bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversible redox control of plant vacuolar H+–ATPase activity is related to disulfide bridge formation in subunit E as well as subunit A
Authors: Tavakoli Nastaran , Kluge Christoph , Golldack Dortje , Mimura Tetsuro , Dietz Karl‐Josef
Published: March 2003

The plant vacuolar proton pump can be subjected to reversible redox regulation in vitro. The redox‐dependent activity change involves disulfide bridge formation not only in Vatp A, as reported for bovine V–ATPase, but also in the stalk subunit Vatp E. Microsomal membranes isolated from barley leaves were analysed for their activity of bafilomycin‐sensitive ATP hydrolysis and proton pumping using quinacrine fluorescence quenching in vesicle preparations. ATP hydrolysis and proton pumping activity were inhibited by H2O2. H2O2‐deactivated ATPase was reactivated by cysteine and glutathione. The glutathione concentration needed for half maximal reactivation was 1 mmol l−1. The activity loss was accompanied by shifts in electrophoretic mobility of Vatp A and E which were reversed upon reductive reactivation. The redox‐dependent shift was also seen with recombinant Vatp E, and was absent following site‐directed mutagenesis of either of the two cys residues conserved throughout all plant Vatp E sequences. V–ATPase was also inhibited by oxidized thioredoxin. These results support the hypothesis that tuning of vacuolar ATPase activity can be mediated by redox control depending on the metabolic requirements.
The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes
Authors: Ballvora Agim , Ercolano Maria Raffaella , Weiß Julia , Meksem Khalid , Bormann Christina Angelika , Oberhagemann Petra , Salamini Francesco , Gebhardt Christiane
Published: March 2003

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single‐gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single‐gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine‐rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.
A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket
Authors: Carlsson Anders S. , LaBrie Samuel T. , Kinney Anthony J. , Von Wettstein‐Knowles Penny , Browse John
Published: March 2003

The fab1 mutant of Arabidopsis is partially deficient in activity of β‐ketoacyl‐[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low‐temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild‐type, the Leu207Phe protein showed a 10‐fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size‐exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.
Activation tagging identifies a gene fromPetunia hybridaresponsible for the production of active cytokinins in plants
Authors: Zubko Elena , Adams Christopher J. , Macháèková Ivana , Malbeck Jiri , Scollan Claire , Meyer Peter
Published: March 2003

Cytokinins (CKs) are phytohormones that play an important role in plant growth and development. Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKsin planta. In an activation tagging experiment we have identified a petunia line that showed CK‐specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering. This phenotype correlated with the enhanced expression of a gene we labelledSho(Shooting). Sho, which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK‐specific effects when expressed in other plant species. In contrast to theiptgene fromAgrobacterium, which primarily increases zeatin levels,Shoexpression in petunia and tobacco especially enhances the levels of certain N6‐(Δ2‐isopentenyl) adenosine (2iP) derivatives. Our data suggest thatShoencodes a plant enzyme whose activity is sufficient to produce active CKs in plants.
Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double‐strand breaks
Authors: Tamura Katsunori , Adachi Yugo , Chiba Keiko , Oguchi Keiko , Takahashi Hideo
Published: March 2003

In higher organisms such as mammals and plants, DNA double‐strand breaks (DSBs) are repaired preferentially by non‐homologous end joining (NHEJ) rather than by homologous recombination. The NHEJ pathway is mediated by Ku, a heterodimer of ≈70 and 80 kDa subunits, which contributes to various aspects of the metabolism of DNA ends in eukaryotic cells. On the basis of their predicted sequence similarity to human Ku70 and Ku80, cDNAs encoding the first plant homologues of these proteins (AtKu70 and AtKu80, respectively) have now been isolated from Arabidopsis thaliana. AtKu70 and AtKu80 share 28.6 and 22.5% amino acid sequence identity with human Ku70 and Ku80, respectively. Yeast two‐hybrid analysis demonstrated that AtKu70 and AtKu80 form a heterodimer, and electrophoretic mobility‐shift assays revealed that this heterodimer binds to double‐stranded telomeric and non‐telomeric DNA sequences, but not to single‐stranded DNA. The AtKu heterodimer also possesses single‐stranded DNA‐dependent ATPase and ATP‐dependent DNA helicase activities. Reverse transcription and the polymerase chain reaction revealed that AtKu70 and AtKu80 genes are expressed widely but at low levels in plant tissues. The expression of these two genes in cultured cells was markedly increased in response to the generation of DSBs by bleomycin or methylmethane sulfonate. These results suggest that the evolutionarily conserved Ku70–Ku80 heterodimer functions in DSB repair by the NHEJ pathway in A. thaliana.
OsPNH1 regulates leaf development and maintenance of the shoot apical meristem in rice
Authors: Nishimura Asuka , Ito Momoyo , Kamiya Noriko , Sato Yutaka , Matsuoka Makoto
Published: March 2003

