bluuebunny/crossref_metadata_2025 huggingface dataset:
Ozone treatment rapidly activates MAP kinase signalling in plants
Authors: Samuel Marcus A. , Miles Godfrey P. , Ellis Brian E.
Published: March 2003

Brief exposure to ozone, a potent cross‐inducer of plant stress responses, leads within minutes to activation of an ERK‐type MAP kinase (approximately 46 kDa) in tobacco. This activation process is calcium‐dependent and can be blocked both by free radical quenchers and by a specific inhibitor of MEK‐1 (MAPKK). Hydrogen peroxide and superoxide anion radicals can substitute for ozone as the activation stimulus, which does not appear to require salicylate as an intermediary. The properties of the ozone‐induced MAPK suggest that it may be SIPK (salicylate‐induced protein kinase), a tobacco MAPK that is activated by a variety of stress treatments. The ability of ozone to activate SIPK indicates that this protein kinase acts as a very early transducer of redox stress signals in plant cells.
A novel composite locus of Arabidopsis encoding two polypeptides with metabolically related but distinct functions in lysine catabolism
Authors: Tang Guiliang , Zhu Xiaohong , Tang Xiaohu , Galili Gad
Published: March 2003

Both plants and animals catabolise lysine via saccharopine by two consecutive enzymes, lysine‐ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305–1316). By expressing several epitope‐tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3′ non‐coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism
Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana
Authors: Seo Mitsunori , Koiwai Hanae , Akaba Shuichi , Komano Teruya , Oritani Takayuki , Kamiya Yuji , Koshiba Tomokazu
Published: March 2003

Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOα, AOβ and AOγ, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOδ, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO

δ was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOδ. These results indicate that AOδ is encoded by AAO3. AOδ produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 μm), and the oxidation product was determined by gas chromatography–mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOδ, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.
A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells
Authors: Takeuchi Masaki , Ueda Takashi , Sato Ken , Abe Hiroshi , Nagata Toshiyuki , Nakano Akihiko
Published: March 2003

Protein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited. We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants. In this study, we have established a transient expression system of GFP‐fusion proteins in tobacco and Arabidopsis cultured cells. By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER‐to‐Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER. The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin. These results not only provide the first evidence that the Sar1 GTPase functions in the ER‐to‐Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.
Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus†
Authors: Gutensohn Michael , Schulz Burkhard , Nicolay Peter , Flügge Ulf‐Ingo
Published: March 2003

Two Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter‐GUS plants and in situ hybridisations as well as immunoblot analysis. The atToc34 and atToc33 genes are expressed in green as well as non‐green tissues and are developmentally regulated. Despite these similarities, however, the two Arabidopsis Toc34 homologues are differentially expressed in various plant organs. To gain more insight into the in vivo function of both proteins, antisense plants were created. While antisense plants of atToc33 are characterized by a pale yellowish phenotype, antisense plants of atToc34 show a weaker phenotype. Protein interaction studies using an in vitro translated precursor protein and heterologously expressed atToc34 and atToc33 proteins showed a direct GTP‐dependent interaction, but demonstrated different affinities of the two atToc proteins towards the precursor protein. Thus, our results indicate a more specialized function for both atToc34 and atToc33, suggesting specificity for certain imported precursor proteins.
NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures
Authors: Clarke Andrew , Desikan Radhika , Hurst Roger D. , Hancock John T. , Neill Steven J.
Published: March 2003

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant–pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO‐induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase‐like activity in Arabidopsis cells, while the caspase‐1 inhibitor, Ac‐YVAD‐CMK, blocked NO‐induced cell death. A well‐established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO‐induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell‐permeable cGMP analogue, 8Br‐cGMP, although 8Br‐cGMP alone did not induce cell death or potentiate NO‐induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO‐induced cell death in Arabidopsis. In‐gel protein kinase assays showed that NO activates a potential mitogen‐activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2‐induced cell death, did not inhibit the effects of NO.
Multiple pathways for Cre/lox‐mediated recombination in plastids
Authors: Hajdukiewicz Peter T. J. , Gilbertson Larry , Staub Jeffrey M.
Published: March 2003

