bluuebunny/crossref_metadata_2025 huggingface dataset:
Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype
Authors: Liu Chun‐ming , McElver John , Tzafrir Iris , Joosen Ronny , Wittich Peter , Patton David , Van Lammeren Andre A. M. , Meinke David
Published: March 2003

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T‐DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.
Insights into the evolution of self‐compatibility in Lycopersicon from a study of stylar factors
Authors: Kondo Katsuhiko , Yamamoto Mayu , Itahashi Ryoko , Sato Takanori , Egashira Hiroaki , Hattori Tsukaho , Kowyama Yasuo
Published: March 2003

To elucidate the molecular basis of loss of self‐incompatibility in Lycopersicon, S‐RNases and HT‐proteins were analysed in seven self‐compatible (SC) and three self‐incompatible (SI) taxa. No or low stylar RNase activity was a common feature in most SC taxa examined, in contrast to the uniformly high levels of activity found in all SI species. The S‐RNase gene is most likely deleted in the four red‐fruited SC taxa (L. esculentum, L. esculentum var. cerasiforme, L. pimpinellifolium and L. cheesmanii) because S‐RNase genes could not be amplified from genomic DNA. S‐RNase genes could, however, be amplified from the genomes of the three green‐fruited SC taxa examined. L. chmielewskii and L. hirsutum f. glabratum show a decreased accumulation of transcripts, possibly reflecting changes in the 5′ flanking regions of the S‐RNase genes. The remaining green‐fruited SC species, L. parviflorum, has a functional S‐RNase gene in its genome that is expressed at high levels in the style, suggesting a genetic factor responsible for the low S‐RNase activity. Together these results argue for several independent mutations in the S‐RNase gene over the course of Lycopersicon diversification, and that loss of S‐RNase function is unlikely to the primary cause of the loss of self‐incompatibility. We also examined the HT‐B genes that play a role in self‐incompatibility. HT‐B transcripts were markedly reduced in the styles of all the SC taxa examined. A scenario is described where a mutation causing reduced transcription of HT‐B in an ancestral SI species was central to the loss of self‐incompatibility in Lycopersicon.
Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes
Authors: Walz Christina , Juenger Melanie , Schad Martina , Kehr Julia
Published: March 2003

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS‐PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.
Comparative analysis of plastid transcription profiles of entire plastid chromosomes from tobacco attributed to wild‐type and PEP‐deficient transcription machineries
Authors: Legen Julia , Kemp Sabine , Krause Kirsten , Profanter Birgit , Herrmann Reinhold G. , Maier Rainer M.
Published: March 2003

Transcription of plastid chromosomes in vascular plants is accomplished by at least two RNA polymerases of different phylogenetic origin: the ancestral (endosymbiotic) cyanobacterial‐type RNA polymerase (PEP), of which the core is encoded in the organelle chromosome, and an additional phage‐type RNA polymerase (NEP) of nuclear origin. Disruption of PEP genes in tobacco leads to off‐white phenotypes. A macroarray‐based approach of transcription rates and of transcript patterns of the entire plastid chromosome from leaves of wild‐type as well as from transplastomic tobacco lacking PEP shows that the plastid chromosome is completely transcribed in both wild‐type and PEP‐deficient plastids, though into polymerase‐specific profiles. Different probe types, run‐on transcripts, 5′ or 3′ labelled RNAs, as well as cDNAs, have been used to evaluate the array approach. The findings combined with Northern and Western analyses of a selected number of loci demonstrate further that frequently no correlation exists between transcription rates, transcript levels, transcript patterns, and amounts of corresponding polypeptides. Run‐on transcription as well as stationary RNA concentrations may increase, decrease or remain similar between the two experimental materials, independent of the nature of the encoded gene product or of the multisubunit assembly (thylakoid membrane or ribosome). Our findings show (i) that the absence of photosynthesis‐related, plastome‐encoded polypeptides in PEP‐deficient plants is not directly caused by a lack of transcription by PEP, and demonstrate (ii) that the functional integration of PEP and NEP into the genetic system of the plant cell during evolution is substantially more complex than presently supposed.
The topological specificity factor AtMinE1 is essential for correct plastid division site placement in Arabidopsis
Authors: Maple Jodi , Chua Nam‐Hai , Møller Simon G.
Published: March 2003

