bluuebunny/crossref_metadata_2025 huggingface dataset:
Male gametophyte development in bread wheat (Triticum aestivum L.): molecular, cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogeny
Authors: Wang Aiming , Xia Qun , Xie Wenshuang , Dumonceaux Tim , Zou Jitao , Datla Raju , Selvaraj Gopalan
Published: March 2003

Bread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self‐pollinating plant with bisexual flowers that produce short‐lived pollen. Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds. We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther‐specific biochemical function. These eight‐exon genes, found at only one copy per haploid complement in this large genome, express specifically within the sporophytic tapetum cells. The presence of TAA1 mRNA and protein was evident only at specific stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated‐microspores. This temporal regulation matched the assembly of wall‐impregnated sporopollenin, a phenylpropanoid‐lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature. Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum. Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly specific sexual interactions at the pollen–pistil interface. This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general.
Six active phage‐type RNA polymerase genes in Nicotiana tabacum
Authors: Hedtke Boris , Legen Julia , Weihe Andreas , Herrmann Reinhold G. , Börner Thomas
Published: March 2003

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7‐enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP‐fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino‐terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual‐targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid‐encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non‐viral plant mRNA that is exclusively translated from a non‐AUG codon.
The ABC transporter SpTUR2 confers resistance to the antifungal diterpene sclareol
Authors: Van Den Brûle Sybille , Müller Axel , Fleming Andrew J. , Smart Cheryl C.
Published: March 2003

PDR5‐like proteins represent one group of the ABC superfamily of transporters. Members of this group are present in plants and, due to the function of PDR5‐related proteins in fungi in the excretion of xenobiotics (including antifungal agents), it has been proposed that they might play a similar role in plants in the response to and detoxification of herbicides and fungicides. However, until now no functional data has been presented showing an altered plant response to any herbicide or fungicide as a result of manipulating the expression of a PDR5‐like gene in plants. In this paper, we show that the plant SpTUR2 PDR5‐like ABC transporter is localised to the plasma membrane and that expression of this protein in Arabidopsis leads to the acquisition of resistance to the diterpenoid antifungal agent sclareol. These data both define a possible endogenous substrate for this transporter and highlight the potential of manipulating plant chemical resistance via modulating the expression of specific PDR5‐like transporters.
Germination and storage reserve mobilization are regulated independently in Arabidopsis
Authors: Pritchard Sarah L. , Charlton Wayne L. , Baker Alison , Graham Ian A.
Published: March 2003

The phytohormone abscisic acid (ABA) inhibits the germination of many seeds, including Arabidopsis, but the mechanism for this is not known. In cereals, ABA inhibits the expression of genes involved in storage reserve mobilization. We have found that in Arabidopsis ABA decreases transcription from the promoters of marker genes for β‐oxidation and the glyoxylate cycle, essential pathways for the conversion of storage lipid (triacylglycerol) into sucrose. Thirty per cent of stored lipid is broken down over 6 days following imbibition of ABA‐treated seed. Sucrose levels in ABA‐treated seeds, rather than decreasing as under normal growth conditions, actually double during the 3 days following imbibition. This sucrose is derived from triacylglycerol as demonstrated by two mutants disrupted in the conversion of triacylglycerol into sucrose, kat2 and icl1, which do not accumulate sucrose in the presence of ABA. We conclude that the ABA block on germination is not a consequence of inhibition of storage lipid mobilization. Two independent programmes appear to operate, one that is blocked by ABA, governing developmental growth resulting in germination; and a second that governs storage lipid mobilization which is largely ABA‐independent.
Does proline accumulation play an active role in stress‐induced growth reduction?
Authors: Maggio Albino , Miyazaki Saori , Veronese Paola , Fujita Tomomichi , Ibeas José I. , Damsz Barbara , Narasimhan Meena L. , Hasegawa Paul M. , Joly Robert J. , Bressan Ray A.
Published: March 2003

An interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a Δ1‐pyrroline‐5‐carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modifications are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline‐overaccumulating cells in mild hyperosmotic stress.
Virus‐induced gene silencing in tomato
Authors: Liu Yule , Schiff Michael , Dinesh‐Kumar S. P.
Published: March 2003

We have previously demonstrated that a tobacco rattle virus (TRV)‐based vector can be used in virus‐induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up‐regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction‐ and ligation‐free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large‐scale functional analysis of tomato ESTs.
Functional characterization of a heavy metal binding protein CdI19 from Arabidopsis
Authors: Suzuki Nobuaki , Yamaguchi Yube , Koizumi Nozomu , Sano Hiroshi
Published: March 2003