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to ≈10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker‐free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre‐mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome ‘hot spot’ of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans‐splicing events in higher plant plastids.
NtSET1, a member of a newly identified subgroup of plant SET‐domain‐containing proteins, is chromatin‐associated and its ectopic overexpression inhibits tobacco plant growth
Authors: Shen Wen‐Hui
Published: March 2003

The SET‐ and chromo‐domains are recognized as signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. This paper reports the identification of a novel subgroup of SET‐domain‐containing proteins in tobacco and Arabidopsis, which show highest homologies with the Drosophila position‐effect‐variegation repressor protein SU(VAR)3–9 and the yeast centromer silencing protein CLR4. The tobacco SET‐domain‐containing protein (NtSET1) was fused to the green fluorescence protein (GFP) that serves as a visual marker for localization of the recombinant protein in living cells. Whereas control GFP protein alone was uniformly dispersed within the nucleus and cytoplasm, the NtSET1‐GFP fusion protein showed a non‐uniform localization to multiple nuclear regions in interphase tobacco TBY2 cells. During mitosis, the NtSET1‐GFP associated with condensed chromosomes with a non‐random distribution. The NtSET1 thus appears to have distinct target regions in the plant chromatin. Overexpression of the NtSET1‐GFP in transgenic tobacco inhibited plant growth, implicating the possible involvement of the NtSET1 in transcriptional repression of growth control genes through the formation of higher‐order chromatin domains.
Functional importance of conserved domains in the flowering‐time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants
Authors: Robson Frances , Costa M. Manuela R , Hepworth Shelley R , Vizir Igor , Pin˜eiro Manuel , Reeves Paul H , Putterill Joanna , Coupland George
Published: March 2003

CONSTANS promotes flowering of Arabidopsis in response to long‐day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO‐Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO‐Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B‐boxes, which act as protein–protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B‐boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co‐3 allele, that has a mutation in the second B‐box, delayed flowering of wild‐type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.
RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria
Authors: Farré Jean‐Claude , Araya Alejandro
Published: March 2003

Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self‐splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon‐binding and intron‐binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site‐directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1‐IBS1 and EBS2‐IBS2 interactions and reveal a new secondary‐structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.
Vacuolar membrane localization of the Arabidopsis‘two‐pore’ K+ channel KCO1
Authors: Czempinski Katrin , Frachisse Jean‐Marie , Maurel Christophe , Barbier‐Brygoo Helene , Mueller‐Roeber Bernd
Published: March 2003

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus‐infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5′ region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying β‐glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5′ leader intron. The same overall structure was identified in two genes encoding AtKCO1‐like K+ channels from Solanum tuberosum (StKCO1α and StKCO1β). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co‐fractionate with tonoplast markers (Nt‐TIPa, vATPase). In fluorescence images from transgenic AtKCO1–GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.
Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X‐QUAC, the main anion conductance in xylem‐parenchyma cells of barley roots
Authors: Köhler Barbara , Wegner Lars H. , Osipov Viktor , Raschke Klaus
Published: March 2003

We report here that NO3– in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X‐QUAC. Properties of this conductance were investigated on xylem‐parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch‐clamp technique. Chord conductances were minimal around − 40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mm Cl– in the bath was replaced by NO3–, a shift in the midpoint potential of the depolarisation‐activated gate by about − 60 mV from 43 to − 16 mV occurred (Km = 3.4 mm). No such effect was seen when chloride was replaced by malate. Addition of 10 mm NO3– to the pipette solution and reduction of [Cl–] from 124 to 4 mm (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X‐QUAC activation, nor was it affected by changes in external [K+]. If only the NO3– effect on gating was considered, an increase of the NO3– concentration in the xylem sap to 5 mm would result in an enhancement of NO3– efflux by about 30%. Although the driving force for NO3– efflux would be reduced simultaneously, NO3– efflux into the xylem through X‐QUAC would be maintained with high NO3– concentrations in the xylem sap; a situation which occurs for instance during the night.
cDNA microarray analysis of gene expression during Fe‐deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe‐deficient barley roots
Authors: Negishi Takashi , Nakanishi Hiromi , Yazaki Junshi , Kishimoto Naoki , Fujii Fumiko , Shimbo Kanako , Yamamoto Kimiko , Sakata Katsumi , Sasaki Takuji , Kikuchi Shoshi , Mori Satoshi , Nishizawa Naoko K.
Published: March 2003