In plant cells, plastids divide by binary fission involving a complex pathway of events. Although there are clear similarities between bacterial and plastid division, limited information exists regarding the mechanism of plastid division in higher plants. Here we demonstrate that AtMinE1, an Arabidopsis homologue of the bacterial MinE topological specificity factor, is an essential integral component of the plastid division machinery. In prokaryotes MinE imparts topological specificity during cell division by blocking division apparatus assembly at sites other than midcell. We demonstrate that overexpression of AtMinE1 in E. coli results in loss of topological specificity and minicell formation suggesting evolutionary conservation of MinE mode of action. We further show that AtMinE1 can indeed act as a topological specificity factor during plastid division revealing that AtMinE1 overexpression in Arabidopsis seedlings results in division site misplacement giving rise to multiple constrictions along the length of plastids. In agreement with cell division studies in bacteria, AtMinE1 and AtMinD1 show distinct intraplastidic localisation patterns suggestive of dynamic localisation behaviour. Taken together our findings demonstrate that AtMinE1 is an evolutionary conserved topological specificity factor, most probably acting in concert with AtMinD1, required for correct plastid division in Arabidopsis.
Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas
Authors: Schroda Michael , Beck Christoph F. , Vallon Olivier
Published: March 2003

We have shown previously that the HSP70A (A) promoter, when fused upstream of other promoters, significantly improves their performance in driving transgene expression in Chlamydomonas. Here, we employed the bacterial resistance gene ble, driven by the RBCS2 (R) promoter or an AR promoter fusion, to determine, by which mechanism(s) the A promoter may exert its enhancing effect. We observed that transformation rates of AR‐ble constructs were significantly higher than those of R‐ble constructs. However, ble mRNA levels in pools of transformants generated with either construct type were the same. Co‐transformation experiments revealed that the R‐ble transgene was silenced in 80% of the transformants, whereas this fraction was reduced to 36% in transformants harbouring the AR‐ble transgene. We conclude that the A promoter acts by decreasing the probability that a transgene becomes transcriptionally silenced. We mapped two elements within the A promoter that are responsible for this effect. The core of the first element appears to be located between nucleotides − 7 and + 67 relative to the HSP70A transcriptional start site. Its activity is strongly dependent on its spatial setting with respect to the R promoter and is increased by upstream sequences (− 196 to − 8). The second element is independent of the first and is located to the region from − 754 to − 197. Its activity is spacing‐independent and additive to the first element.
Genotype‐dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa (Medicago sativa)
Authors: Ivashuta Sergey , Naumkina Marina , Gau Mitsuru , Uchiyama Kazuhiro , Isobe Sachiko , Mizukami Yuko , Shimamoto Yoshiya
Published: March 2003

In a search for cold‐regulated genes that are differentially expressed in alfalfa genotypes of contrasting freezing tolerance, we screened 1036 arrayed cDNA clones. The screening resulted in isolation of cDNA clones, which demonstrated dramatic differences in expression between hardy and un‐hardy alfalfa varieties. Detailed analysis revealed that these cDNAs represent parts of novel non‐coding repetitive elements carrying long‐terminal repeats (LTR) and other retroelement‐like features. Despite strong expression under low temperatures, DNA templates remained highly methylated, and a drug‐induced decrease in methylation did not activate transcription under normal temperatures. We identified that these repetitive elements represent a large family and could insert into, or be adjacent to, the unrelated polyprotein sequences of putative retrotransposons. These retrotransposons also showed low temperature‐induced transcriptional activation; however, this activation was not genotype‐dependent. The retroelements described in this study are the first retroelement characterized in the Medicago genus. Furthermore, they represent the only known example of genotype‐specific cold‐induced transcriptional activation of multiple copies of a repetitive element whose expression is associated with a genotype difference in cold acclimation.
Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence
Authors: Jing Hai‐Chun , Sturre Marcel J. G. , Hille Jacques , Dijkwel Paul P.
Published: March 2003