Heavy metals are potentially highly toxic for organisms. Plants possess the ability to minimize damage but the underlying molecular mechanisms have yet to be detailed. Screening Cd‐responsive genes in Arabidopsis, we previously identified a gene encoding a putative metal binding protein CdI19, which, upon introduction into yeast cells, conferred marked toleration of Cd exposure. Here we describe that bacterially expressed CdI19 directly interacts with Cd at its CXXC motif, as revealed by circular dichroism analysis, and that it is exclusively localized at plasma membranes, as revealed by heterologous expression of fusion product with a green fluorescent protein in BY2 cells. Northern blot analyses indicated that CdI19 transcripts were induced not only by Cd, but also by dicationic forms of Hg, Fe and Cu. Histochemical assays using transgenic Arabidopsis expressing the CdI19 promoter::GUS showed CdI19 to be expressed in petiole, hypocotyl, peduncle and vascular bundles in root tissues. Overexpression of the CdI19 cDNA conferred Cd tolerance in transgenic Arabidopsis. These results suggest that CdI19 plays an important role in the maintenance of heavy metal homeostasis and/or in detoxification by endowing plasma membranes with the capacity to serve as an initial barrier against inflow of free heavy metal ions into cells.
Distribution of 1000 sequenced T‐DNA tags in the Arabidopsis genome
Authors: Szabados László , Kovács Izabella , Oberschall Attila , Ábrahám Edit , Kerekes Irén , Zsigmond Laura , Nagy Réka , Alvarado Martha , Krasovskaja Inga , Gál Mónika , Berente Anikó , Rédei George P. , Haim Amit Ben , Koncz Csaba
Published: March 2003

Induction of knockout mutations by T‐DNA insertion mutagenesis is widely used in studies of plant gene functions. To assess the efficiency of this genetic approach, we have sequenced PCR amplified junctions of 1000 T‐DNA insertions and analysed their distribution in the Arabidopsis genome. Map positions of 973 tags could be determined unequivocally, indicating that the majority of T‐DNA insertions landed in chromosomal domains of high gene density. Only 4.7% of insertions were found in interspersed, centromeric, telomeric and rDNA repeats, whereas 0.6% of sequenced tags identified chromosomally integrated segments of organellar DNAs. 35.4% of T‐DNAs were localized in intervals flanked by ATG and stop codons of predicted genes, showing a distribution of 62.2% in exons and 37.8% in introns. The frequency of T‐DNA tags in coding and intergenic regions showed a good correlation with the predicted size distribution of these sequences in the genome. However, the frequency of T‐DNA insertions in 3′‐ and 5′‐regulatory regions of genes, corresponding to 300 bp intervals 3′ downstream of stop and 5′ upstream of ATG codons, was 1.7–2.3‐fold higher than in any similar interval elsewhere in the genome. The additive frequency of insertions in 5′‐regulatory regions and coding domains provided an estimate for the mutation rate, suggesting that 47.8% of mapped T‐DNA tags induced knockout mutations in Arabidopsis.
NPP1, a Phytophthora‐associated trigger of plant defense in parsley and Arabidopsis
Authors: Fellbrich Guido , Romanski Annette , Varet Anne , Blume Beatrix , Brunner Frédéric , Engelhardt Stefan , Felix Georg , Kemmerling Birgit , Krzymowska Magdalena , Nürnberger Thorsten
Published: March 2003

Activation of non‐cultivar‐specific plant defense against attempted microbial infection is mediated through the recognition of pathogen‐derived elicitors. Previously, we have identified a peptide fragment (Pep‐13) within a 42‐kDa cell wall transglutaminase from various Phytophthora species that triggers a multifacetted defense response in parsley cells. Many of these oomycete species have now been shown to possess another cell wall protein (24 kDa), that evoked the same pattern of responses in parsley as Pep‐13. Unlike Pep‐13, necrosis‐inducing Phytophthora protein 1 (NPP1) purified from P. parasitica also induced hypersensitive cell death‐like lesions in parsley. NPP1 structural homologs were found in oomycetes, fungi, and bacteria, but not in plants. Structure–activity relationship studies revealed the intact protein as well as two cysteine residues to be essential for elicitor activity. NPP1‐mediated activation of pathogen defense in parsley does not employ the Pep‐13 receptor. However, early induced cellular responses implicated in elicitor signal transmission (increased levels of cytoplasmic calcium, production of reactive oxygen species, MAP kinase activation) were stimulated by either elicitor, suggesting the existence of converging signaling pathways in parsley. Infiltration of NPP1 into leaves of Arabidopsis thaliana Col‐0 plants resulted in transcript accumulation of pathogenesis‐related (PR) genes, production of ROS and ethylene, callose apposition, and HR‐like cell death. NPP1‐mediated induction of the PR1 gene is salicylic acid‐dependent, and, unlike the P. syringae pv. tomato DC3000(avrRpm1)‐induced PR1 gene expression, requires both functional NDR1 and PAD4. In summary, Arabidopsis plants infiltrated with NPP1 constitute an experimental system that is amenable to forward genetic approaches aiming at the dissection of signaling pathways implicated in the activation of non‐cultivar‐specific plant defense.
A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone‐like activity of a chloroplast‐localized small heat shock protein
Authors: Gustavsson Niklas , Kokke Bas PA. , Härndahl Ulrika , Silow Maria , Bechtold Ulrike , Poghosyan Zaruhi , Murphy Denis , Boelens Wilbert C. , Sundby Cecilia
Published: March 2003

