caused TRALI in an untransfused male volunteer (Dooren et al. Lancet 1998; 358: 1601–1602). We therefore ascertained the incidence of class II Ab in sera in donors implicated in TRALI using commercial ELISA kits.

Sera from 32 donors were tested for HLA class II antibodies using GTI B‐Screen ELISA kits. All of these sera had previously been tested for leucocyte‐reactive Ab using the GLAM assay, and for HLA class I Ab by LCT and ELISA (GTI QuikScreen® kits). The following reaction patterns were obtained:

Hence HLA class II Ab were detected in 8/32 (25%) of sera implicated in TRALI. We suspect that they are more frequent than neutrophil‐specific Ab in TRALI donors: we identified the latter in only 8/123 (6·5%) donors tested in the course of investigating 31 TRALI cases, i.e. in 6/21 (28%) donors who were LCT‐GLAM+ and in 2/9 (22%) who were LCT+GLAM+.
Anti‐HPA1a antibodies induce CD62P and fibrinogen binding
Authors: Koren D. , Janisiw M. , Eichelberger B. , Panzer S.
Published: March 2003

B ackground 

Alloantibodies to platelet antigens may affect platelet function by delivering stimulatory or inhibitory signals or by occupying platelet receptor‐ligand molecules. So far, reports from the literature are controversial.

M ethods 

We investigated CD62P and CD63 expression, as well as fibrinogen‐binding on platelets that have been incubated with sera containing anti‐HPA‐1a (NAIT, n = 11; PTP, n = 5) by flow cytometry. HPA‐1a/a platelets from HLA‐matched donors were incubated with antisera and then stimulated with TRAP‐6 (20 µm and 50 µm) to study stimulatory or inhibitory effects.

R esults 

NAIT: TRAP‐6 20 µm and 50 µm induced CD62P expression was enhanced by 2 and 4 sera, respectively. Fibrinogen binding was induced by 6 sera and enhanced if with TRAP‐6 20 µm and 50 µm by 3 and 8 sera, respectively.

PTP: 2 sera induced CD62P and all 5 augmented its TRAP‐6 50 µm induced expression. Fibrinogen binding was induced by 4 sera and all 5 sera enhanced its TRAP‐6 50 µm induced binding. There were no changes in the CD63 expression.

C onclusions 

HPA‐1a antibodies activate platelets to release CD62P and support fibrinogen binding. These effects are stronger in PTP than in NAIT.
Anti‐D (WinRho‐SD) is associated with more than one mechanism of action in autoimmune thrombocytopenic purpura (AITP)
Authors: Semple J. W. , Speck E. R. , Kim M. , Blanchette V. , Freedman J.
Published: March 2003

We have shown that the treatment of children with chronic AITP with anti‐D is associated with a transient elevation of several pro‐and anti‐inflammatory serum cytokines followed by a gradual reduction in leucocyte phagocytosis and increase in platelet count (Semple et al. Blood 92: 178a, 1998). To further study these observations, we developed an in vitro phagocytosis assay using fluorescently labelled opsonized bacteria and flow cytometry. Results show that when anti‐D was titrated into whole peripheral RhD + blood, the phagocytic activity of individual granulocytes and monocytes was transiently increased whereas the percentage of cells able to be recruited to phagocytosis was significantly inhibited within 30 min and lasted for up to 8 h. This was not seen in RhD‐blood. In control experiments using intravenous gammaglobulin (IVIg), similar finding were observed but to a lesser degree compared with anti‐D. When the anti
d‐treated peripheral blood was analysed for intracellular cytokines by flow cytometry, monocytes produced detectible levels of IL‐1 receptor antagonist, IL‐6 and TNF‐within 1 h after the addition of anti‐D. These in vitro results appear to mimic the in vivo observations in children with chronic AITP and suggest that anti‐D may transiently activate cells of the RES which is followed by a gradual reduction in phagocytic potential. Thus, like IVIg, anti‐D may have more than one mechanism of action.
Frequency determination of RhD specific B‐lymphocytes in RhD antagonism
Authors: Mulder A. , Eijsink C. , Van Dijk M. E. I. Franke‐. , Kardol M. J. , Van Marion J. , Ten Asbroek M. , Doxiadis I. I. N. , Claas F. H. J. , Brand A.
Published: March 2003

Immunoprophylaxis with Rh‐D immunoglobulin has been proven effective for the prevention of haemolytic disease of the newborn (HDN) in > 90% of cases. At present, anti RhD immunoglobulin is prepared from plasma of volunteer RhD‐donors who are immunized with RhD+ erythrocytes, while human monoclonal RhD antibodies (RhD‐HuMAbs) are in development. Selection of donors is entirely based on the progression of plasma RhD antibody titre during the course of repeated boosting. Donor selection would improve if a predictive test were available for estimating the number of B‐lymphocytes involved in RhD antibody secretion.