The onset of leaf senescence is controlled by leaf age and ethylene can promote leaf senescence within a specific age window. We exploited the interaction between leaf age and ethylene and isolated mutants with altered leaf senescence that are named as onset ofleafdeath (old) mutants. Early leaf senescence mutants representing three genetic loci were selected and their senescence syndromes were characterised using phenotypical, physiological and molecular markers. old1 is represented by three recessive alleles and displayed earlier senescence both in air and upon ethylene exposure. The etiolated old1 seedlings exhibited a hypersensitive triple response. old2 is a dominant trait and the mutant plants were indistinguishable from the wild‐type when grown in air but showed an earlier senescence syndrome upon ethylene treatment. old3 is a semi‐dominant trait and its earlier onset of senescence is independent of ethylene treatment. Analyses of the chlorophyll degradation, ion leakage and SAG expression showed that leaf senescence was advanced in ethylene‐treated old2 plants and in both air‐grown and ethylene‐treated old1 and old3 plants. Epistatic analysis indicated that OLD1 might act downstream of OLD2 and upstream of OLD3 and mediate the interaction between leaf age and ethylene. A genetic model was proposed that links the three OLD genes and ethylene into a regulatory pathway controlling the onset of leaf senescence.
Paternal mRNA and protein synthesis coincides with male chromatin decondensation in maize zygotes
Authors: Scholten Stefan , Lörz Horst , Kranz Erhard
Published: March 2003

Decondensation of the male genome after fertilization is a prerequisite for replication and transcription. Cytological analysis has revealed decondensation of the male chromatin to commence immediately after karyogamy and progress rapidly, pointing to an early start of transcription. To investigate early transcription from the paternal genome in maize zygotes, we generated transgenic plants containing green fluorescent protein (GFP) under control of the 35S promoter. Single transgenic sperm cells from these plants were used to fertilize isolated wild‐type egg cells in vitro. These sperm cells did not contain gfp transcripts. Appearance of gfp mRNA, 4 h after fertilization, was coincident with decondensation of the male chromatin, and clearly demonstrates early accessibility to the transcriptional machinery of at least a part of the male genome. Translational activity in early zygotes was evident 6 h after fertilization, as demonstrated by measurable levels of GFP fluorescence signal. Using a similar strategy, we also demonstrated activity of the paternal genome early in endosperm development. These findings may exclude any global mechanism of silencing the entire paternal genome over this period, and make an almost immediate paternal contribution to zygote and early endosperm development conceivable. These data are also considered in the perspective of current views of genome activation in the zygotes and young embryos of animals.
Characterization of Arabidopsis thaliana AtFKBP42 that is membrane‐bound and interacts with Hsp90
Authors: Kamphausen Thilo , Fanghänel Jörg , Neumann Dieter , Schulz Burkhard , Rahfeld Jens‐U.
Published: March 2003

The twisted dwarf1 (twd1) mutant from Arabidopsis thaliana was identified in a screen for plant architecture mutants. The TWD1 gene encodes a 42 kDa FK506‐binding protein (AtFKBP42) that possesses similarity to multidomain PPIases such as mammalian FKBP51 and FKBP52, which are known to be components of mammalian steroid hormone receptor complexes. We report here for the first time the stoichiometry and dissociation constant of a protein complex from Arabidopsis that consists of AtHsp90 and AtFKBP42. Recombinant AtFKBP42 prevents aggregation of citrate synthase in almost equimolar concentrations, and can be cross‐linked to calmodulin. In comparison to one active and one inactive FKBP domain in FKBP52, AtFKBP42 lacks the PPIase active FKBP domain. While FKBP52 is found in the cytosol and translocates to the nucleus, AtFKBP42 was predicted to be membrane‐localized, as shown by electron microscopy.
Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem
Authors: Asano Takayuki , Masumura Takehiro , Kusano Hiroaki , Kikuchi Shoshi , Kurita Akihiro , Shimada Hiroaki , Kadowaki Koh‐ichi
Published: March 2003

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene‐expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non‐redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non‐redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.
Molecular analysis of herbivore‐induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides)
Authors: Peters Darren J. , Constabel C. Peter
Published: March 2003

In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single‐copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes.
T‐DNA recombination and replication in maize cells
Authors: Zhao Xiaoxia , Coats Isabelle , Fu Ping , Gordon‐Kamm Bill , Lyznik L. Alexander
Published: March 2003