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR‐like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast‐localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast‐localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR‐catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time‐scale, was ultimately DTT‐dependent and led to recovery of Hsp21 conformation and chaperone‐like activity, both of which are lost upon methionine sulfoxidation (Härndahl et al., 2001). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.
An EDS1 orthologue is required for N‐mediated resistance against tobacco mosaic virus
Authors: Peart Jack R. , Cook Graeme , Feys Bart J. , Parker Jane E. , Baulcombe David C.
Published: March 2003

In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide‐binding site, leucine‐rich‐repeats and amino‐terminal similarity to animal Toll and Interleukin‐1 (so‐called TIR‐NBS‐LRR proteins). EDS1 is not required by NBS‐LRR proteins that possess an amino‐terminal coiled‐coil motif (CC‐NBS‐LRR proteins). Using virus‐induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR‐NBS‐LRR protein, was EDS1‐dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC‐NBS‐LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR‐NBS‐LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.
The role of invertases and hexose transporters in controlling sugar ratios in maternal and filial tissues of barley caryopses during early development
Authors: Weschke Winfriede , Panitz Reinhard , Gubatz Sabine , Wang Qing , Radchuk Ruslana , Weber Hans , Wobus Ulrich
Published: March 2003

To analyse carbohydrate metabolism and its role during early seed development of barley we characterised genes encoding two cell wall‐bound invertases (HvCWINV1 and HvCWINV2) and two putative hexose transporter‐like genes (HvSTP1 and HvSTP2). No typical vacuolar invertase gene could be identified. Instead, a gene encoding sucrose:fructan 6‐fructosyltransferase (HvSF6FT1), an enzyme with soluble acid invertase activity, was isolated and characterised. Furthermore, enzyme activities and sugar levels were measured. HvSF6FT1‐mRNA levels and acid soluble invertase activity are highest in the maternal pericarp 1–2 days after flowering (DAF). HvSF6FT1 is strongly expressed in regions flanking the main vascular bundle and to a lower extent in filial endospermal transfer cells, which persist until maturity and never accumulate starch. In contrast, cell wall‐bound invertase HvCWINV2 is expressed early in development mainly in the style region and later on in pericarp areas which transiently accumulate starch and undergo degradation later in development. The hexose transporter HvSTP2 shows a temporal and spatial expression pattern similar to HvCWINV2. Transcripts of HvCWINV1 have been localised within the first row of endospermal cells and in the outermost area of the nucellar projection as well as in endospermal transfer cells before starch filling; the same regions of the endosperm are labelled with a hexose transporter HvSTP1‐probe. HvSTP1 is expressed at very low levels within the pericarp but much higher in the syncytial endosperm at 3 DAF and in endospermal transfer cells 7 DAF. The temporal and spatial association of HvCWINV1 and HvSTP1 expression indicates that hexoses liberated by the invertase within the endospermal cavity are taken up by the transporter to be delivered into the central uncellularised space of the endosperm to supply mitotically active endosperm cells with hexoses. The results are discussed and compared with published data on the role of soluble sugars as signal molecules in seed developmental processes.
A novel ER‐derived compartment, the ER body, selectively accumulates a β‐glucosidase with an ER‐retention signal in Arabidopsis
Authors: Matsushima Ryo , Kondo Maki , Nishimura Mikio , Hara‐Nishimura Ikuko
Published: March 2003