To acquire Fe from soil, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots. The secretion of MAs increases in response to Fe deficiency, and shows a distinct diurnal rhythm. We used a microarray that included 8987 cDNAs of rice EST clones to examine gene expression profiles in barley roots during Fe‐deficiency stress. Approximately 200 clones were identified as Fe‐deficiency‐inducible genes, of which seven had been identified previously. In order to meet the increased demand for methionine to produce MAs, Fe‐deficiency enhances the expression of genes that participate in methionine synthesis, as well as recycling methionine through the Yang cycle. Of these 200 genes, approximately 50 exhibited different transcription levels in Fe‐deficient roots at noon and at night. Northern blot analysis of time course experiments confirmed that five of these genes exhibited a diurnal change in their level of expression. The diurnal changes in the expression of these genes suggest that polar vesicle transport is involved in the diurnal secretion of MAs.
Nitrate signalling on the nitrate reductase gene promoter depends directly on the activity of the nitrate transport systems in Chlamydomonas
Authors: Llamas Angel , Igeño María Isabel , Galván Aurora , Fernández Emilio
Published: March 2003

Nitrate signalling on the nitrate reductase (Nia1) gene promoter from Chlamydomonas reinhardtii has been studied by using a construct of the Nia1 promoter transcriptionally fused to the Chlamydomonas arylsulphatase gene as a reporter in strains bearing different sets of nitrate/nitrite transport genes. The high‐affinity nitrate transport (HANT) system I is required for efficient signalling by nitrate, even at submicromolar concentrations of the anion. In addition, the autogenous regulation of nitrate reductase has been found to depend on the presence of system I. The low‐affinity nitrate transport system III promoted signalling optimally on the promoter at millimolar nitrate concentrations. The HANT system IV, which is insensitive to ammonium and active at low CO2, allowed nitrate signalling at micromolar concentrations even in the presence of ammonium, suggesting that the balance of these two effectors controls Nia1 transcription. Our data indicate that nitrate signalling on the Nia1 gene promoter occurs intracellularly and depends on the activity of nitrate transporters.
In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side‐chains and abnormal periderm development
Authors: Oomen Ronald J. F. J. , Doeswijk‐Voragen Chantal H. L. , Bush Maxwell S. , Vincken Jean‐Paul , Borkhardt Bernhard , Van Den Broek Lambertus A. M. , Corsar Julia , Ulvskov Peter , Voragen Alphons G. J. , McCann Maureen C. , Visser Richard G. F.
Published: March 2003

Rhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule‐bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side‐chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.
Improvement of the movement and host range properties of a plant virus vector through DNA shuffling
Authors: Toth Rachel L. , Pogue Gregory P. , Chapman Sean
Published: March 2003

Virus expression vectors based on the tobacco mosaic virus (TMV) genome are powerful tools for foreign gene expression in plants. However, the inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. To improve these properties of TMV vectors, the gene encoding the 30‐kDa movement protein was subjected to mutagenesis and DNA shuffling. A vector that expresses the green fluorescent protein was used to allow simple visual discrimination of mutants with enhanced movement phenotypes. An initial round of mutagenesis produced 53 clones with a faster local movement phenotype. Two subsequent rounds of DNA shuffling produced additional clones that showed further increased rates of cell‐to‐cell movement and degrees of systemic invasion in restrictive hosts. Surprisingly, sequence analysis of the best performing shuffled genes revealed alterations resulting in coding and silent changes in the movement protein gene. Separation of these coding and silent alterations into distinct gene backgrounds revealed that each contributes to improved movement protein function to differing degrees. The resulting vectors demonstrate that the complex activities of the movement protein genes of viruses can be evolved to have improved movement phenotypes, as evidenced by cell‐to‐cell and systemic invasion. The experiments produced improved vectors that will be of use both for in planta functional screening and for therapeutic protein production and demonstrated the power of shuffling for plant virus vector improvement.
Conformational features of crystal‐surface cellulose from higher plants
Authors: Viëtor Remco J. , Newman Roger H. , Ha Marie‐Ann , Apperley David C. , Jarvis Michael C.
Published: March 2003