T‐DNA recombination and replication was analyzed in ‘black mexican sweet’ (BMS) cells transformed with T‐DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T‐DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T‐DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T‐DNA molecules. Circularization of T‐DNA by the FLP/FRTsite‐specific recombination system and/or homologous recombination was not necessary to recover circular T‐DNAs. However, replicating T‐DNAs appeared to be suitable substrates for site‐specific and homologous recombination. Among 33 T‐DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T‐DNAs was highly heterogenous. Since no circular T‐DNA molecules were detected in treatments withoutrep, it suggested that T‐DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T‐DNA molecules were indicative of illegitimate recombination and were similar to left‐border recombination of T‐DNA into the genome after Agro‐mediated plant transformation. However, recombination between T‐DNA molecules differed from T‐DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T‐DNA molecules did not enhance genomic random integration of T‐DNA in the experimental configuration used for this study.
Two different novel cis‐acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark‐induced senescence
Authors: Simpson Sean D. , Nakashima Kazuo , Narusaka Yoshihiro , Seki Motoaki , Shinozaki Kazuo , Yamaguchi‐Shinozaki Kazuko
Published: March 2003

Many plant genes have been shown to be induced by water stress and function in stress tolerance. The erd1 gene has been shown to be upregulated in response to both water stress and etiolation. Promoter studies using the erd1 promoter region fused to the luciferase (LUC) reporter gene in Arabidopsis thaliana were performed to identify the putative cis elements involved. Results indicated that the cis elements, responsible for gene expression during dehydration and etiolation, are separately located in two discrete portions of the erd1 promoter. Base substitution analysis showed that a 14‐bp region from −599 to −586, and a myc recognition motif from −466 to −461 are necessary for the induction of LUC activity in dehydrated plants. On the other hand, base substitution analysis revealed that both an abscisic acid responsive element (ABRE)‐like sequence (from −199 to −195) and an ACGT sequence (from −155 to −152) are required for an etiolation‐induced increase in LUC activity. LUC activity measurements from etiolated transgenic plants incubated in either water, N6‐benzyleadenine (BA), or a 1% sucrose solution found that while BA was able to delay the increase in LUC activity seen in water‐treated plants, no increase in LUC activity was seen in plants incubated in sucrose. These results indicate that the erd1 promoter contains two different regulatory systems that are involved in upregulation by dehydration stress and dark‐induced senescence.
The DNA‐binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low‐temperature responsive genes in barley is modulated by temperature
Authors: Xue Gang‐Ping
Published: March 2003

An HvCBF2 cDNA was isolated from barley leaves. It encoded a protein containing an AP2 DNA‐binding domain homologous to C‐repeat (CRT)/dehydration‐responsive element (DRE) binding factors (CBF/DREB1). In contrast to the previously reported cold‐inducible CBF/DREB1 genes, HvCBF2 was expressed in barley leaves under non‐stress conditions. Only a transient increase in the HvCBF2 transcript level was observed during cold treatment. Transactivation analysis showed that HvCBF2 was a transcriptional activator, capable of activating expression of a reporter gene driven by a low‐temperature and drought‐responsive HVA1s promoter in barley leaves. The activity of HvCBF2 as a transcriptional activator was upregulated by low temperature. DNA‐binding analysis revealed that HvCBF2 did not bind to the CRT/DRE motif at 30°C. A low, but detectable, binding activity was observed at 25°C and the binding activity gradually increased as the temperature decreased. The binding activity at 0°C was the highest and more than 10 times higher than that at 25°C. The activation and inactivation of HvCBF2 activity were reversible and were achieved in a cell‐free system simply by temperature change. Analysis of the binding sequence showed that HvCBF2 bound to a (G/a)(T/c)CGAC core motif, where the lower‐case letters are less efficient bases. These data suggest that HvCBF2 is a transcription factor interacting with the core CRT/DRE motif containing a preferred sequence of GTCGAC and its DNA‐binding activity is regulated by temperature. This represents a new type of activation mechanism for transcriptional activators.
Mutations in a gene for plastid ribosomal protein S6‐like protein reveal a novel developmental process required for the correct organization of lateral root meristem in Arabidopsis
Authors: Horiguchi Gorou , Kodama Hiroaki , Iba Koh
Published: March 2003

Arabidopsis rfc3 mutants have previously been isolated with an altered fatty acid composition of membrane lipids. In this study, rfc3 was found to have a sucrose‐conditional defect in the patterning of distal elements in the lateral root meristem. By utilizing this feature, a sucrose‐sensitive process important for lateral root development was localized to the growing portion of rfc3 primary root. Because lateral root formation occurs at a later stage, this finding suggests the existence of an RFC3‐dependent, non‐primordium autonomous signal playing a role in the organization of lateral root meristem. Map‐based cloning of RFC3 gene revealed that it encodes a plastid‐localized ribosomal protein S6‐like protein and provides a potential link between control of plastid gene expression and LR development.
Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco
Authors: Dat James F. , Pellinen Riikka ,   Tom Beeckman, Van De Cotte Brigitte , Langebartels Christian , Kangasjärvi Jaakko , Inzé Dirk , Van Breusegem Frank
Published: March 2003