The ER body is a novel compartment that is derived from endoplasmic reticulum (ER) in Arabidopsis. In contrast to whole seedlings which have a wide distribution of the ER bodies, rosette leaves have no ER bodies. Recently, we reported that wound stress induces the formation of many ER bodies in rosette leaves, suggesting that the ER body plays a role in the defense system of plants. ER bodies were visualized in transgenic plants (GFP‐h) expressing green fluorescent protein (GFP) with an ER‐retention signal, HDEL. These were concentrated in a 1000‐g pellet (P1) of GFP‐h plants. We isolated an Arabidopsis mutant, nai1, in which fluorescent ER bodies were hardly detected in whole plants. We found that a 65‐kDa protein was specifically accumulated in the P1 fraction of GFP‐h plants, but not in the P1 fraction of nai1 plants. N‐terminal peptide sequencing revealed that the 65‐kDa protein was a β‐glucosidase, PYK10, with an ER‐retention signal, KDEL. Immunocytochemistry showed that PYK10 was localized in the ER bodies. Compared with the accumulation of GFP‐HDEL, which was associated with both cisternal ER and ER bodies, the accumulation of PYK10 was much more specific to ER bodies. PYK10 was one of the major proteins in cotyledons, hypocotyls and roots of Arabidopsis seedlings, while PYK10 was not detected in rosette leaves that have no ER bodies. These findings indicated that PYK10 is the main component of ER bodies. It is possible that PYK10 produces defense compounds when plants are damaged by insects or wounding.
An Arabidopsis mutant defective in UV‐B light‐mediated responses
Authors: Suesslin Christina , Frohnmeyer Hanns
Published: March 2003

In plants, low fluences of ultraviolet‐B radiation (UV‐B; 280–320 nm) trigger the activation of protection mechanisms and various photomorphogenic responses. We established a novel screen to isolate Arabidopsis T‐DNA mutants (uli, UV‐B light insensitive) with reduced sensitivity towards UV‐B light‐mediated inhibition of hypocotyl growth. One of the mutants, uli3, shows also a reduced sensitivity of UV‐B‐induced gene expression events and was, therefore, chosen for further investigation. The ULI3 gene encodes an 80‐kDa protein with potential domains for heme‐ and diacylglycerol‐binding. In transiently transfected protoplasts, the ULI3:GFP fusion protein is localised in the cytoplasm but also adjacent to membranes. In etiolated Arabidopsis seedlings irradiated by UV‐B, ULI3 mRNA expression is strongly up‐regulated. This is caused by elevated transcription as demonstrated using stable transformants where a GUS‐reporter was driven by the ULI3‐promoter. ULI3 is preferentially expressed in the outer cell layers in leaves, stems and flowers, but not in roots. There is evidence that ULI3 represents a specific component involved in UV‐B‐mediated signal transduction in higher plants.
A genome‐wide identification of E2F‐regulated genes in Arabidopsis
Authors: Ramirez‐Parra Elena , Fründt Corinne , Gutierrez Crisanto
Published: March 2003

The completion of the Arabidopsis genomic sequence offers the possibility to extract global information about regulatory mechanisms. Here, we describe a data mining strategy in combination with gene expression analysis to identify bona fide genes regulated by the E2F transcription factor. Starting with a genome‐wide search of chromosomal sites containing E2F‐binding sites, we studied in depth two of the most abundant E2F‐binding sites within the Arabidopsis genome and identified over 180 potential E2F target genes. Among them and in addition to cell cycle‐related genes, we have also identified genes belonging to other functional categories, e.g. transcription, stress and defense or signaling. We have determined the expression levels of genes selected from different categories under two experimental situations. Using cultured cells partially synchronized with aphidicolin, we found that most potential E2F targets identified in silico show a cell cycle‐regulated expression pattern with a peak in early/mid S‐phase. In addition, we used Arabidopsis transgenic plants expressing a DP gene containing a truncated DNA‐binding domain, which likely has a dominant‐negative effect on AtE2Fa, b and c (also named AtE2F3, 1 and 2, respectively), which require DP for efficient DNA binding. Contrary to the up‐regulation observed in early/mid S‐phase‐cultured cells, the expression of a large number of potential E2F targets was decreased in the transgenic plants. Our results strongly support that the RBR/E2F pathway plays a crucial role in regulating the expression of the genes identified in this study.
Loss of non‐host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid
Authors: Van Wees Saskia C. M. , Glazebrook Jane
Published: March 2003

In plants carrying the NahG transgene, salicylate hydroxylase converts salicylic acid (SA) to catechol. Arabidopsis NahG plants are defective in non‐host resistance to Pseudomonas syringae pv. phaseolicola strain 3121 (Psp), suggesting that resistance requires SA signaling. However, several mutants with defects in SA signaling, including eds1, pad4, eds5, sid2, and npr1, remain resistant to Psp, demonstrating that susceptibility of NahG plants is not due to absence of SA. SA synthesis is blocked in sid2NahG double mutants, but resistance to Psp is retained. Therefore, it must be the degradative action of NAHG on SA that causes the loss of resistance of NahG to Psp. Treatment of plants with catechol compromised Psp resistance suggesting that the effect of NahG on resistance results from catechol production. Application of catalase to NahG or catechol‐treated wild‐type plants partially restored resistance to Psp, suggesting that the deleterious effect of catechol results from inappropriate production of hydrogen peroxide. These results indicate that conclusions about SA requirements based solely on phenotypes of NahG plants should be re‐evaluated.
Fatal Clostridium perfringens sepsis from a pooled platelet transfusion
Authors: McDonald  , Hartley  , Orchard  , Hughes  , Brett  , Hewitt  , Barbara
Published: March 2003

