bluuebunny/crossref_metadata_2025 huggingface dataset:
Choosing the best estimate of gestational age from routinely collected population‐based perinatal data
Authors: Blair Eve , Liu Yingxin , Cosgrove Peter
Published: July 2004

Obtaining gestational data of acceptable validity on whole populations is a considerable challenge, which must be met in order to further epidemiological investigation involving perinatal factors. As the means of estimating gestational duration multiply, routinely available population data pertaining to gestational duration may be conflicting. This exacerbates its reputation for unreliability, which is due primarily to the generally occult nature of conception and secondly to the propensity for data entry errors of the data from which gestational duration is estimated. However, the key to improving reliability may paradoxically lie in the increasing variety of methods for estimating gestational duration, because agreement between independent observations increases the reliability that can be placed on estimates of factors that are inherently difficult to measure.

This paper demonstrates that the acceptability of population data for gestational duration can be improved by simple rectification of recurring data entry errors and demanding compatibility of two independent estimates of gestational duration. Compared with the previous algorithm that relied primarily on last menstrual period data, the new algorithm, that considers both antenatal and neonatal indicators of gestational duration, decreased the proportion of births in a geographically defined birth cohort 1986–99 (n = 354 216) without a gestational estimate, from 0.5% to 0.03%, halved the number of births of >400 g estimated to occur before 20 weeks, and almost eliminated gestational estimates of >45 weeks.
Influence of inter‐pregnancy interval on preterm delivery
Authors: Arafa Mostafa A. , Alkhouly Alaa , Youssef Manal E.
Published: July 2004

The influence of pregnancy spacing on preterm delivery (<37 weeks gestation) was examined in a group of mothers attending three Maternal and Child Health centres in three different districts in the city of Alexandria, Egypt between October 2001 and July 2002. All pregnant mothers attending the centres were interviewed or had their medical records reviewed. They were followed up until delivery and were questioned about current and past obstetric history and their medical history; their haemoglobin level was measured. The inter‐pregnancy interval was calculated as the number of months between the delivery date of the women's last previous live infant and the date of the last menstrual period before the current pregnancy. There were 1202 pregnant women eligible for inclusion. The inter‐pregnancy intervals were divided into five categories: <12, 12–36, 37–48, 49–60 and >60 months. We calculated the rates of preterm deliveries for each inter‐pregnancy interval. Unadjusted odds ratios were estimated and stepwise logistic regression analysis was then used to adjust for all confounding factors. The rate of preterm delivery was 9.4%, and the lowest risk occurred in women who had an inter‐pregnancy interval >60 months; 7.3% of those women gave birth to preterm infants. The rate of preterm delivery increased with increasing inter‐pregnancy interval until 49–60 months; however, the association between inter‐pregnancy interval and preterm delivery was not statistically significant whether using the crude or adjusted odds ratios.
Trends in neural tube defects in Western Australia in Indigenous and non‐Indigenous populations
Authors: Bower Carol , Eades Sandra , Payne Jan , D’Antoine Heather , Stanley Fiona
Published: July 2004

Neural tube defects (NTD) were 43% more common in Indigenous than in non‐Indigenous infants in Western Australia in the 1980s, and there has been a fall in NTD overall in Western Australia since promotion of folate and voluntary fortification of food has occurred. In order to investigate whether the fall had occurred in both indigenous and non‐Indigenous infants, data on NTD (births and terminations) were obtained from the WA Birth Defects Registry, and on all births from the Maternal and Child Health Research Data Base. Knowledge of folate was asked in a survey of indigenous women interviewed postpartum. Before the promotion of folate (1980–92), there has been a 42% increase in NTD in Indigenous compared with non‐Indigenous infants (prevalence ratio (PR) = 1.42 [95% confidence interval (CI) 1.04, 1.94]); while in the most recent period (1996–2000), the prevalence in Indigenous infants was almost twice that of non‐Indigenous infants (PR 1.98 [CI 1.25, 3.15]). Fifty‐five per cent of Indigenous women knew about folate in pregnancy. Similar to sudden infant death syndrome, this study has highlighted health promotion that has been successful in reducing the risk of a childhood condition overall, but has failed to be effective for Indigenous children.
Association between severe nausea and vomiting in pregnancy and lower rate of preterm births
Authors: Czeizel Andrew E. , Puhó Erzsébet
Published: July 2004

Nausea and vomiting in pregnancy has been associated with some reduction in miscarriage in other studies. However the relationship between nausea and vomiting during pregnancy and preterm birth/low birthweight in newborns is inconsistent; therefore the objective of the study was to clarify this possible association. The population‐based large control (without any defects) data set of the Hungarian Case‐Control Surveillance System of Congenital Abnormalities was evaluated.