In transgenic tobacco plants with reduced catalase activity, high levels of hydrogen peroxide (H2O2) can accumulate under photorespiratory conditions. Such a perturbation in H2O2 homeostasis induced cell death in clusters of palisade parenchyma cells, primarily along the veins. Ultrastructural alterations, such as chromatin condensation and disruption of mitochondrial integrity, took place before cell death. Furthermore, enhanced transcript levels of mitochondrial defense genes accompanied these mitochondrial changes. Pharmacological data indicated that the initiation and execution of cell death require de novo protein synthesis and that the signal transduction pathway leading to cell death involved changes in ion homeostasis, (de)phosphorylation events and an oxidative burst, as observed during hypersensitive responses. This oxidase‐dependent oxidative burst is essential for cell death, but it is not required for the accumulation of defense proteins, suggesting a more prominent role for the oxidative burst in abiotic stress‐induced cell death.
Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves
Authors: Fryer Michael J. , Ball Louise , Oxborough Kevin , Karpinski Stanislaw , Mullineaux Philip M. , Baker Neil R.
Published: March 2003

In Arabidopsis leaves, high light stress induces rapid expression of a gene encoding a cytosolic ascorbate peroxidase (APX2), whose expression is restricted to bundle sheath cells of the vascular tissue. Imaging of chlorophyll fluorescence and the production of reactive oxygen species (ROS) indicated that APX2 expression followed a localised increase in hydrogen peroxide (H2O2) resulting from photosynthetic electron transport in the bundle sheath cells. Furthermore, leaf transpiration rate also increased prior to APX2 expression, suggesting that water status may also be involved in the signalling pathway. Abscisic acid stimulated APX2 expression. Exposure of ABA‐insensitive mutants (abi1‐1, abi2‐1) to excess light resulted in reduced levels of APX2 expression and confirmed a role for ABA in the signalling pathway. ABA appears to augment the role of H2O2 in initiating APX2 expression. This regulation of APX2 may reflect a functional organisation of the leaf to resolve two conflicting physiological requirements of protecting the sites of primary photosynthesis from ROS and, at the same time, stimulating ROS accumulation to signal responses to changes in the light environment.
Developmental variation in stem‐cell markers from human fetal liver and umbilical cord blood leukocytes
Authors: Kilpatrick  , Atkinson  , Palmer  , Murphy  , Turner
Published: March 2003

Fetal tissues containing haematopoietic stem cells (HSC) are of potential value for allogeneic transplantation and gene therapy. Flow cytometry was used to investigate CD34+ cells from human fetal livers and umbilical cord (placental) blood (UCB). CD34+ cells, expressed as a proportion of CD45‐positive leukocytes, were much more abundant in fetal livers (mean 38%) than in UCB (mean 0.3%), but fetal liver cells had lower proportions of CD34+HLA‐DR+ and CD34+ CD38+ subsets. In fetal liver, there was a strong and highly significant inverse correlation between CD34+ cells (as a proportion of total leukocytes) and gestational age; no such relationship was found for subsets of CD34+ cells coexpressing CD38 or CDw90 (Thy‐1), but CD34+HLA‐DR+ cells were less abundant in first‐ compared to second‐trimester livers. In UCB, a trend towards decreasing CD34+ cells (as a proportion of total leukocytes) with increasing gestational age in late pregnancy was also observed. The composition of fetal leukocytes changes during development, and therefore the timing of fetal HSC harvesting could be of relevance to transplantation outcome.
Blood transfusion and total joint replacement surgery: T helper 2 (TH2) cytokine secretion and clinical outcome
Authors: Kirkley  , Cowles  , Pellegrini  , Harris  , Boyd  , Blumberg
Published: March 2003