Native cellulose in higher plants forms crystalline fibrils a few nm across, with a substantial fraction of their glucan chains at the surface. The accepted crystal structures feature a flat‐ribbon 21 helical chain conformation with every glucose residue locked to the next by hydrogen bonds from O‐3′ to O‐5 and from O‐2 to O‐6′. Using solid‐state NMR spectroscopy we show that the surface chains have a different C‐6 conformation so that O‐6 is not in the correct position for the hydrogen bond from O‐2. We also present evidence consistent with a model in which alternate glucosyl residues are transiently or permanently twisted away from the flat‐ribbon conformation of the chain, weakening the O‐3′ − 0‐5 hydrogen bond. Previous molecular modelling and the modelling studies reported here indicate that this ‘translational’ chain conformation is energetically feasible and does not preclude binding of the surface chains to the interior chains, because the surface chains share the axial repeat distance of the 21 helix. Reduced intramolecular hydrogen bonding allows the surface chains to form more hydrogen bonds to external molecules in textiles, wood, paper and the living plant.
Transgene integration in aspen: structures of integration sites and mechanism of T‐DNA integration
Authors: Kumar Sandeep , Fladung Matthias
Published: March 2003

To obtain insight into the mechanism of transferred DNA (T‐DNA) integration in a long‐lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T‐DNA/plant junctions, 20 left T‐DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T‐DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T‐DNA left and right ends are treated mechanistically differently during the T‐DNA integration process. Comparison of the genomic target sites prior to integration to the T‐DNA revealed that the T‐DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T‐DNA. Short sequence similarities between recombining strands near break points, in particular for the left T‐DNA end, were observed in most of the lines analysed. These results confirm the well‐accepted T‐DNA integration model based on single‐stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T‐DNA integration was also identified in nine transgenic lines, in which the right border of the T‐DNA was partly truncated. Such integration events are described via a model for the repair of genomic double‐strand breaks in somatic plant cells based on synthesis‐dependent strand‐annealing. This report in a long‐lived tree system provides major insight into the mechanism of transgene integration.
Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissues
Authors: Rech Philippe , Grima‐Pettenati Jacqueline , Jauneau Alain
Published: March 2003

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [−340/−124] as essential for vascular cambium/xylem‐specific expression. In plants transformed with the smallest promoter region [−124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [−124/+117] region contained cis‐elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol‐rich tissues.
Heterogeneity of the blood group Ax allele: genetic recombination of common alleles can result in the Ax phenotype
Authors: Olsson  , Chester
Published: March 2003

The Ax phenotype is an important subgroup of the ABO blood group system. Its inheritance does not always follow Mendelian rules and recent studies suggested that different alleles can result in this phenotype. This suggestion has been explored by cloning and sequencing exons 6 and 7 of the ABO gene and the intervening intron from members of six unrelated families expressing the Ax phenotype.

Two families showed the previously described T646A ‘Ax’ mutation as the only deviation from the consensus A1 allele. In two other families the Ax phenotype was inherited as two different recombinational gene products. Combination of exon 6 derived from A or B/O2 alleles with exon 7 from the O1v allele created two novel alleles that have four O1v‐characteristic nucleotide substitutions in exon 7, including T646A. Sequencing and analysis of polymorphisms in intron 6 defined the crossing‐over zones of these hybrid alleles. Southern blot confirmed the hybrid formation by detecting ABO‐related polymorphisms ≈ 1.35 kb downstream from the ABO reading frame.