Of 38 151 controls, 3869 (10.1%) had medically recorded and treated nausea and vomiting during pregnancy. The proportion of mothers who later delivered girls was higher in this group. Mothers with nausea and vomiting in pregnancy had a somewhat (0.3 week) longer gestational age and a lower proportion of preterm births (6.4% vs. 9.5%). This finding may be connected with the favourable hormonal milieu (larger placenta, higher level of chorionic gonadotrophin and oestrogens) of pregnant women with nausea and vomiting.
Partner violence during pregnancy and risk of adverse pregnancy outcomes
Authors: Coker Ann L. , Sanderson Maureen , Dong Beili
Published: July 2004

The purpose of this cross‐sectional study was to investigate the association between partner physical or emotional abuse during pregnancy and pregnancy outcomes including perinatal death, low birthweight and preterm delivery. Women, aged 18–65, who attended one of two large primary care practices from 1997–98 were recruited for this study. Ever pregnant women were asked the frequency of abuse during each pregnancy and details of the pregnancy outcomes. Information regarding abuse during pregnancy and pregnancy outcomes was available for 755 women surveyed who reported a live birth or late fetal death, 14.7% indicated that an intimate partner was violent or abusive toward them during a pregnancy (274 of 1862 pregnancies). Abuse during pregnancy was significantly associated with an increased risk of perinatal death (adjusted relative risk [aRR] = 2.1, 95% confidence interval [CI] 1.3, 3.4) and, among live births, with preterm low birthweight (aRR = 2.4; 95% CI 1.5, 4.0) and term low birthweight (aRR = 1.9; 95% CI 1.0, 3.4). Greater abuse frequency was associated with increased risk. Abuse during pregnancy was associated with perinatal deaths and preterm low birthweight deliveries.
The Walker Project: a longitudinal study of 48 000 children born 1952–1966 (aged 36–50 years in 2002) and their families
Authors: Libby Gillian , Smith Anne , McEwan Neil F. , Chien Patrick F. W. , Greene Stephen A. , Forsyth J. Stewart , Crombie Iain K. , Macdonald Thomas M. , Morris Andrew D.
Published: July 2004

The Walker cohort is a database of over 48 000 birth records that has recently become available. It contains meticulously recorded details of pregnancy, labour, birth and care before discharge for babies born in hospital in Dundee, Scotland between 1952 and 1966. These babies accounted for 75% of all births in Dundee at this time. Over 34 000 (73%) of these subjects can be identified and this presents the opportunity to link this birth information with a large number of current health‐outcome databases covering both primary and secondary care. Further, it allows linkage of records across siblings and over two and, in future, three generations. The number of birth records available and linkage to current databases make this a unique birth cohort with huge potential for the investigation of the fetal origins of adult disease.
Arabidopsis Yellow Stripe‐Like2 (YSL2): a metal‐regulated gene encoding a plasma membrane transporter of nicotianamine–metal complexes
Authors: DiDonato Raymond J. , Roberts Louis A. , Sanderson Tamara , Eisley Robynn Bosler , Walker Elsbeth L.
Published: July 2004

The Yellow Stripe‐Like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow Stripe1 (YS1), the transporter responsible for the primary uptake of iron from the soil. YS1 transports iron that is complexed by specific plant‐derived Fe(III) chelators called phytosiderophores (PS). Non‐grass species of plants neither make nor use PS, yet YSL family members are found in non‐grass species (monocot, dicot, gymnosperm, and moss species) including Arabidopsis thaliana. YSLs in non‐grasses have been hypothesized to transport metals complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. Here we show that Arabidopsis YSL2 (At5g24380) transports iron and copper when these metals are chelated by NA. YSL2 is expressed in many cell types in both roots and shoots, suggesting that diverse cell types obtain metals as metal–NA complexes. YSL2 transcription is regulated by the levels of iron and copper in the growth medium. Based on its expression pattern, a major function of the YSL2 appears to be in the lateral movement of metals in the vasculature.
The chloroplastic protein translocation channel Toc75 and its paralog OEP80 represent two distinct protein families and are targeted to the chloroplastic outer envelope by different mechanisms
Authors: Inoue Kentaro , Potter Daniel
Published: July 2004