Surgery and blood transfusions have both been reported to cause decreases in various measures of cell‐mediated immunity. A study of in vitro T helper lymphocyte type 2 (Th2) cytokine secretion after major joint replacement surgery was performed because these cytokines (IL4 and IL10) generally down‐regulate cellular immune function. Th1 cytokines such as IL2 tend to up‐regulate cellular immunity. Forty‐three patients undergoing elective joint replacement surgery had pre‐ and multiple post‐operative levels of IL2, IL4 and IL10 secretion measured and analysed with regard to demographic and clinical outcome data. Total joint replacement alone without allogeneic transfusions led to substantial increases in peak mean IL4 (2.1 times the pre‐operative level) and IL10 secretion in vitro (4.3‐fold) compared with much more modest increases in IL2 (1.36‐fold) (P < 0.0001 for changes from baseline for each cytokine). In 14 patients who received allogeneic transfusions, these changes were greater than those in recipients of only autologous blood for IL4 (5.0‐fold; P = 0.0036 vs. no allogeneic transfusion) and IL10 (15.7‐fold; P = 0.079) but not for IL2 (1.38‐fold; P = 0.38). The dramatic increase in Th2 cytokine secretion and minimal change in Th1 cytokine secretion after total joint replacement, with or without allogeneic transfusions, was seen regardless of type of anaesthetic, duration of surgery or whether knee or hip replacement occurred. These changes in cytokine patterns may contribute to the decreases in cellular immune function seen after surgery. Allogeneic transfusions but not autologous transfusions appear to exacerbate this immune deviation toward a T helper 2 (Th2) type response, and thus probably contribute to down‐regulation of cellular immunity in the setting of joint replacement surgery.
Erythropoietin to minimize perioperative blood transfusion: a systematic review of randomized trials
Authors: Laupacis  , Fergusson  ,   for The International Study of Peri‐operative Transfusion (ISPOT) Investigators
Published: March 2003

Our aim was to perform a systematic review to determine the efficacy and side‐effects of erythropoietin, given with or without autologous predonation, to patients undergoing orthopaedic or cardiac surgery.

A number of studies have been done to determine whether erythropoietin minimizes exposure to perioperative allogeneic red cell transfusion. A systematic review of all randomized trials will provide the best estimate of the efficacy and side‐effects of erythropoietin therapy.

All randomized trials of erythropoietin in cardiac or orthopaedic surgery that reported the proportion of patients receiving perioperative allogeneic transfusion were included. The efficacy of erythropoietin was evaluated in subgroups of patients depending upon the route of administration, dose of erythropoietin, the type of control and the methodological quality of the study report.

The odds ratio for the proportion of patients transfused with allogeneic blood in studies of erythropoietin to augment autologous donation was 0.42 (95% confidence limits 0.28–0.62; P < 0.0001) for orthopaedic surgery and 0.25 (95% CI 0.08–0.82; P = 0.02) for cardiac surgery. The odds ratio for erythropoietin alone was 0.36 (95% CI 0.24–0.56; P = 0.0001) in orthopaedic surgery and 0.25 (95% CI 0.06–1.04; P < 0.06) in cardiac surgery. The route of administration, dose of erythropoietin, type of control and methodological quality of the study report had no statistically significant effect upon the odds ratios. Although there was no convincing evidence that erythropoietin used alone increased the frequency of thrombotic complications, some studies found an excess of events in erythropoietin‐treated patients, and the number of patients studied was relatively small.

Erythropoeitin, when given alone or to augment autologous donation, decreased exposure to perioperative allogeneic transfusion in orthopaedic and cardiac surgery. Further studies are required to definitively establish the safety of erythropoietin alone, to determine the optimal dose of perioperative erythropoietin, and to compare its efficacy and cost‐effectiveness with other methods of minimizing perioperative transfusion.
Quality assurance by a commercial flow cytometry method of leucodepletion of whole blood donations: initial application of universal testing and proposals for a batch‐release sampling plan
Authors: Barclay  , Walker  , Gibson  , McColl  , Turner
Published: March 2003

We have used the Becton‐Dickinson LeucoCOUNT test to monitor residual leucocytes in whole blood by flow cytometry following leucodepletion filtration. This test was found to be quick, robust and reliable, and allowed measurement of residual leucocytes down to 2.25 × 104 leucocytes per unit, which was found to match approximately the limit of filter proficiency. The results of testing > 1000 units showed a lognormal distribution with means between log(10) 4.864 (0.73 × 105) and log(10) 5.016 (1.04 × 105) leucocytes per unit in the three different homogeneous groups of filtered units studied. The numbers of units with residual leucocytes exceeding the 5 × 106 upper threshold were 1/577, 2/457 and 0/87 in these groups. The filtration processes were validated according to the published BEST working party guidelines and were well within the 99% confidence and 95% tolerance target for the 5 × 106 upper threshold set for the UK. A sampling plan based on British Standard BS 6001 with elimination of outliers by the extreme studentized deviate (Grubbs' test) was evaluated on random samples from these groups and has been adopted locally as a prospective batch‐release criterion for release of leucodepleted blood.
Perception of risk of blood transfusion: knowledge, group membership and perceived control
Authors: Ferguson E. , Farrell K. , Lowe K. C. , James V.
Published: March 2003

. The aim was to explore the perceptions of risk and choice behaviour associated with blood transfusion, within four key societal groups, namely general practitioners (GPs), anaesthetists, health/lifestyle journalists and blood donors.