The Arabidopsis PINHEAD/ZWILLE (PNH/ZLL) gene is thought to play an important role in the formation of the shoot apical meristem (SAM) and in leaf adaxial cell specification. To investigate the molecular mechanisms of rice development, we have isolated a rice homologue of PNH/ZLL, called OsPNH1. Around the SAM, OsPNH1 was strongly expressed in developing leaf primordia, specifically in the presumptive vascular domains, developing vascular tissues, a few cell‐layers of the adaxial region, and future bundle sheath extension cells. In the SAM, only weak expression was observed in the central region, whereas strong expression was detected in the mid‐vein region of leaf founder cells in the peripheral SAM domain. We produced transgenic rice plants containing the antisense OsPNH1 strand. The antisense OsPNH1 plants developed malformed leaves with an altered vascular arrangement and abnormal internal structure. These plants also formed an aberrant SAM with reduced KNOX gene expression. We examined the subcellular localization of the OsPNH1‐GFP fusion protein and found that it was localized in the cytoplasm. On the basis of these observations, we propose that OsPNH1 functions not only in SAM maintenance as previously thought, but also in leaf formation through vascular development.
Tomato LeAGP‐1 arabinogalactan‐protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis
Authors: Zhao Zhan Dong , Tan Li , Showalter Allan Marshall , Lamport Derek Thomas Anthony , Kieliszewski Marcia Jane
Published: March 2003

Functional analysis of the hyperglycosylated arabinogalactan‐proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O‐Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino‐oligosaccharides (arabinosides) and clustered, non‐contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline‐rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence‐driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP‐1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)‐LeAGP‐1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP‐1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)‐anchor signal sequence; the other lacking a 12‐residue internal lysine‐rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP‐1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp‐polysaccharides to Hyp‐arabinosides plus non‐glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O‐glycosylation profile of an HRGP from its DNA sequence.
Characterization of a HKT‐type transporter in rice as a general alkali cation transporter
Authors: Golldack Dortje , Su Hua , Quigley Francoise , Kamasani Uma R. , Muñoz‐Garay Carlos , Balderas Enrique , Popova Olga V. , Bennett John , Bohnert Hans J. , Pantoja Omar
Published: March 2003

We report the characterization of rice OsHKT1 (Oryza sativa ssp. indica) homologous to the wheat K+/Na+‐symporter HKT1. Expression of OsHKT1 in the yeast strain CY162 defective in K+‐uptake restored growth at mM and µM concentrations of K+ and mediated hypersensitivity to Na+. When expressed in Xenopus oocytes, rice OsHKT1 showed uptake characteristics of a Na+‐transporter but mediated transport of other alkali cations as well. OsHKT1 expression was analysed in salt‐tolerant rice Pokkali and salt‐sensitive IR29 in response to external cation concentrations. OsHKT1 is expressed in roots and leaves. Exposure to Na+, Rb+, Li+, and Cs+ reduced OsHKT1 transcript amounts in both varieties and, in some cases, incompletely spliced transcripts were observed. By in situ hybridizations the expression of OsHKT1 was localized to the root epidermis and the vascular tissue inside the endodermis. In leaves, OsHKT1 showed strongest signals in cells surrounding the vasculature. The repression of OsHKT1 in the two rice varieties during salt stress was different in various cell types with main differences in the root vascular tissue. The data suggest control over HKT expression as a factor that may distinguish salt stress‐sensitive and stress‐tolerant lines. Differences in transcript expression in space and time in different lines of the same species appear to be a component of ion homeostasis correlated with salt sensitivity and tolerance.
A role for HKT1 in sodium uptake by wheat roots
Authors: Laurie Sophie , Feeney Kevin A. , Maathuis Frans J. M. , Heard Peter J. , Brown Sherralyn J. , Leigh Roger A.
Published: March 2003