The remaining two families expressed the Ax phenotype via an allele having A2‐specific mutations. Thus, a heterogeneous molecular background leads to the serologically defined Ax phenotype and may well explain the different modes of inheritance observed.
Massive haemolysis in a group A recipient of a group O peripheral blood stem cell allogeneic transplant
Authors: Tiplady C. W. , Fitzgerald J. M. , Jackson G. H. , Conn J. S. , Proctor S. J.
Published: March 2003

. A 28‐year‐old man with lymphoblastic lymphoma received G‐CSF mobilized stem cells from his HLA identical sister, who had been taking methotrexate for psoriasis until 1 month prior to harvest. The recipient's blood group was A Rh D positive and donor's group O Rh D positive. Engraftment and major haemolysis were evident by day 9. From day 9 to day 13 he received 17 units of blood (approximately 3 L of red cells) at a time when his calculated red cell volume was 1 L. This massive transfusion requirement was not explained by his clinical condition and led us to consider factors that may have influenced the degree of haemolysis. The stem cell graft contained 2·85 × 106 CD34+ cells kg−1 and we speculate there was B cell hyperactivity following the withdrawal of methotrexate in the donor and this went unchecked by the omission of methotrexate in the GVHD prophylaxis of the recipient. We have also considered the phenomenon of bystander haemolysis, previously unreported in this situation, as haemolysis of transfused group O blood must have also occurred. The case also illustrates the importance of transfusing donor type red cells and recipient type fresh frozen plasma (FFP) and platelets into minor mismatched transplant patients. The decision to revert to donor type FFP and platelets should only be made when the direct antiglobulin test is negative and the appropriate isohaemagglutinins are no longer demonstrable.
Towards new partnerships for health development in developing countries: the contractual approach as a policy tool
Authors: Carrin Guy , Jancloes Michel , Perrot Jean
Published: March 2003

What roles for government but also for society's groups in the advancement of public health in developing countries? This paper focuses on the need to adopt the contractual approach as a powerful policy tool and sketches the contours of a policy framework for good contracting. A short historical review of health system changes leads up to a discussion of the the current emergence of a multitude of actors, the forging of alliances between the various partners, examples on how significant health policy benefits might be secured through contracting, and the implications of building alliances, such as defining and assigning accountability to the contracting partners.
How useful are anthropometric, clinical and dietary measurements of nutritional status as predictors of morbidity of young children in central Africa?
Authors: Tonglet René
Published: March 2003

Summary

objective  To identify useful predictors of morbidity of young children in central Africa.

method  Population‐based follow‐up study in Northern Kivu, Congo, of 842 children under two years of age who completed weekly follow‐up interviews and health examinations during three months. Main outcome measures were crude and adjusted effects of summary measures of nutritional status on one‐month cumulative incidence of malaria, respiratory illness, and diarrhoea.

results  Anthropometric indicators appeared to perform badly in predicting morbidity. In contrast, nonanthropometric variables such as growth as judged by the caretaker, child's diet at the time of examination, and occurrence of disease in the month preceding the interval of observation were useful.

conclusions  In the context of the ‘Sick Child Initiative’, simple tests and diagnostic tools to improve quality of both prevention and cure in first‐level facilities need to be identified. Focusing on non‐anthropometric indicators should be encouraged to offer a comprehensive appraisal of health status to all children.
The burden of mucocutaneous conditions and the association with HIV‐1 infection in a rural community in Uganda
Authors: Mayanja Billy , Morgan Dilys , Ross Amanda , Whitworth James
Published: March 2003

Summary objective To determine the prevalence of mucocutaneous conditions and their association with HIV‐1 infection in a rural community in Uganda.

methods In a prospective cohort study, participants were recruited from a large population study and invited to attend a clinic every 3 months for a detailed medical interview and a thorough physical examination. All findings including mucocutaneous findings were coded onto a standard questionnaire.

results By the end of 1996, 436 participants had provided 1450 person years of observation (pyo); 646 pyo in HIV‐positives and 804 pyo in HIV‐negatives. Overall, 70% of participants had a skin condition during follow‐up, and although skin conditions were significantly more common in HIV‐positive subjects, the background level in HIV‐negative subjects was high (77.3% and 63.6%, respectively). Herpes zoster, thin/sparse hair, maculo‐papular rash and prurigo were significantly more common in the HIV‐positives. Kaposi sarcoma, palmar/plantar rash and herpes zoster had positive predictive values for HIV infection of over 80%. Oral conditions were found in over 40% of participants and were significantly more common in HIV‐positive subjects. Oral candidiasis and Kaposi sarcoma were significantly more frequent among HIV‐positives.