A male patient with acute myeloid leukaemia received a pooled platelet preparation prepared by Opti‐pressTM system on the last day of its shelf life. The patient collapsed after two‐thirds of the contents had been transfused. Clostridium perfringens was isolated from the platelet bag within 18 h of the acute event. Metronidazole, gentamicin and Clostridium antiserum were then administered in addition to the broad spectrum antibiotics started previously. However, the patient died 4 days after the platelets were transfused. The cause of death was given as cardiovascular shock, entirely compatible with an overwhelming bacteraemic and septic episode. A coroner's verdict of accidental death due to transfusion of a contaminated unit of platelets was recorded.

On subsequent investigation Cl. perfringens type A serotype PS68,PS80 (identical to that found in the platelet bag) was cultured from the venepuncture site of the arm of one of the donors who contributed towards the platelet pool. The donor had two young children and frequently changed nappies. Faecal contamination of the venepuncture site was the suspected source for the transmission of Cl. perfringens, an organism commonly found in the soil and intestinal tract of humans. This case dramatically highlights the consequences of transfusing a bacterially contaminated unit. It is vital that such incidents are investigated and reported so that the extent of transfusion‐associated bacterial transmission can be monitored and preventative measures taken if possible.
Transfusion of white‐cell‐containing allogeneic blood components and postoperative wound infection: effect of confounding factors
Authors: Vamvakas  , Carven
Published: March 2003

Randomized controlled trials (RCTs) of the relationship between allogeneic transfusion and post‐operative bacterial infection at any site have generated discordant results, but have suggested an association of transfusion with wound infection. To examine the specific association of perioperative transfusion with wound infection, we reviewed the records of 964 consecutive patients undergoing elective colorectal cancer resection at our hospital. Diagnoses of wound infection were made retrospectively by the Centers for Disease Control criteria, and transfusion was defined as number of units of white‐cell‐containing allogeneic blood components received. The probability of wound infection in association with the transfusion was calculated following adjustment for the effects of 12 confounders that had not been previously considered in combination. These factors related to severity of illness, difficulty of operation and risk of wound infection. Wound infection developed in 39 (11.4%) transfused patients, as compared to 24 (3.9%) untransfused subjects (P < 0.0001). In the multivariate analysis, there was a trend suggesting an adverse transfusion effect, which amounted to a 7% increase in the risk of wound infection per unit of red cells or platelets transfused (relative risk [RR] = 1.07; 95% confidence interval for RR = 0.98–1.16; P = 0.1241). This marginally significant effect was related to post‐operative (P = 0.1274), rather than perioperative (P = 0.3061), transfusion. We conclude that allogeneic transfusion may perhaps be associated with a small increase in the risk of post‐operative wound infection, but this small effect can be established only by RCTs enrolling several thousand patients. The modest magnitude of the effect may have been responsible for the disagreements among the published RCTs.
The expression of the NB1 antigen on myeloid precursors and neutrophils from children and umbilical cords
Authors: Stroncek  , Shankar  , Litz  , Clement
Published: March 2003

The neutrophil‐specific antigen NB1 is expressed by neutrophils from 97% of healthy adults. However, membrane expression of this molecule is unique in that it is found on only a subpopulation of neutrophils present in NB1‐positive adults. We have investigated the ontogeny of NB1 antigen expression by haematopoietic progenitor cells to determine the stage and pattern of antigen expression during granulocytic cell differentiation. In addition, we examined whether the ontogeny and frequency of granulocytic cells expressing the NBl antigen might vary in subjects according to age.

A monoclonal antibody (MoAb) specific for NB1 (1B5) and flow cytometry was used to assess the frequency and characteristics of the NB1‐positive cells found in umbilical cord blood (n = 11), children (n = 37), healthy adults (n = 46) and patients with chronic myelogenous leukaemia (n = 8). We also used flow cytometry to isolate NB1‐positive and NB1‐negative bone marrow and peripheral blood cells from various tissue sources. The separated subpopulations were then analysed by Wright stain and light microscopy.