Toc75 is postulated to form the protein translocation channel in the chloroplastic outer envelope membrane. Proteins homologous to Toc75 are present in a wide range of organisms, with the closest homologs occurring in cyanobacteria. Therefore, an endosymbiotic origin of Toc75 has been postulated. Recently, a gene encoding a paralog to Toc75 was identified in Arabidopsis and its product was named atToc75‐V. In the present study, we characterized this new Toc75 paralog, and investigated extensively the relationships among Toc75 homologs from higher plants and bacteria in order to gain insights into the evolutionary origin of the chloroplastic protein translocation channel. First, we found that the native molecular weight of atToc75‐V is 80 kDa and renamed it (AtOEP80) Arabidopsis thalianaouter envelope protein of 80 kDa. Second, we found that AtOEP80 and Toc75 utilize different mechanisms for their targeting to the chloroplastic envelope. Toc75 is directed with a cleavable bipartite transit peptide partly via the general import pathway, whereas AtOEP80 contains the targeting information within its mature sequence, and its targeting is independent of the general pathway. Third, we undertook phylogenetic analyses of Toc75 homologs from various organisms, and found that Toc75 and OEP80 represent two independent gene families that are most likely derived from cyanobacterial sequences. Our results suggest that Toc75 and OEP80 diverged early in the evolution of plastids from their common ancestor with modern cyanobacteria.
Roles of the AtErcc1 protein in recombination
Authors: Dubest Sandra , Gallego Maria E. , White Charles I.
Published: July 2004

Atercc1, the recently characterized Arabidopsis homologue of the Ercc1 (Rad10) protein, is a key component of nucleotide excision repair as part of a structure‐specific endonuclease which cleaves 5′ to UV photoproducts in DNA. This endonuclease also acts in removing overhanging non‐homologous DNA ‘tails’ in synapsed recombination intermediates. We have previously demonstrated this recombination function of the Arabidopsis thaliana Xpf homologue, AtRad1p, and show here that recombination between plasmid DNA substrates containing non‐homologous tails is specifically reduced 12‐fold in atercc1 mutant plants compared with the wild type. Furthermore, using chromosomal tandem‐repeat recombination substrates, we show that AtErcc1p is required for bleomycin induction of mitotic recombination in the chromosomal context. This work thus confirms both the specific and general recombination roles of the Atercc1 protein in recombination in Arabidopsis.
Functional identification of rice syn‐copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products
Authors: Xu Meimei , Hillwig Matthew L. , Prisic Sladjana , Coates Robert M. , Peters Reuben J.
Published: July 2004

Rice produces a number of phytoalexins, and at least one allelopathic agent, from syn‐copalyl diphosphate (CPP), representing the only known metabolic fate for this compound. Thus, the class II terpene synthase that converts the universal diterpenoid precursor geranylgeranyl diphosphate to syn‐CPP catalyzes the committed step in biosynthesis of these natural products. Here the extensive sequence information available for rice was coupled to recombinant expression and functional analysis to identify syn‐copalyl diphosphate synthase (OsCPSsyn). In addition, OsCPSsyn mRNA was found to be specifically induced in leaves by conditions that stimulate phytoalexin biosynthesis. Therefore, transcription of OsCPSsyn seems to be an important regulatory point for controlling the production of these defensive compounds. Finally, alignments carried out with OsCPSsyn revealed that class II terpene synthases exhibit a sequence conservation pattern substantially different from that of the prototypical class I enzymes. One particularly notable feature is the specific conservation of the functionally cryptic ‘insertional’ sequence element in class II terpene synthases, indicating that this region is important for the corresponding cyclization reaction.
TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana
Authors: Baudry Antoine , Heim Marc A. , Dubreucq Bertrand , Caboche Michel , Weisshaar Bernd , Lepiniec Loïc
Published: July 2004

Genetic analyses have demonstrated that together with TTG1, a WD‐repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two‐ and three‐hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.
Application of a high‐throughput HPLC‐MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality
Authors: Jander Georg , Norris Susan R. , Joshi Vijay , Fraga Michele , Rugg Arthur , Yu Shaoxia , Li Lily , Last Robert L.
Published: July 2004