conclusion HIV infection increases the already high burden of mucocutaneous diseases in this rural population. We identified some conditions that are more common in HIV and others that can be used as indicators of HIV infection.
Cortisol metabolism, cortisol sensitivity and the pathogenesis of leprosy reactions
Authors: Rook Graham A. W. , Baker Robert
Published: March 2003

Summary The concentration of cortisol in a tissue is regulated by a reversible enzyme ‘shuttle’ that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti‐inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation.
Antibiotic dispensing by drug retailers in Kathmandu, Nepal
Authors: Wachter David A. , Joshi Mohan P. , Rimal Binaya
Published: March 2003

Summary objectives  To assess over‐the‐counter antimicrobial dispensing by drug retailers in Kathmandu, Nepal, for rationality, safety, and compliance with existing government regulations.

methods  Standardized cases of dysuria in a young adult male and acute watery diarrhoea in a child were presented by a mock patient to retailers at 100 randomly selected pharmacies. Questions asked by retailers and advice and medications given at their initiative were recorded.

results  All retailers engaged in diagnostic and therapeutic behaviour beyond their scope of training or legal mandate. Historical information obtained by retailers was inadequate to determine the nature or severity of disease or appropriateness of antimicrobial therapy. 97% (95% CI = 91.5–99.4%) of retailers dispensed unnecessary antimicrobials in diarrhoea, while only 44% (95% CI = 34.1–54.3%) recommended oral rehydration therapy and only 3% (95% CI = 0.6–8.5%) suggested evaluation by a physician. 38% (95% CI = 28.5–48.2%) gave antimicrobials in dysuria, yet only 4% (95% CI = 1.1–9.9%) adequately covered cystitis. None covered upper urinary tract or sexually transmitted infections, conditions which could not be ruled out based on the interviews, and only 7% (95% CI = 2.9–13.9%) referred for a medical history and physical examination necessary to guide therapy.

conclusions  Although legislation in Nepal mandates a medical prescription for purchase of antibiotics, unauthorized dispensing is clearly problematic. Drug retailers in our study did not demonstrate adequate understanding of the disease processes in question to justify their use of these drugs. Risks of such indiscretion include harm to individual patients as well as spread of antimicrobial resistance. More intensive efforts to educate drug retailers on their role in dispensing, along with increased enforcement of existing regulations, must be pursued.
Fascioliasis: sonographic abnormalities of the biliary tract and evolution after treatment with triclabendazole
Authors: Richter Joachim , Freise Stefan , Mull Robert , Millán Juan C. ,   Triclabendazole Clinical Study Group
Published: March 2003

Summary Diagnosis of infection with the liver fluke Fasciola hepatica is usually difficult. Ultrasonography (US) might be a useful diagnostic alternative, and we assessed the value of sequential US in the diagnosis and monitoring of fascioliasis in 76 patients at baseline and for 60 days after treatment with triclabendazole. At baseline, biliary abnormalities were observed in 52 patients. Crescent‐shaped parasites were seen in 11 patients; in 2 cases parasites were spontaneously moving and in 4 patients parasites were motionless. Postprandial examination revealed parasites adhering to the gallbladder wall in a further 5 cases. In 3 further cases, gallbladder contents were mobile but did not sediment downwards after patients changed position. Non‐specific abnormalities were: impaired gallbladder contractility (n= 23), gallbladder tenderness (n= 19), debris (n= 6), calculi (n= 5), wall thickening (n= 2) and bile duct dilatation (n= 12). During day 1–7, Fasciola‐like crescents in the gallbladder or passing through the bile duct were detected in another 15 patients, impaired gallbladder contractility in 16, gallbladder tenderness in 16, and bile duct dilatation in an additional 28 patients. Thirty‐two patients with these US abnormalities experienced colic‐like abdominal pain accompanied by increased alkaline phosphatase in 25 cases. During day 30–60, abnormalities regressed completely in 45 patients; 2/6 triclabendazole failures were evident by detection of living parasites. Biliary tract abnormalities are frequently observed by US, but the detection‐rate of Fasciola hepatica is disappointingly low despite the parasite's relatively large size. US findings must therefore be interpreted together with other clinical measurements. The visualization of parasites being expelled through the dilated common bile duct allowed the causal interpretation of post‐therapeutic abdominal pain and increase of liver enzymes. When triclabendazole is given on suspicion, visualization of worm expulsion and bile duct dilatation by US may be used to confirm diagnosis.
Trachoma: Can trichiasis be treated with a sticking‐plaster? A randomized clinical trial in China
Authors: Graz B. , Xu J. M. , Yao Z. S. , Han S. R. , Kok A.
Published: March 2003