We have developed a B cell precursor assay (BCPF) for specific antibody producing cells. Immunomagnetically isolated CD19+ B‐cells are cultured for 10 days in limiting dilution format and supernatants are tested for the presence of agglutinating antibodies. We validated this method using B‐cells from women immunized by pregnancy or deliberate immunization. RhD specific BCPF values were found to range from 42 to 16000 per 106 B‐cells in immunized women. We are currently correlating serum RhD antibody agglutination titres with RhD‐BCPF titres.

This method will be useful for: (i) prediction of donors who will or will not develop high titreed RhD antibodies upon boosting; (ii) evaluation of the efficiency of immunoprophylaxis in the presence of passive antibodies; (iii) evaluation of the downregulating effects of immunosuppressive drugs or agents on B‐cells; (iv) assessing donor suitability for RhD‐HuMAb hybridoma development.
HPA‐1, ‐2 and ‐3 genotyping using the TaqMan™ reaction
Authors: Hurd C. , Ocheja A. , Cheetham‐Wilkinson C. , Ouwehand W.
Published: March 2003

Human platelet alloantigen (HPA) genotyping is widely used (i) in the diagnosis and management of the alloimmune thrombocytopenias; (ii) in the HPA typing of donor panels for the provision of HPA‐matched blood products; and (iii) in the study of HPA and cardiovascular disease association.

We describe HPA‐1, ‐2 and ‐3 genotyping by allelic discrimination using the Taqman™ reaction. The assay combines the PCR reaction, using platelet glycoprotein‐specific primers, with the release of a reporter fluorophore from an allele‐specific oligonucleotide probe by the 5′−3′ exonuclease activity of Taq polymerase. By using two differently labelled allele‐specific probes and in situ measurement of released fluorescence, the genotype of each biallelic system can be determined in a single PCR reaction without additional post‐PCR manipulation.

The TaqMan™ assay has been compared with the PCR using allele specific primers (PCR‐SSP) evaluated by ethidium bromide staining in agarose gels. Parallel testing of 253 genomic DNA samples showed that the TaqMan™ assay to be more consistent and to be independent of subjective interpretation errors. Subjective reading of weak bands accounted for the misinterpretation of the PCR‐SSP evaluated on ethidium bromide gels. The TaqMan™ reaction is readily adaptable to high throughput genotyping by automation of the dispensing and data handling steps.
Platelet gel as a new routine method to improve wound healing and tissue regeneration
Authors: Sacchi M. C. , Maresca P. , Tartuferi L. , Bellanda M. , Micheletti P. , Riva S. , Borzini P. , Levis A. , Rosti G.
Published: March 2003

I ntroduction 

Previous data showed that platelets contain growth factors (PDGF, FGF, EGF, TGF‐β) that stimulate capillary endothelial migration (angiogenesis), fibroblast proliferation and migration, and collagen synthesis. Nowadays the platelet gel is considered a useful and available tool to increase either the quality or the final quantity of new‐conformed bone and to stimulate repair of chronic ulcers resulting in accelerated granulation tissue formation and epithelization. By thinking about the importance to use the platelet gel in surgery and in the treatment of ulcers, an efficient, reliable, reproducible, standardized, easy, safe and economic technique has been recently set up, which permits to obtain quickly an autologous platelet gel in laboratory so it can be used as routine in oral, implant, maxillofacial surgery and in attempt to accelerate the healing of chronic wounds.

P atients and M
ethods 

All patients were given informed consent before inclusion in the study. PC, prepared starting from a low quantity of whole blood (60 mL), is activated by a Batroxabine‐calcium gluconate blend to obtain a platelet gel.

R esults 

We observed, after about 5 weeks from the first application, the recovery of three of the nine treated patients suffering from chronic wounds to lower limb. Till now, the excellent initial results obtained consist in an improvement in the cleansing, in a reduction in ulcer sizes and, for all patients, in an improvement in the painful symptomatology. Furthermore, in implant surgery it has been demonstrated that the application of a platelet gel into bone graft produces, five months after the surgery, a neo‐formed bone considerably radiopaque as pointed out by the T.C. exam and confirmed by the hystological analysis.
Retrospective follow‐up of recipients and donors of blood donations reactive for anti‐HBc or for single HCV antibodies
Authors: Howell D. R. , Webster M. H. , Barbara J. A. J
Published: March 2003