The high affinity potassium transporter, HKT1 from wheat was introduced into Florida wheat in sense and antisense orientation under control of a ubiquitin promoter. Ten transgenic lines expressing the transgene were identified and two of these showed strong down‐regulation of the native HKT1 transcript. One line (271) was expressing the antisense construct and the other (223) was expressing a truncated sense construct. The two lines were examined further for phenotype relating to cation transport. Membrane depolarisations were measured in low (0.1 mm) K+ and high (100 mm) NaCl. Under these conditions there was no difference between line 271 and the control at low K+, but at high Na+ there was a rapid depolarisation that was significantly larger in control plants. 22Na uptake was measured in this line and there was a significant decrease in uptake at 100 mm NaCl in the transgenic line when compared with the control. The two transgenic lines were grown at high NaCl (200 mm) and analysed for growth and root sodium content. Lines 271 and 223 showed enhanced growth under salinity when compared with the control and had lower sodium in the root. Secondary ion mass spectrometry (SIMS) analysis of transverse sections of the root showed that Na+ and K+ were strongly localised to stelar regions when compared with other ions, and that the Na+ : K+ ratios were reduced in salt‐stressed transgenic tissue when compared with the control.
Construction of a ‘unigene’ cDNA clone set by oligonucleotide fingerprinting allows access to 25 000 potential sugar beet genes
Authors: Herwig Ralf , Schulz Britta , Weisshaar Bernd , Hennig Steffen , Steinfath Matthias , Drungowski Mario , Stahl Dietmar , Wruck Wasco , Menze Andreas , O'Brien John , Lehrach Hans , Radelof Uwe
Published: March 2003

Access to the complete gene inventory of an organism is crucial to understanding physiological processes like development, differentiation, pathogenesis, or adaptation to the environment. Transcripts from many active genes are present at low copy numbers. Therefore, procedures that rely on random EST sequencing or on normalisation and subtraction methods have to produce massively redundant data to get access to low‐abundance genes. Here, we present an improved oligonucleotide fingerprinting (ofp) approach to the genome of sugar beet (Beta vulgaris), a plant for which practically no molecular information has been available. To identify distinct genes and to provide a representative ‘unigene’ cDNA set for sugar beet, 159 936 cDNA clones were processed utilizing large‐scale, high‐throughput data generation and analysis methods. Data analysis yielded 30 444 ofp clusters reflecting the number of different genes in the original cDNA sample. A sample of 10 961 cDNA clones, each representing a different cluster, were selected for sequencing. Standard sequence analysis confirmed that 89% of these EST sequences did represent different genes. These results indicate that the full set of 30 444 ofp clusters represent up to 25 000 genes. We conclude that the ofp analysis pipeline is an accurate and effective way to construct large representative ‘unigene’ sets for any plant of interest with no requirement for prior molecular sequence data.
An Arabidopsis thaliana knock‐out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished
Authors: Schneider Anja , Häusler Rainer E. , Kolukisaoglu Üner , Kunze Reinhard , Van Der Graaff Eric , Schwacke Rainer , Catoni Elisabetta , Desimone Marcelo , Flügge Ulf‐Ingo
Published: March 2003

The Arabidopsis thaliana tpt‐1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T‐DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)‐specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6‐phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6‐bisphosphatase. Despite its regulatory role in the feed‐forward control of sucrose biosynthesis, variations in the fructose 2,6‐bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt‐1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1‐1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt‐1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady‐state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.
Prenylcysteine α‐carboxyl methyltransferase expression and function in Arabidopsis thaliana
Authors: Narasimha Chary S. , Bultema Rebecca L. , Packard Clay E. , Crowell Dring N.
Published: March 2003

Farnesylated proteins undergo a series of post‐translational modifications, including carboxyl terminal isoprenylation, proteolysis, and methylation. In Arabidopsis thaliana, protein farnesylation has been shown to be necessary for negative regulation of ABA signaling. However, the role of post‐isoprenylation protein processing in ABA signal transduction has not been described. Here, we show that the A. thaliana genome contains two distinct genes on chromosome V, AtSTE14A and AtSTE14B, which encode functional prenylcysteine α‐carboxyl methyltransferases. AtSTE14B encodes a methyltransferase with lower apparent Kms for prenylcysteine substrates and higher specific activities than the previously described AtSTE14A‐encoded methyltransferase. Furthermore, whereas AtSTE14A transcription is restricted to root and shoot tips, young leaves, and vascular tissue, AtSTE14B transcription is observed in all organs except hypocotyls and petioles. Pharmacological inhibitors of prenylcysteine α‐carboxyl methyltransferase activity cause increased ABA sensitivity, seed dormancy, and stomatal closure, consistent with the hypothesis that prenylcysteine α‐carboxyl methylation is necessary for negative regulation of ABA signaling. These results suggest that carboxyl methylation, which is a reversible and potentially regulated step in the processing, targeting, and function of isoprenylated plant proteins, may be an important biochemical target for introducing altered ABA sensitivity and drought tolerance into plants.
Overexpression of Bax inhibitor suppresses the fungal elicitor‐induced cell death in rice (Oryza sativa L.) cells
Authors: Matsumura H. , Nirasawa S. , Kiba A. , Urasaki N. , Saitoh H. , Ito M. , Kawai‐Yamada M. , Uchimiya H. , Terauchi R.
Published: March 2003