Trachoma is the most frequent cause of preventable blindness in the world. At the trichiasis/entropion stage, lid surgery is recommended, but many patients only use epilation, which does not prevent loss of vision. We developed a new treatment that should be more accessible than lid surgery and more effective than epilation: a sticking plaster that forces eyelashes back to their correct position. The first randomized controlled trial was conducted in Shanghai with 57 patients to compare the plaster method with epilation. After 3 months of follow‐up, with no attrition, 67% of those treated by the new method presented a good clinical status, vs none of those treated by epilation (P < 0.001). The new treatment was well tolerated and lid function remained normal. Although our results show overwhelming benefit of this new, simple treatment for trachoma at the trichiasis stage, more research is needed at the primary health care level and in other settings to determine the potential use of the new method on a large scale and by nonspecialists.

RésuméLa trachome est la première cause de cécitéévitable dans le monde, avec 146 millions de personnes atteintes. Au stade trichiasis/entropion, le traitement chirurgical est recommandé, mais pour beaucoup de patients l'épilation des cils reste le seul traitement, insuffisant pour éviter la perte de la vue. Nous avons développé une nouvelle méthode de traitement, qui se veut plus accessible que la chirurgie et plus efficace que l'épilation: la contention des cils par un sparadrap double‐face. Le premier essai clinique randomisé est présenté, comparant le nouveau traitement à l'épilation, conduit à Shanghai en milieu universitaire avec 57 patients. Après trois mois de suivi, sans perte, 67% des patients traités par contention avaient un status oculaire clinique bon, contre 0% dans le groupe épilation (P < 0.001). Le traitement par contention est bien toléré et il permet de battre des cils normalement. Nos résultats démontrent donc l'efficacité de ce nouveau traitement du trachome au stade trichiasis, d'usage simple et bon marché. Des essais dans des centres de soins primaires et dans d'autres cultures sont nécessaires pour préciser l'usage possible du nouveau traitement par des non‐spécialistes et sur une large échelle.
Malaria infection and morbidity in infants in relation to genetic polymorphisms in Tanzania
Authors: Stirnadel Heide A. , StÃ¶ckle Marcel , Felger Ingrid , Smith Tom , Tanner Marcel , Beck Hans‐Peter
Published: March 2003

To investigate the effects of host polymorphisms on malaria morbidity and infection, the frequency distributions of TNF α promotor gene and sickle cell trait were studied in infants in an area highly endemic for Plasmodium falciparum transmission in Tanzania. Differences in parasite prevalence, density, febrile episodes with and without parasitaemia, PCV levels and the frequencies of different MSP‐2 parasite infections were assessed by genotype. The frequency of the TNF α promotor allele 2 was 0.09, and the trait was in Hardy–Weinberg equilibrium. There were no differences in malariametric indices between infants with the normal TNF α promoter gene and those who were heterozygous for this trait. Infants heterozygous for the TNF α promotor gene appeared to have fewer febrile episodes when they were free of parasites. The two infants homozygous for the TNF α promoter allele 2 had both a much higher incidence of fever, independently of parasitaemia, than the average for the other genotypes. The frequency of the sickle cell allele S was 0.06 and the trait was also in Hardy–Weinberg equilibrium. Infants heterozygous for the sickle cell trait had significantly lower parasite densities, but similar prevalence, and MSP‐2 infections compared to infants with normal haemoglobin. PCV levels, and the incidence of febrile episodes both with and without parasitaemia were also similar. In contrast to the sickle cell trait, the TNF α promotor polymorphism appeared not to have any protective effect on malaria in this study, and its importance in other unspecified fever‐causing diseases in this population needs further investigation.