The size of the NB1‐positive neutrophil subpopulation in 46 healthy adults (56 ± 19%) was identical to that found for neutrophils from 36 children ranging in age from 8 months to 18 years (56 ± 11%). In contrast, expression of the NB1 antigen by the neutrophils present in umbilical cord blood (91 ± 3%, n = 11) was significantly greater than that in adults (P < 0.002) or children (P < 0.002).

We also examined the size of the NB1‐positive subpopulation among neutrophils from eight patients with chronic myelogenous leukaemia (CML). The NB1‐positive subset in CML subjects (29.5 ± 22.4%) was significantly less that in healthy adults (P < 0.02) or children (P < 0.02).

Marrow cells from eight adults were similarly separated and analysed. We found that 69 ± 17% of segmented and band forms of neutrophils, 70 ± 2% of metamyelocytes and 61 ± 23% of myelocytes were NB1‐positive. In fetal bone marrow, 86 ± 9% of the segmented and band forms, 82 ± 10% of the metamyelocytes and 3 ± 4% of myelocytes were NB1‐positive.

In conclusion, neutrophil‐specific antigen NB1 is first expressed at the myelocyte stage of myeloid differentitation. In adult bone marrow, the percentages of myelocytes, metamyelocytes and segmented or band cells that expressed this antigen were similar and comparable in magnitude to the frequency of NB1‐positive neutrophils found in the circulation. Although the size of the NBl‐positive neutrophil subpopulation was the same in healthy adults and children, it was significantly increased in umbilical cord blood, and in fetal marrow cells.
IgG anti‐Jka/Jkb antibodies are unlikely to fix complement
Authors: Yates  , Howell  , Overfield  , Voak  , Downie  , Austin
Published: March 2003

Antibodies in the Kidd blood group system show a great deal of serological variability, are notoriously elusive and hence evoke difficulties in detection. However, they have been regularly reported as causing severe immediate or delayed haemolytic transfusion reactions and this clinical potential has been largely attributed to their complement binding ability.

In initial investigations on 43 anti‐Jka/Jkb sera with a range of titres of IgG antibody only a few seemed to fix complement, though following repeated tests on 20 of these sera a further five were shown to bind complement, making a total of 12 (27.9%) showing evidence of complement binding. Twenty‐three sera were unavailable for re‐testing. Subsequent tests revealed that only those sera which showed direct agglutination or were positive with an anti‐IgM reagent in an indirect antiglobulin test (IAT) fixed complement. Evaluations on the IgG fractions of six selected potent anti‐Jka sera failed to reveal any complement‐fixing ability although all the original sera bound complement avidly and contained variable amounts of IgM antibody, some at very low subagglutinating levels.

These findings challenge past perceptions and give cause for reflection on the changing methodologies and strategies which could unduly compromise the detection of these potentially clinically damaging antibodies.
Quality and standardization in blood component preparation with an automated blood processing technique
Authors: Van Rhenen  , Vermeij  , De Voogt  , Bernes  , Payrat
Published: March 2003

The use of automated blood processors in combination with bottom and top blood containers has been found to improve the standardization and quality of blood components. A study was performed to validate a new type of processor (Optipress® II) and compare its performance with a first generation processor (Optipress® I).

Primary separation on the Optipress II was investigated on 570 mL (± 10%) of anticoagulated blood in a nonpaired study. In addition, the quality of the products in routine production was compared between the results of the Optipress I and Optipress II. The whole blood units were kept overnight at room temperature (20 ± 2 °C). Separation was performed under conditions to obtain 55 mL buffy coats with a 50% haematocrit (ht). Platelet concentrate preparation was investigated in a paired study and compared to the routine manual method using PAS II additive solution. Parameters studied were volume, red cell, white cell and platelet counts, ht, haemoglobin (hb, total and free).

Primary separation was more efficient in the Optipress II because the platelet count was lower in the erythrocyte concentrates (P < 0.0001), platelets were lower in plasma (P < 0.0001) and platelet counts were higher in buffy coats (P < 0.0001). Buffy coat volume showed less variation (Optipress II VC = 4%, Optipress I VC = 7.4%). Secondary separation did not show differences between the Optipress II and manual method but was advantageous because of the automatic termination of the procedure.