The study comprised a series of cross‐sectional survey assessments incorporating a one‐way between groups. Eighty‐eight GPs, 141 anaesthetists, 20 journalists and 250 blood donors participated. The main outcome measures were perceptions of risk associated with blood transfusion (and nine other hazards, e.g. smoking) and whether or not participants, given the choice, would choose to have a transfusion of donated blood or a blood substitute. Level of knowledge formed a third main outcome variable.

In terms of rank order, all respondent groups rated the risk of transfusion relatively realistically. That is, they perceived it as very low compared, for example, with smoking or driving a car. Levels of perceived risk about transfusion were associated primarily with subjective knowledge (a sense of what it is felt that people know) rather than objective (what people actually know). This relationship indicated that the more people felt was known, the less risk they perceived. Furthermore, participants were up to five times more likely to choose a blood substitute if they perceived a high level of risk associated with blood transfusion. All groups (especially journalists and blood donors) were under‐confident with respect to their objective knowledge about transfusion (i.e. they knew more than they thought they did).

Transfusion risk was perceived as relatively low. Objective knowledge was not associated with perceived risk for a transfusion. It is argued that facts on blood and transfusion should be presented in a manner that is congruent with the type of information being presented.
Epitope mapping of four novel CD44 monoclonal antibodies using surface plasmon resonance and soluble CD44
Authors: Masson D. , Vusio P. , Loirat M. J. , Spring F. , Anstee D. J. , Denis M. G. , Lustenberger P. , Blanchard D.
Published: March 2003

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule. The molecular diversity of this glycoprotein is generated by both post‐translational modification and the differential use of alternatively spliced exons which play a critical role in determining the exact conformation of the molecule. CD44 isoforms are found in many tissues and in soluble form in plasma. Soluble CD44 was purified by a two‐step purification combining ion exchange and immuno‐affinity chromatography. Our aim was to check that all the known antigenic epitopes are present on sCD44, which could thus be used for the mapping of new anti‐CD44 antibodies. Competitive binding assays using reference antibodies and novel anti‐CD44 monoclonal antibodies were performed by real‐time biospecific interaction analysis. Reference mAbs identified on soluble CD44 the three distinct epitopes previously defined using red blood cell membrane CD44. From the four novel CD44 mAbs, two mAbs (NaM198‐6B5, NaM198‐10B4) mapped to epitope group 1, whereas the others (NaM10‐8F4, NaM77‐9D6) mapped to epitope group 2. Immunopurified sCD44 obtained from the plasma of healthy donors appears to be a usable tool for the mapping of anti‐CD44 mAbs.
A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell‐associated C3d
Authors: Stroncek D. F. , Njoroge J. M. , Procter J. L. , Childs R. W. , Miller J.
Published: March 2003

Summary. This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)‐associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti‐C3d, anti‐IgG, anti‐IgM and anti‐IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti‐IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti‐C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti‐IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post‐transplant, the flow cytometry assays for anti‐IgG and/or anti‐C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC‐associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.
The early sporogonic cycle of Plasmodium falciparum in laboratory‐infected Anopheles gambiae: an estimation of parasite efficacy
Authors: Gouagna Louis Clément , Gouagna Louis Clément , Mulder Bert , Mulder Bert , Noubissi Elisabeth , Noubissi Elisabeth , Tchuinkam Timoléon , Tchuinkam Timoléon , Jan Peter Verhave  , Boudin Christian , Boudin Christian , Boudin Christian
Published: March 2003