Treatment of suspension‐cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension‐cultured cells treated with the elicitor. Among the downregulated genes in the elicitor‐treated cells, a BI‐1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI‐1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.
Hepatitis C in blood transfusion recipients identified at the Oxford Blood Centre in the national HCV look‐back programme
Authors: Dike  , Christie  , Kurtz  , Teo
Published: March 2003

After the introduction in September 1991 of donor screening for hepatitis C, 95 potentially infectious blood donors who had given blood before this date were identified at the Oxford blood centre. Three hundred and ninety‐nine blood components issued previously from these donors were identified in the course of the national HCV look‐back programme. Of 399 questionnaires sent to hospital blood banks 392 were returned, identifying 290 recipients of whom 177 (61%) had died, and 113 (39%) were still alive 4–13 years after transfusion. One hundred and four recipients were traced and tested. Forty‐nine recipients were not HCV infected. Forty‐four of 58 (76%) recipients who received blood from donors found to be HCV RNA positive after September 1991 gave positive test results for HCV RNA. Eleven of 58 showed only antibody (anti‐HCV), and 3/58 who had apparently received infectious blood showed no sign of past infection. The 11 who showed anti‐HCV only, together with the three who showed no sign of past infection despite strong evidence of receiving HCV RNA‐positive blood, had a mean age at transfusion of 27 years, compared with mean age at transfusion of 46 years in the 44 recipients with persistent HCV infection. Virus genotyping in 33/44 HCV RNA‐positive recipients revealed five different genotypes. These did not seem to influence the outcome. Virus genotypes in 31 donor–recipient pairs showed complete concordance. Liver biopsies in 23/44 RNA‐positive recipients showed minimal inflammation in four, mild in eight and moderate in 11. Liver fibrosis, Ishak grades 1–3, was present in 16/23 recipients. One other male recipient, not subjected to a liver biopsy, developed a hepatocellular carcinoma which caused his death at the age of 71, 8 years after transfusion.
Serious adverse events after HPA‐incompatible platelet transfusions in an alloimmunized patient with leukaemia
Authors: Sirén  , Koponen  , Kekomäki
Published: March 2003

We describe an alloimmunized female patient who developed serious adverse reactions when receiving HPA‐incompatible platelet transfusions. She had received 13 transfusions with random platelets before the first allergic reactions. Antibodies against both the human leucocyte antigens (HLA) and several human platelet antigens (HPA) were detected at the time of transfusions. When the patient received HLA‐ and HPA‐compatible platelets, no reactions followed the transfusions and platelet count increments were good. When she was transfused with platelets from donors with one foreign HLA antigen, her reactions were fever, chills and headache and the response to platelet transfusions was poor. When the platelets were HLA compatible but HPA incompatible, the reactions were repeatedly rapid pulse, shortness of breath, tightness of chest and wheezing interpretable as anaphylactoid reactions. Platelet count increments were satisfactory. When rare side‐effects occur after transfusion, detailed immunohaematological studies are indicated.
Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant
Authors: Mody  , Lazarus  , Semple  , Freedman
Published: March 2003

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube‐H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube‐H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube‐H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6–24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube‐H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin‐induced in vitro platelet activation after 6–24 h, in both whole blood and isolated platelets.
Hepatitis C Lookback Programme: a single hospital experience
Authors: Pawson  , Rajan  , Hazlehurst  , Dusheiko  , Miller  , Hewitt  , Mehta
Published: March 2003