Further improvement of standardization in blood component preparation is possible with an automated blood processor, leading to improvement of the quality of blood products for patient care.
Rh phenotype prediction by DNA typing and its application to practice
Authors: Flegel  , Wagner  , Müller  , Gassner
Published: March 2003

The complexity of the RHD and RHCE genes, which is the greatest of all blood group systems, confounds analysis at the molecular level. RH DNA typing was introduced in 1993 and has been applied to prenatal testing. PCR‐SSP analysis covering multiple polymorphisms was recently introduced for the screening and initial characterization of partial D. Our objective is to summarize the accrued knowledge relevant to the approaches to Rh phenotype prediction by DNA typing, their possible applications beyond research laboratories and their limitations. The procedures, results and problems encountered are highly detailed. It is recommended that DNA typing comprises an analysis of more than one polymorphism. We discuss future directions and propose a piecemeal approach to improve reliability and cost‐efficiency of blood group genotyping that may eventually replace the prevalent serology‐based techniques even for many routine tasks. Transfusion medicine is in the unique position of being able to utilize the most extensive phenotype databases available to check and develop genotyping strategies.
Evaluation of an enzyme‐linked immunosorbent assay kit (GTI PakPlus®) for the detection of antibodies against human platelet antigens
Authors: Lucas  , Rogers
Published: March 2003

Twenty‐six serum samples from 24 patients were investigated for the presence of platelet‐specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet‐specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme‐linked immunosorbent assay — GTI PakPlus® (GTI kit). Two alkaline phosphatase‐conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti‐IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti‐HPA‐1a [n = 3], anti‐HPA‐3a [n = 1], anti‐HPA‐3b [n = 1] and anti‐HPA‐5b [n = 3]). Greater signal‐to‐background ratios were achieved in the GTI kit with the anti‐IgG conjugate but five antibody specificities (anti‐HPA‐1a [n = 1], anti‐HPA‐3a [n = 1], anti‐HPA‐3b [n = 1], anti‐HPA‐5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal‐to‐background ratio in the majority of sera tested. These findings re‐emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.
True HIV seroprevalence in Indian blood donors
Authors: Choudhury  , Ayagiri  , Ray
Published: March 2003

The National AIDS Control Organization (NACO), the apex body for controlling AIDS in India, projected that HIV seroprevalence would increase from 7/1000 in 1995 to 21.2/1000 in 1997. A high incidence (8.2%) of HIV was observed in blood donors. This study was carried out to find out the true HIV positivity in Indian blood donors. Blood donors from our centre were followed for more than 5 years to determine the true HIV seroprevalence and our result was compared with similar studies from India. Voluntary and relative blood donors who visited the SGPGIMS, Lucknow, since 1993 to June 1998 were included. They were screened for HIV 1/2 by ELISA kits (WHO approved). First‐time HIV‐positive samples were preserved frozen for further study (stage‐I). They were repeated in duplicate and retested with other kits. If found positive, the sample was labelled as ELISA positive (stage‐II). ELISA‐positive samples were confirmed by Western Blot (WB) at stage‐III. A total of 65 288 donors were included and 834 (12.8/1000) were reactive at stage‐I. But 1.1/1000 donors were found to be ELISA positive at stage‐II, and 0.28/1000 donors were positive by WB at stage‐III. The ‘seropositivity’ rate from the NACO was significantly (P < 0.001) higher than our study. There were five similar Indian studies and seropositivity rate varied from 0.72/1000 (using ELISA and WB) to 5.5/1000 (using ELISA alone). The ‘seropositivity’ rate from the NACO was significantly (P < 0.001) higher than all these studies. HIV seroprevalence in the present study is lower (P < 0.001) than other Indian figures. The present and other studies confirmed that the projected HIV seroprevalence (82/1000) in Indian blood donors was high. The NACO result was based on one‐time ELISA screening reports from zonal blood testing centres which also receive samples from paid donors donating in commercial blood banks. The HIV prevalence of blood donors (and national prevalence) is to be reassessed.
Low ferritin levels indicate the need for iron supplementation: strategy to minimize iron‐depletion in regular blood donors
Authors: Alvarez‐Ossorio  , Kirchner  , Klüter  , Schlenke
Published: March 2003

Iron deficiency is a common problem in regular blood donors which can be prevented by timely iron supplementation. Consequently, these donors should be supplied with oral iron in good time. We evaluated the need to use ferritin rather than or in addition to haemoglobin to screen iron deficiency in blood donors. To this end, serum ferritin was measured routinely every 10th donation in 632 long‐term and 171 first‐time donors. Furthermore, donors with ferritin < 15 μg L−1 were supplemented with iron. The supplementation efficiency was assessed by follow‐up haemoglobin levels over the course of five donations in blood donors with high donation frequency.  Our results showed that ferritin decreases after 10 donations and with the increase of donation frequency. In 26% of regular donors, ferritin levels were < 15 μg L−1 and 12% of them were anaemic due to low haemoglobin. After iron supplementation, haemoglobin was raised rapidly in donors with initially low haemoglobin, and thus donor deferment was never indicated. In conclusion, regular ferritin measurement is a useful indicator for iron depletion in blood donors. Our data suggested the usefulness of ferritin screening in first‐time donors and regular donors with low haemoglobin levels within the normal range.
Human plasma and Tirilazad mesylate protect stored human erythrocytes against the oxidative damage of gamma‐irradiation
Authors: Sharifi  , Dzik  , Sadrzadeh
Published: March 2003