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20–90 min for standard methods) HPLC‐MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate‐derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.
Plant growth‐promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1‐independent and jasmonic acid‐dependent signaling pathway
Authors: Ryu Choong‐Min , Murphy John F. , Mysore Kiankumar S. , Kloepper Joseph W.
Published: July 2004

Arabidopsis thaliana ecotype Columbia plants (Col‐0) treated with plant growth‐promoting rhizobacteria (PGPR) Serattia marcescens strain 90‐166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90‐166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90‐166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90‐166 naturally produces SA under iron‐limited conditions. Col‐0 and NahG plants treated with the SA‐deficient mutant, 90‐166‐1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col‐0 plants. Another PGPR mutant, 90‐166‐2882, caused reduced disease severity in Col‐0 and NahG plants. In a time course study, strain 90‐166 reduced virus accumulation at 7 but not at 14 and 21 days post‐inoculation (dpi) on the non‐inoculated leaves of Col‐0 plants. NahG and npr1‐1 plants treated with strain 90‐166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90‐166‐treated fad3‐2 fad7‐2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90‐166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.
Two genes encoding Arabidopsis halleri MTP1 metal transport proteins co‐segregate with zinc tolerance and account for high MTP1 transcript levels
Authors: Dräger Dörthe B. , Desbrosses‐Fonrouge Anne‐Garlonn , Krach Christian , Chardonnens Agnes N. , Meyer Rhonda C. , Saumitou‐Laprade Pierre , Krämer Ute
Published: July 2004

The zinc hyperaccumulator plant Arabidopsis halleri is able to naturally accumulate 100‐fold higher leaf zinc concentrations when compared with non‐accumulator species such as the closely related A. lyrata and A. thaliana, without showing toxicity symptoms. A novel member of the cation diffusion facilitator (CDF) protein family, an A. halleri metal tolerance protein 1 (MTP1), and the homologous A. thaliana Zn transporter (ZAT)/AtMTP1 metal‐specifically complement the zinc hypersensitivity of a Saccharomyces cerevisiae zrc1 cot1 mutant strain. A fusion of the AhMTP1 protein to green fluorescent protein (GFP) localizes to the vacuolar membrane of A. thaliana protoplasts. When compared with A. lyrata and A. thaliana, the total MTP1 transcript levels are substantially higher in the leaves and upregulated upon exposure to high zinc concentrations in the roots of A. halleri. The high MTP1 transcript levels in A. halleri can be primarily attributed to two genetically unlinked genomic AhMTP1 gene copies. The two corresponding loci co‐segregate with zinc tolerance in the back‐cross 1 generation of a cross between the zinc‐tolerant species A. halleri and the zinc‐sensitive species A. lyrata. In contrast, a third MTP1 gene in the genome of A. halleri generates only minor amounts of MTP1 transcripts and does not co‐segregate with zinc tolerance. Our data suggests that zinc tolerance in A. halleri involves an expanded copy number of an ancestral MTP1 gene, encoding functional proteins that mediate the detoxification of zinc in the cell vacuole. At the transcript level, MTP1 gene copies of A. halleri are regulated differentially and in response to changes in zinc supply.
Stable high‐level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions
Authors: Butaye Katleen M.J. , Goderis Inge J.W.M. , Wouters Piet F.J. , Pues Jonathan M.‐T.G. , Delauré Stijn L. , Broekaert Willem F. , Depicker Ann , Cammue Bruno P.A. , De Bolle Miguel F.C.
Published: July 2004

Basic and applied research involving transgenic plants often requires consistent high‐level expression of transgenes. However, high inter‐transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high‐level transgene expression is obtained using Arabidopsis thaliana post‐transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a β‐glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin‐dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S‐driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T‐DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12‐fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR‐based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high‐throughput screening of transgene‐based phenotypes as well as for obtaining extremely high transgene expression in plants.
High throughput T‐DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics
Authors: Sallaud Christophe , Gay Céline , Larmande Pierre , Bès Martine , Piffanelli Pietro , Piégu Benoit , Droc Gaétan , Regad Farid , Bourgeois Emmanuelle , Meynard Donaldo , Périn Christophe , Sabau Xavier , Ghesquière Alain , Glaszmann Jean Christophe , Delseny Michel , Guiderdoni Emmanuel
Published: July 2004