As part of the national Hepatitis C (HCV) Lookback Programme, HCV‐infected donors donating blood after September 1991 were identified and the fate of their previous donations received at a single hospital were traced; 123 of 160 implicated blood components were traceable and transfused. Only 19 recipients were alive and traceable and were tested for HCV. Nine of the 14 recipients (64%) of HCV‐positive donations and 2 of 5 recipients (40%) of HCV‐indeterminate donations had evidence of HCV infection. Neither the number of donor exposures nor the type of component was predictive of recipient HCV status. Three recipients have chronic active hepatitis. The Hepatitis C Lookback Programme successfully identifies some but not all cases of transfusion‐transmitted HCV. Transfusion records, particularly in the medical case notes, should be substantially improved. Many of the traced recipients are young, so that identification of HCV is of great importance.
Platelet storage in PAS‐2 or autologous plasma: impact on functional parameters
Authors: Bunescu A. , Hild M. , Lundahl J. , Egberg N.
Published: March 2003

Currently, several platelet additive solutions for long‐term platelet storage have been introduced. The aim of this study was to compare the deterioration of functional status of platelets stored for up to 5 days in autologous plasma (AP) only, with platelet stored in PAS‐2, a salt solution containing acetate, citrate and sodium chloride. Change in platelet adhesion, aggregation and activation was measured by flow cytometric technique. In addition, β‐Thromboglobulin (β‐TG), lactate and glucose were determined. After 5 days of storage, the expression of P‐Selectin was significantly higher, the production of lactate and the consumption of glucose were significantly lower, in platelets stored in PAS‐2 than in autologous plasma. No significant differences were detected on day 5 between the two groups with regard to fibrinogen, von Willebrand factor binding capacity, or to β‐TG release. It can be concluded that neither storage medium was consistently better for the parameters tested. However, it must be emphasized that platelets stored in autologous plasma exhibited less lesion, in terms of P‐Selectin expression compared with platelets stored in PAS‐2.
The impact of the new tick‐box questionnaire, and the personal donor interview, on donor deferrals in the East of Scotland
Authors: Norris A. , Galea G.
Published: March 2003

The impact of a new tick‐box questionnaire (TBQ) and personal donor interview (PDI) on donor and recipient safety was assessed over an 18‐month and a 13‐month period, respectively, by prospectively studying individual donors prior to and after the introduction of the new methodology. A ‘hit’ was defined as an instance where the TBQ or PDI prompted a donor to divulge information which they would not otherwise have divulged, with the new information having an impact on donor eligibility. There was a ‘hit’ rate of 0·19% for TBQ and 0·65% for PDI. Of these donors, 33% in the TBQ category and 14% of PDIs were reinstated, 24% and 32%, respectively, were deferred because of a malaria/chagas risk, and 16% of the ‘hits’ related to donor safety issues. When assessing recipient safety, particularly risk of a window period viral transmission, PDI is very significantly superior at identifying such donors (14 times better). Such information establishes the important safety aspects of these interventions and requires that further work be done to see whether PDIs, in particular, may be better targeted to specific groups of donors.
Common human leucocyte antigen haplotypes in Indians – its implications in finding unrelated compatible bone marrow donors
Authors: Ghosh K. , Shankarkumar U. , Mohanty D.
Published: March 2003

Summary All over the world there have been efforts to make unrelated stem cell registries to cater for patients who do not have a compatible family donor and are in need of allogenic stem cell transplantation. Donors of Indian origin are poorly represented all these registries. Approximately 10 million Indians live outside India, and a substantial number of them live in the developed West. Hence when some of these patients of Indian origin need unrelated stem cell donor, they search the available National Marrow Donor Program (NMDP) registry database and invariably do not get a human leucocyte antigen (HLA) matched donor. They come to India, where a haphazard search for the donor ensues and it invariably ends in failure. Hence, we have compiled the data of our laboratory and other published reports in population HLA studies from India and abroad to show several common haplotypes from these studies. It is hoped that this will give the busy clinician an idea whether a search for prospective unrelated stem cell donor for an Indian patient is likely to succeed or not.
Monitoring transfusion practice – a computerized procedure
Authors: Titlestad K. , Kristensen T. , Jorgensen J. , Georgsen J.
Published: March 2003

Summary As the description of transfusion practice over time requires the handling of massive amounts of data, and because the majority of records have until now been paper‐based, little is known about the epidemiology of blood transfusion. Furthermore, most previous studies have collected data manually from patient records or from computerized registers created solely for study purposes.