Transfusion‐associated graft‐versus‐host disease (TA‐GVHD) is a serious condition that under certain circumstances can be lethal in immunosuppressed patients. The risk of TA‐GVHD can be reduced in this population by gamma irradiation (γRad) of blood components. γRad results in production of reactive oxygen species which can damage red blood cells (RBC). Tirilazad mesylate (TM) is a member of the 21‐aminosteroids (Lazaroids) family and is a powerful antioxidant. We investigated the ability of TM and human plasma (which contain powerful antioxidants) to protect stored human RBC against the oxidative damage of γRad. Fresh intact packed RBC obtained from the normal donors, with and without autologous plasma or TM (0.05 mg mL−1 RBC), were exposed to γRad (50 Gy) and stored for 28 days at 4 °C. Oxidative damage was assessed by osmotic fragility at 65 m
m NaCl concentration (expressed by percentage haemolysis in 65 m
m NaCl solution) and lipid peroxidation (measured by thiobarbituric acid reactive substances, TBARS). Our results showed that storage and irradiation of untreated intact RBC increased the osmotic fragility at 65 m
m NaCl concentration (65.8 ± 1.3 vs. 51.20 ± 0.87% haemolysis; irradiated vs. controls, respectively; P = 0.002) and lipid peroxidation (TBARS = 4.47 ± 0.12 vs. 3.45 ± 0.09 μm L−1 RBC; irradiated vs. controls, respectively; P = 0.001). TM protected the intact RBC against radiation‐induced haemolysis (35.8 ± 5.0 vs. 65.8 ± 1.3% haemolysis; treated vs. untreated irradiated RBC, respectively; P = 0.02) and lipid peroxidation (TBARS = 2.91 ± 0.2 vs. 4.47 ± 0.12 μm L−1 RBC; treated vs. untreated irradiated RBC, respectively; P = 0.005). Addition of autologous plasma to packed RBC significantly reduced the extent of radiation‐induced haemolysis by more than six‐fold (12.45 ± 0.26 vs. 65.8 ± 2.2% haemolysis; irradiated RBC with versus without plasma, respectively; P = 0.0001). In conclusion, these results show that irradiation and storage of blood damages RBC via oxidative processes and addition of autologous plasma and/or TM protects RBC against such damage and possibly enhances their storage and survival.
The computer crossmatch: a safe alternative to the serological crossmatch
Authors: Chapman J. F. , Milkins C. , Voak D.
Published: March 2003

. The crossmatch has evolved from including a wide range of techniques through a test purely to eliminate ABO incompatibility (immediate spin) to computer crossmatching in which no serological testing is carried out and validation ensures the correct ABO/RhD type blood is issued. The crossmatch was always considered to be the most important feature of the compatibility test and in particular the antiglobulin phase; however, there are potential risks associated with serological and computer crossmatching including technical and procedural errors. The use of immediate spin and computer crossmatch change the emphasis for safety of the compatibility test from the crossmatch to the antibody screen. UK guidelines have now been published describing the features necessary for the introduction of computer crossmatching. Computer crossmatching is used by many institutions in various countries. It is considered safe practice and brings benefits to the laboratory and the patient.

Compatibility testing is only one element of the blood transfusion procedure; the others are equally as important and include correct patient identification at the time of collection of the blood sample and at the administration of the blood transfusion.
Refractory hyperhaemolysis in a patient with β‐thalassaemia major
Authors: Grainger J. D. , Makar Y. , McManus A. , Wynn R.
Published: March 2003

. We report the case of a 1‐year‐old girl with newly diagnosed β‐thalassaemia major. Following an initial blood transfusion with phenotypically matched blood, she developed a haemolytic anaemia which progressed with subsequent transfusions.

The Direct Antiglobulin test (DAT) was strongly positive with C3d and weakly with IgG. The only free antibodies detected were a weak anti‐H and a weak cold auto‐antibody, which did not exhibit a wide thermal range. The indirect Donath–Landsteiner and Ham's tests were negative.

There was no sustained clinical response to steroids, immunoglobulin infusions or splenectomy. An HLA identical sibling donor was available for allogeneic bone marrow transplantation (BMT) and the haemolysis resolved during the immunosuppressive transplant conditioning.

Such hyperhaemolysis without significant red cell alloantibodies has previously been reported in patients with sickle cell anaemia, but only rarely in patients with β‐thalassaemia major.