A library of 29 482 T‐DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T‐DNA left border from the first 12 707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T‐DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T‐DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T‐DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T‐DNA inserts along the recently released 12 rice pseudomolecules confirmed this non‐uniform chromosome distribution. T‐DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T‐DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E‐value <1.00 e−05). To illustrate the in silico reverse genetic approach, identification of 83 T‐DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD‐ZIP, Zinc‐finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7–10% of the rice gene complement is already tagged by T‐DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.
OsYSL2 is a rice metal‐nicotianamine transporter that is regulated by iron and expressed in the phloem
Authors: Koike Shintaro , Inoue Haruhiko , Mizuno Daichi , Takahashi Michiko , Nakanishi Hiromi , Mori Satoshi , Nishizawa Naoko K.
Published: July 2004

We identified 18 putative yellow stripe 1 (YS1)‐like genes (OsYSLs) in the rice genome that exhibited 36–76% sequence similarity to maize iron(III)‐phytosiderophore transporter YS1. Of particular interest was OsYSL2, the transcripts of which were not detected in the roots of either iron‐sufficient or iron‐deficient plants, but dramatic expression was induced in the leaves by iron deficiency. Based on the nucleotide sequence, OsYSL2 was predicted to encode a polypeptide of 674 amino acids containing 14 putative transmembrane domains. OsYSL2:green fluorescent protein (GFP) was localized in the plasma membrane of onion epidermal cells. Promoter:β‐glucuronidase (GUS) analysis revealed that OsYSL2 was expressed in companion cells in iron‐sufficient roots. GUS activity was increased in companion cells, but no GUS staining was observed in epidermal or cortex cells, even in iron‐deficient roots. In the leaves and leaf sheaths of iron‐sufficient rice, GUS staining was observed in phloem cells of the vascular bundles. In iron‐deficient leaves, the OsYSL2 promoter was active in all tissues with particularly strong GUS activity evident in companion cells. The phloem‐specific expression of the OsYSL2 promoter suggests that OsYSL2 is involved in the phloem transport of iron. Strong OsYSL2 promoter activity was also detected in developing seeds. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL2 transported iron(II)‐nicotianamine (NA) and manganese(II)‐NA, but did not transport iron(III)‐phyosiderophore. These results suggest that OsYSL2 is a rice metal‐NA transporter that is responsible for the phloem transport of iron and manganese, including the translocation of iron and manganese into the grain.
Dynamic Nucleation of Golgi Apparatus Assembly from the Endoplasmic Reticulum in Interphase HeLa Cells
Authors: Kasap Murat , Thomas Stephanie , Danaher Erin , Holton Virginia , Jiang Shu , Storrie Brian
Published: July 2004

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self‐assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5′‐diphosphate‐restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5′‐diphosphate‐restricted Sar1p endoplasmic reticulum‐exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.
Continual Expression of Rab5(Q79L) Causes a Ligand‐Independent EGFR Internalization and Diminishes EGFR Activity
Authors: Dinneen Jennifer L. , Ceresa Brian P.
Published: July 2004

The amount of cell‐surface Epidermal Growth Factor Receptor (EGFR) available to secreted ligand (EGF) dictates a cell's ability to mediate cell proliferation, differentiation or migration. Multiple factors regulate EGFR cell‐surface expression including the rates of protein synthesis and protein degradation, and the endocytic trafficking of both stimulated and unstimulated EGFR. Rab5 is a 25 kDa protein that is localized to the plasma membrane and the early endosome. Its exact molecular function, however, remains controversial. We have used stable and transient expression systems in HeLa cells to examine the consequence of continual, overexpression of wild‐type and activated mutants of rab5 on EGFR localization and signaling. Continual expression of constitutively activated mutants of rab5 causes a ligand‐independent redistribution of EGFRs into intracellular vesicles that can not be blocked with an antagonistic antibody. The net result is a decrease in the level of cell‐surface EGFRs available for ligand stimulation. Thus, rab5 activation regulates EGFR signaling by facilitating the internalization of the unliganded EGFR.
The in vitro quality of washed, prestorage leucocyte‐depleted red blood cell concentrates
Authors: Weisbach V. , Riego W. , Strasser E. , Zingsem J. , Ringwald J. , Zimmermann R. , Eckstein R.
Published: July 2004

Background and Objectives  No data are currently available on the quality of washed prestorage leucocyte‐depleted red blood cell concentrates (RCCs).