We describe a procedure for monitoring transfusion practices based exclusively upon the collection of data from the existing computerized registers. During the calendar years 1997 and 1998, more than 750 000 data records were collected from the blood transfusion registers, diagnosis and procedure registers, and clinical biochemistry registers at two university hospitals. Linking of the data required a unique and consistent identification of all data records. This was made possible by the national Personal Identification Number. In this way more than 98% of all transfusions could be identified and approximately 95% (77 950) could be connected to a principal diagnosis.

The described procedure creates new opportunities for studies of transfusion practices both on a large scale and in richness of detail, and we propose to establish a network where transfusion practices data are available and comparable for individual departments, hospitals, whole regions, and possibly nations.
Recombinant forms of glycophorin C as a tool for characterization of epitopes for new murine monoclonal antibodies with anti‐glycophorin C specificity
Authors: Jaskiewicz E. , Czerwinski M. , Uchikawa M. , Murata S. , Miyazaki T. , Ikeda H. , Lisowska E.
Published: March 2003

Summary Glycophorin C (GPC) and glycophorin D (GPD) are minor but important components of human RBC membranes. They carry the high‐frequency antigens Ge2, Ge3 and Ge4 of the Gerbich blood group system. The epitopes for five new monoclonal antibodies (MoAbs) with anti‐GPC specificity were characterized. Two antibodies (4G11 and 5B11) reacted with glycosylated N‐terminal epitopes, and three reacted with internal epitopes of GPC. Pepscan analysis showed that the MoAb RB11 required for binding the EPDP sequence, occurring twice in GPC polypeptide chain. The MoAb 7F11 recognized the sequence 13PLSLEPDP20, and the MoAb RB8 did not react with synthetic peptides. Further characterization of the internal epitopes was performed in fluorescence‐activated cell sorter (FACS) with the use of recombinant GPC and its variant forms transiently expressed on COS‐7 cells. The results indicated that the MoAb RB11 recognized distinctly its target sequence EPDP only in a normal GPC molecule. The reactivity of the MoAb 7F11 with the PLSLEPDP sequence was confirmed and found to be enhanced by the O‐glycan at the Ser15 residue. The MoAb RB8 recognized the glycopeptidic epitope in proximity to the Ser15 residue, requiring the presence of O‐glycan. The combination of immunochemical techniques with the use of the recombinant forms of GPC has made it possible to define the role of sugar chains in the recognition of peptidic epitopes in glycosylated antigen and sheds new light on the Gerbich system antigens.
US NAT yield: where are we after 2 years?
Authors: Stramer S. L.
Published: March 2003

Summary. The US blood supply has been tested for human immunodeficiency virus‐1 (HIV‐1) and hepatitis C virus (HCV) using nucleic acid amplification testing (NAT) of pools of small number of samples since early 1999. Since the implementation of NAT under an investigational new drug (IND) application, the results for the yield and false positivity have been remarkably consistent for greater than 2 years of testing even among multiple programmes using two different test methodologies and manufacturers: Gen‐Probe/Chiron transcription‐mediated amplification (TMA) and Roche polymerase chain reaction. All programmes in the US and Canada use NAT as a criterion for cellular as well as frozen product release. The focus of this paper is to provide an update of the programmes in the US and Canada, provide data in support of p24 antigen replacement by HIV‐1 NAT and discuss the projections of residual risk of HIV, HCV and hepatitis B virus (HBV) following NAT and the associated cost/benefit.
Platelet concentrates derived from buffy coat and apheresis: biochemical and functional differences
Authors: Böck M. , Rahrig S. , Kunz D. , Lutze G. , Heim M. U.
Published: March 2003

Summary Today, platelet concentrates are generally produced from whole blood by differential centrifugation (buffy coat‐derived platelet concentrates – PCs) or by plateletpheresis (apheresis‐derived platelet concentrates – APCs). As PCs are characterized by a lower number of platelets than APCs, four to six PCs are customarily combined in order to obtain an equivalent dose. In the 1970s and 1980s, the use of PCs exceeded that of APCs by far; in contrast, since the beginning of the 1990s, APCs comprise more than half of all transfused platelets. However, the selection of PCs or APCs for transfusion to thrombocytopenic patients is still a matter of debate.