This study investigated the successive losses in the parasite densities of Plasmodium falciparum stages during the early sporogony in laboratory‐reared Anopheles gambiae infected by membrane feeding with blood from naturally infected gametocyte carriers (>50 gametocytes/mm3). The developmental stages of P. falciparum in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post‐infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid‐size oocysts count, classic microscopy examination was used at day 7 post‐infection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in A. gambiae. Thirty‐seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100%; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid‐size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid‐size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid‐size oocysts) and between round forms and mid‐size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (yield Y1 = 41.6%) and between ookinetes and young oocysts (Y2 = 61.4%). By contrast, no significant decrease was observed between young oocysts and mid‐size oocysts (Y3 = 91.2%). The overall yield of the early sporogonic cycle (from round form to oocyst at day 7) was equal to 25.7%, indicating that almost 3/4 of the total parasites were lost during the early step of the sporogonic cycle, from 3 h post‐infection to day 7.
Diagnostic quality in rural health centres in Burkina Faso
Authors: Krause G. , Schleiermacher D. , Borchert M. , Benzler J. , Heinmüller R. , Ouattara K. , Coulibaly S. , Diasso I. , Ilboudo A. , Diesfeld H.‐J.
Published: March 2003

Summary

objective  To study the quality of diagnostic practice in rural Burkina Faso.

method  In 9 health centres of 3 districts, 313 outpatient consultations were observed, and 417 diagnoses by 15 nurses were analysed. Criteria for evaluation of patient history and clinical examination were based on the diagnostic guidelines distributed by the Ministry of Health.

results  In only 20% of the diagnoses the nurses took a sufficient history and in only 40% they conducted a sufficient clinical examination. In 21% patients underwent no clinical examination at all. Only 12% of all diagnoses were based on sufficient history‐taking and adequate clinical examinations. The individual elements of clinical examination were performed correctly in 82% of cases. The variation between nurses was immense, but no correlation could be found with regard to their basic training. However, nurses who had received the diagnostic guidelines examined patients more carefully than those who had not. Larger numbers of patients per day are not associated with shorter nurse‐patient contact, and neither is sufficiency of patient history associated with duration of the consultation.

conclusion The low diagnostic quality of the outpatient consultations in the studied area indicates that this issue has been neglected in national public health initiatives. But examination skills are good and diagnostic guidelines may have had a positive effect on the diagnostic quality.
Cryptic speciation in Triatoma sordida (Hemiptera: Reduviidae) from the Bolivian Chaco
Authors: Noireau  , Gutierrez  , Zegarra  , Flores  , Brenière  , Cardozo  , Dujardin
Published: March 2003

Eight natural Bolivian populations of two closely related species of Triatominae, Triatomasordida and T. guasayana, were analysed by multilocus enzyme electrophoresis at 20 loci. Both species were readily separated and no natural hybrid was observed. Among the silvatic sample of T. sordida, strong departure from panmixia within and between loci was detected in two sites of the Chaco, suggesting two reproductively separate populations easily recognized at Idh2 and Mdh2 loci. Genetic distance between them was in agreement with the hypothesis of distinct species. However, the detection of 3% of putative hybrids suggested a recent evolutionary divergence.
Human IgE responses to rSm22.6 are associated with infection intensity rather than age per se, in a recently established focus of Schistomiasis mansoni
Authors: Webster M. , Roberts M. , Fulford A. J. C. , Marguerite M. , Gallisot M‐C. , Diagne M. , Niang M. , Riveau G. , Capron A. , Dunne D. W.
Published: March 2003

In studies of schistosomasis mansoni‐endemic communities, individuals with IgE responses to a 22 kD adult worm antigen (rSm22.6) suffered lower intensities of reinfection after treatment. It is of interest to define the factors that lead to the production of rSm22.6‐specific IgE because it is a marker for resistant individuals and it may be involved in the development of resistance to reinfection. In endemic populations rSm22.6‐specific IgE increases linearly with age. However, it is not possible to distinguish between age per se and ‘history of infection’ in endemic populations because individuals are exposed to the parasite at an early age. We have, therefore, quantified pre‐ and post‐treatment isotype responses to rSm22.6 in a comparatively ‘epidemic’ Senegalese community where the patients were infected at different ages and where pre‐treatment intensity of infection can be taken as a reasonable measure of antigen exposure. Post‐treatment isotype responses to rSm22.6 correlated positively with pre‐treatment intensities of infection but were not shown to be related to age. IgG1, IgG4 and IgE responses to rSm22.6 were significantly higher after treatment with the difference increasing with the pre‐treatment level of infection. These results from a recently established focus of infection suggest that isotype responses to rSm22.6 are antigen‐exposure dependent rather than dependent on age per se.