Materials and Methods  Five groups of RCCs stored in additive solution (SAG‐M) were washed. The groups differed in the age of RCCs (2–5 days or 11–15 days), the temperature during the washing procedure and a 6‐h storage period (4 °C or room temperature) and the washing solution (saline, SAG‐M or 5% albumin). We measured ATP, 2,3‐diphosphoglycerate (2,3‐DPG), haemolysis, blood cell count, Na+, K+, pH, pO2, pCO2 and lactate, before and after the washing procedure and hourly during the 6‐h postwash storage period.

Results  The erythrocyte ATP content increased by 2–13%, relative to the baseline value, during the washing procedure. The 2,3‐DPG level decreased by 15–35% in 2–6‐day‐old RCCs and by 30–40% in 11–15‐day‐old RCCs (relative to baseline values) during the washing procedure. In RCCs that were washed and stored at room temperature, and in 2‐week‐old RCCs, a further decrease in 2,3‐DPG of up to 40%, relative to the baseline value, was observed during the 6‐h postwash time‐period.

Conclusions  Washing of RCCs stored in SAG‐M results in a considerable, significant loss of erythrocyte 2,3‐DPG, especially in older RCCs. This loss increases in during a 6‐h storage period postwash, even at 4 °C. This loss of erythrocyte quality might well outweigh the benefits of washed SAG‐M RCCs during massive transfusion in neonates.
The serological and genetic basis of the cis‐AB blood group in Korea
Authors: Cho D. , Kim S.‐H. , Jeon M.‐J. , Choi K.‐L. , Kee S.‐J. , Shin M.‐G. , Shin J.‐H. , Suh S.‐P. , Yazer M. H. , Ryang D.‐W.
Published: July 2004

Background and Objectives  The cis‐AB blood group is rare, although relatively common amongst Koreans. The serological characteristics and genetic basis of Korean cis‐AB blood donors were investigated.

Materials and Methods  Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), designed to detect the cis‐AB01 allele, was performed on 194 AB samples which demonstrated weak or unusual expression of either or both of the A or B antigens.

Results and Conclusions  Sixty cis‐AB01 donors were identified. cis‐AB01/O01 or O02 were the most common genotypes (36/60) detected only in A2B3 donors, and cis‐AB01/B101 (nine of 60) was the least common genotype identified only in A2B donors. Surprisingly cis‐AB01/A102 (15/60) was identified in a variety of phenotypes (A1B3, A1Bx or el, AintB3).
Inactivation of West Nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma‐derived products
Authors: Remington K. M. , Trejo S. R. , Buczynski G. , Li H. , Osheroff W. P. , Brown J. P. , Renfrow H. , Reynolds R. , Pifat D. Y.
Published: July 2004

Background and Objectives  Human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. Recent outbreaks of emergent viral pathogens, such as West Nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma‐derived products. The purpose of this study was to evaluate the efficacy of current viral inactivation methods for West Nile and vaccinia viruses and to reassess the use of model viruses to predict inactivation of similar viral pathogens.

Materials and Methods  Virus‐spiked product intermediates were processed using a downscaled representation of various manufacturing procedures. Virus infectivity was measured before and after processing to determine virus inactivation.

Results  The results demonstrated effective inactivation of West Nile virus, vaccinia virus and a model virus, bovine viral diarrhoea virus, during pasteurization, solvent/detergent treatment and caprylate treatment. Caprylate provided rapid and effective inactivation of West Nile virus, vaccinia virus, duck hepatitis B virus and Sindbis virus. Inactivation of West Nile virus was similar to that of bovine viral diarrhoea virus.

Conclusions  This study demonstrates that procedures used to inactivate enveloped viruses in manufacturing processes can achieve inactivation of West Nile virus and vaccinia virus. In addition, the data support the use of model viruses to predict the inactivation of similar emergent viral pathogens.
Induction of latent human cytomegalovirus by conventional gamma irradiation and prevention by treatment with INACTINE PEN110
Authors: Ohagen A. , Gibaja V. , Horrigan J. , Lunderville D. , Jayarama V. , Marcello J. , Chapman J. , Lazo A.
Published: July 2004

Background and Objectives  Two different leucocyte‐inactivation technologies – gamma irradiation and INACTINE PEN110 – were evaluated for their effects on cell‐associated human cytomegalovirus (CMV).