The present paper compares biochemical and functional properties of both platelet preparations in vitro. Besides plasma parameters (e.g. platelet factor 4 (PF4), P‐selectin, C3a‐desarginin, plasma coagulation factors), platelet function was analysed by aggregometry and the PFA 100 system. APCs are characterized by a better preservation of ADP and collagen‐induced platelet aggregation, and shorter closure times of the PFA 100 test system during storage. The improved primary in vitro haemostatic capacity of APCs is presumed to be owing to a lower cellular activation rate in these preparations. This hypothesis is supported by the higher plasma concentrations of PF4, β‐thromboglobulin and P‐selectin found in PCs compared with APCs. The concentrations of C3a‐desarginin in PCs exceed those in APCs by far. Additionally, thrombin generation is higher in PCs than in APCs.

These data suggest that APCs are characterized by a superior haemostatic capacity over PCs in vitro. However, in vivo studies should be performed to confirm these findings in the patients' circulation also.
Are changes in admission practices for elective surgery posing a transfusion threat to patients?
Authors: Chiganti S. , Regan F.
Published: March 2003

Summary There is an increasing emphasis on reducing in‐patient hospital stay for elective surgical procedures. As a result, pre‐operative stay has shortened, with less time available for performing baseline investigations before surgery, including those required to ensure a supply of safe, compatible blood. We have estimated the frequency of failure to complete ‘group and screen’ (G&S) testing before surgery where indicated, so that neither group‐compatible blood nor crossmatched blood could be provided promptly if an unexpected haemorrhage occurs. Retrospective data were obtained from elective surgical procedures performed over a 3‐week period. Of the 309 procedures performed, 21 patients were exposed to the risk of failure to provide appropriately matched blood had an emergency arisen. (In 20, a G&S was required; in one, a crossmatch was required.) Late samples and lack of information on the request form regarding the date and time of surgery were the main causes. Procedures requiring G&S only as opposed to crossmatch posed the greatest risk. Urgent measures are needed to ensure that, where indicated, a pre‐operative G&S result is available before surgery begins. Educating colleagues, redesigning request forms and improving out‐of‐hour laboratory services and hospital information systems are the main steps needed to reduce this latent risk.
Utility of antibodies against a 22 kD molecule of Dirofilaria immitis in the diagnosis of human pulmonary dirofilariasis
Authors: Perera L. , Pérez‐Arellano J. L. , Cordero M. , Simón F. , Muro A.
Published: March 2003

Summary To assess the characteristics of an ELISA test for the diagnosis of human pulmonary dirofilariasis, we studied the sera of 24 subjects with other helmintoses and of 37 patients suffering from non‐parasitic focal lung diseases, comparing them with negative and positive sera. ELISA and Western blot with complete somatic antigen and ELISA with protein Di22 (specifically recognized in cases of lung dirofilariasis) were performed. With ELISA SA the false positive rate was 25% in cases with other parasitoses and 30% in cases with focal lung diseases. ELISA Di22 decreases this positivity levels. Only 2 cases with visceral larva migrans (8.3%) and a case with lung nodules metastatic from renal adenocarcinoma (2.7%) were positive. ELISA Di22 therefore greatly decreases the false positive rate of ELISA SA.
Obstetric care in southern Tanzania: does it reach those in need?
Authors: Jahn Albrecht , Kowalewski Marga , Kimatta Suleiman S.
Published: March 2003

Summary
objective  To assess whether antenatal care achieves identification and timely referral of high‐risk pregnancies in southern Tanzania.

methods  We compared the risk profiles of pregnant women in general with those attending obstetric care and investigated the reasons for seeking care. The risk profile of inpatients was drawn up through interviews with maternity cases and analysis of their antenatal records at the regional referral hospital (n= 415); population‐based data on the prevalence of specific risk factors were obtained from entries in antenatal care registers (n= 1630) and from literature.

results  A significant risk selection towards obstetric referral level care was observed only for previous caesarean section (prevalence hospital 6.7% all pregnancies 1.5%P < 0.005) and for nulliparity (hospital 42.8% all pregnancies 25.0%P < 0.005). No significant differences were observed for other risk factors such as previous perinatal death height < 150 cm multiple gestation and breech presentation. Prevalence of the risk factors age > 34 years and grand multiparity was significantly lower among hospital users. Coverage of obstetric care was below 50% for all risk factors except previous caesarean section (91.5%)

conclusion  Despite pursuing the risk approach and very good coverage, antenatal care in Tanzania has only limited effect on extending obstetric care to high‐risk mothers. A critical review of the present screening and counselling practices, including a more focused and client‐centred application of risk assessment, is warranted.
Prevalence of GB virus C/hepatitis G virus among blood donors in north‐eastern Brazil
Authors: Goubau Patrick , Andrade Francisco Braga , Liu Hsin‐Fu , Basilio Francisco Placido , Croonen Luc , Barreto‐Gomes Vania A. F.
Published: March 2003