. A retrospective study was undertaken to investigate the infectivity of blood donations that were HCV RIBA indeterminate or reactive for ‘anti‐HBc only’. Samples from 6032 blood donations were tested for anti‐HBc; 17 of them were positive for anti‐HBc but were negative when tested for anti‐HBs. None of the recipients of the red cells from these donations, when tested 9 months after transfusion, had evidence of HBV infection. Samples from 3000 blood donations were tested for HCV antibodies using RIBA‐3; 53 had single lines. Samples taken 9 months after transfusion from the recipients of the red cells from these donations were tested for evidence of infection with hepatitis C, and all were negative.
Prion protein and developments in its detection
Authors: MacGregor I.
Published: March 2003

The theoretical risk of transmission of variant Creutzfeldt–Jakob disease (vCJD) via blood transfusions has led to replacement of UK‐derived plasma for fractionation by plasma sourced outwith the UK and the introduction of leucodepletion of donated blood and its components. Prion protein in an abnormal conformation (PrPsc) has been identified as inextricably linked with the infectivity of transmissible spongiform encephalopathies such as vCJD and in this review some of its properties relevant to its detection are considered. In particular its insolubility in nonionic detergents and its partial resistance to proteinase K digestion have provided methods for separating it from the normal abundant isoform PrPc prior to subsequent detection, usually by means of antibody probes. As yet no antibodies which discriminate between the abnormal and normal isoforms have been identified with the sole exception of one which is not suitable for generally used immunodetection systems. Most detection systems, such as immunohistochemistry or Western blotting, have been optimized for brain or lympho‐reticular tissues. Much less information is available for assays applied to blood or plasma, although some recent publications provide indications of their feasibility. However, a number of obstacles remain to be overcome. These include the issues of assay validation, detection of extremely low levels of PrPsc in the presence of large excesses of PrPc, lack of available standardized reagents, assay specificity and practicality for large‐scale screening use. Progress towards these goals is reviewed.
Traditional medicine to DNA vaccines: the advance of medical research in West Africa
Authors: Greenwood Brian
Published: March 2003

SummaryWest Africa has a rich medical history. Herbal medicine has been practiced for hundreds of years and the establishment of an effective herbal pharmacopoeia was probably the first medical research carried out in West Africa. Arabic medicine was practiced in the countries of the Sahel in the 15th and 16th centuries. The coming of the Europeans focused research on infectious diseases such as malaria, yellow fever and sleeping sickness, to which Europeans were very susceptible and which caused devastating epidemics among the populations of their new colonies. The end of the colonial era saw the establishment of a few large, well‐equipped teaching hospitals but these proved too expensive for the newly independent states of West Africa to run effectively, and the second generation of West African medical schools was based on more modest government hospitals. This led to a change in the focus of research away from the more unusual conditions seen in a specialist referral hospital to an interest in conditions, such as the common infectious diseases, seen more frequently in district hospitals. The advent of the primary health care movement in the 1970s was followed by an increased emphasis on community studies. Molecular biology is likely to have an enormous impact on medicine in general in the coming years. One of the main challenges facing medical researchers in West Africa is how these new technologies can be used most effectively to improve health in countries with limited resources.
Tandemly repeated genomic sequence demonstrates inter‐ and intra‐strain genetic variation in Schistosoma japonicum*
Authors: Drew Alexander C. , Brindley Paul J. , Lewis Fred A. , Liang Yung‐san , Minchella Dennis J. , Minchella Dennis J.
Published: March 2003

Genetic variability within and among four geographical strains of Schistosoma japonicum was examined using a novel repetitive element. The element, termed Sjrh1.0, was isolated from genomic DNA of a Philippine strain of S. japonicum using a combination of restriction fragment PCR and band‐stab PCR. Sjrh1.0 is a tandemly repeated element, the sequence of which appears to be species‐specific, in that it hybridized to DNA from S. japonicum but not to DNA from S. mansoni. Its sequence does not match previously deposited sequences in GenBank. When employed as a probe in Southern hybridization analysis, radiolabelled Sjrh1.0 revealed sex‐specific and strain‐specific differences in genomic DNA of individual worms. We also found individual genetic variation within geographical isolates of the Asian schistosome.
Conference report: Attacking poverty and improving health: a report on the WHO/State of Maryland International Health Congress, Baltimore, September 1997
Authors: Jancloes Michel , Martin John D.
Published: March 2003

An international meeting, ‘Investment Strategies for Healthy Urban Communities’, in Baltimore in September 1997 called on the the business community, city authorities and the health professions to reduce poverty and its adverse health consequences, especially in urban areas, in both the industrialized and developing world. In addition to issuing the Baltimore Charter on partnership for a healthy urban future, the meeting had two main outcomes: the innovative concept of Business for Health, championed by progressive business leaders from Australia, Europe and the United States, to promote business principles to reduce poverty, create enterprises and improve people's health, especially in developing countries; and the establishment by health professionals of an information network between cities and countries on poverty and ill‐health. Two follow‐up meetings in London in December 1997 resulted in an action plan to create networks of health professional groups and representatives of the business community.