Summary We tested 70 blood donors from Fortaleza (Ceara state, Brazil) for GB virus C/hepatitis G virus (GBV/HGV) infection by polymerase chain reaction and detection of antienvelope antibodies. Twenty‐seven (38.6%) showed signs of an active or resolved infection. Sixty‐four percent of those with indications of other blood‐borne viral infections showed signs of GBV‐C/HGV infection also.
Detection of Trypanosoma brucei gambiense in sleeping sickness suspects by PCR amplification of expression‐site‐associated genes 6 and 7
Authors: Kabiri Mostafa , Franco José R. , Simarro Pere P. , Ruiz J. Antonio , Sarsa Mario , Steverding Dietmar
Published: March 2003

Summary We have developed a sensitive and specific method to identify Trypanosoma brucei ssp. using PCR to amplify conserved expression‐site‐associated gene 6 and 7 DNA target sequences. Amplification of 10% of the DNA in a single trypanosome produced sufficient PCR product to be visible as a band in an agarose gel stained with ethidium bromide. We analysed 59 blood samples of serologically positive cases of sleeping sickness by PCR, and directed parasitological examination of tissue fluids. The PCR test detected 87% of the parasitologically positive cases, with a specificity of 97%. In 5 cases, the parasite was demonstrated by the PCR test 4–6 months prior to parasitological detection. This result shows the potential of the assay in early diagnosis of actual T. b. gambiense infections in apparently aparasitaemic sleeping sickness patients.
Some aspects of the natural history of HIV infection
Authors: Coutinho R. A.
Published: March 2003

Summary Studying factors influencing the length of the incubation period of HIV/AIDS is important to our understanding of the natural history of the disease and for the decision when to start with anti‐retroviral therapy. In a multicentre study among HIV‐positive homosexual men with a known date of seroconversion, we found that the median survival time after HIV infection was 12.1 years. Age is an important determinant of the survival: the older the shorter the incubation period and survival. Gender does not seem to play a role, but women appear to have higher CD4 counts than men at seroconversion, AIDS and death. HIV‐positive drug users often die before they ‘reach’ AIDS often from HIV‐related causes e.g. bacterial infections. In a multicentre study we found that such pre‐AIDS mortality is now also found among homosexual men and haemophiliacs but at a much lower level. Most studies show that HIV subtype does not influence the incubation period. On the other hand genetic factors do play an important role.
Evaluation of environmental methods to control snails in an irrigation system in Central Morocco
Authors: Laamrani H. , Khallaayoune K. , Boelee E. , Laghroubi M. M. , Madsen H. , Gryseels B.
Published: March 2003

The Moroccan Ministry of Public Health has launched a programme to eliminate schistosomiasis. One of the components in this process is the control of Bulinus truncatus, the intermediate host snail of Schistosoma haematobium. We evaluated three environmentally safe measures to control B. truncatus in siphon boxes, the main breeding sites for these snails in the Tessaout Amont irrigation system. The first method involved covering the siphon boxes to exclude light and reduce algal growth, the second consisted of increasing the frequency of emptying and cleaning the siphon boxes, and the third method increased water velocity to hinder the establishment of the intermediate hosts. The results showed that covering had a pronounced effect on snail and egg mass density, was accepted by the local community and prevented water contact. Cleaning the siphons three times during the irrigation season led to a reduction in snail density although it was not statistically significant and recolonization was rapid. Increasing water velocity by reducing the dimensions of siphon boxes delayed recolonization, but such a control measure can be applied only in specific situations where it does not pose hydraulic problems. The three interventions were selectively effective against B. truncatus, whereas other snails such as Physa acuta and Lymnaea peregra were hardly affected. Covering, the most promising control measure, could be useful in the Moroccan schistosomiasis eradication programme. However, further investigations are needed to assess its impact on water quality.
The first decade of the Malawi College of Medicine: a critical appraisal
Authors: Muula Adamson S. , Broadhead Robert L.
Published: March 2003

The College of Medicine of the University of Malawi was opened in April 1991. Over almost a decade it has flourished in the face of economic and political constraints, as well as a change in the philosophy of donor support. We review the past, assess the present and look to the future.