Materials and Methods  In vitro CMV‐infected cells were spiked into leucoreduced red blood cell concentrates (RCC) or medium at a final concentration of 0·5–1 × 107 cells/ml to mimic non‐leucoreduced levels of leucocytes. The spiked RCC/medium was divided into three equal units and treated with gamma irradiation at the US Food and Drug Administration (FDA)‐approved dose of 25 Gy, with 0·1% v/v PEN110 at 22 °C for 24 h, or stored at 4 °C as a control. The treated and control cells were recovered and tested using infectivity, viability and polymerase chain reaction (PCR) assays.

Results  Gamma‐irradiated CMV‐infected cells produced active virus, as shown by both infectivity assays and PCR quantification of viral DNA. PCR analysis demonstrated higher CMV DNA levels in gamma‐irradiated, latently infected monocytic THP‐1 cells than untreated control cells. The increased virus production in gamma‐irradiated cells was paralleled by an increased metabolic rate and the development of enlarged multinuclear cells. In contrast, PEN110 treatment terminated virus replication and completely inactivated the infected cell.

Conclusions  These results demonstrate that gamma irradiation, at levels currently used to treat RCC, has the capacity to induce expression of CMV, whereas PEN110 inhibits CMV replication and efficiently inactivates the infected cells.
Recombinant activated factor VII (NovoSeven™): addition to replacement therapy in acute, uncontrolled and life‐threatening bleeding
Authors: Mayo A. , Misgav M. , Kluger Y. , Geenberg R. , Pauzner D. , Klausner J. , Ben‐Tal O.
Published: July 2004

Background and Objectives  Recombinant activated factor VII (rFVIIa, NovoSeven™) has been used off‐label for various conditions. A protocol for its use in acute, uncontrolled life‐threatening bleeding, was devised and employed. A haematologist/transfusion specialist was assigned as a member of the team.

Materials and Methods  The clinical data were reviewed and summarized. A scoring system for the assessment and monitoring of coagulopathy was employed. Each parameter of prothrombin time (PT), activated partial thromboplastin time (aPTT), platelet number and fibrinogen level was allocated points according to the degree of abnormality. Three scoring levels emerged.

Results  Between April 2001 and April 2003, 13 patients received rFVIIa for acute, uncontrolled life‐threatening bleeding. Nine of 13 patients remained alive for 15 days or longer after rFVIIa infusion. All patients who experienced a reduction or cessation of bleeding after rFVIIa infusion, also had a lower coagulopathy score after replacement therapy, prior to rFVIIa infusion, compared with their score at rFVIIa request. There was a reduction in the average use of blood products after rFVIIa infusion. The coagulopathy score was statistically predictive of response to rFVIIa and survival.

Conclusions  In an area where very little data exists, we report the usefulness of rFVIIa. We propose that transfusion replacement should aim to correct coagulopathy before infusion of rFVIIa and that a haematologist/transfusion specialist should be involved in the management of these patients. A prognostically significant coagulopathy scoring system is offered.
Content and functional activity of von Willebrand factor in apheresis plasma
Authors: Burnouf T. , Caron C. , Burkhardt T. , Goudemand J.
Published: July 2004

Background and Objectives  Von Willebrand Factor (VWF) is a complex high‐molecular‐weight (HMW) plasma glycoprotein playing a critical role in primary and secondary haemostasis. Owing to its multimeric structure and sensitivity to proteolysis, VWF can be used as a marker of the impact of collection procedures on the characteristics of plasma for transfusion and for fractionation. We studied VWF content, functional activity and HMW multimers in plasmas collected by five different automated apheresis collection procedures.

Materials and Methods  Five series of 30 plasma units were obtained from volunteer donors at two collection sites using Haemonetics PCS2 machines with Revision (Rev) F, Rev G, high‐separation core (HSC), or filter core (FC) procedures, or Baxter‐Fenwall Autopheresis‐C (Auto‐C). VWF antigen (VWF:Ag), ristocetin cofactor (VWF:RCo) activity and HMW multimers were first determined in 10 randomly selected plasma donations collected with Rev G, HSC, FC and Auto‐C procedures. Then, the same analyses and the collagen binding (VWF:CB) activity were determined in the pools of 30 donations from each of the five procedures and compared with two normal plasma pools (NPP1 and NPP2). A reference plasma (RP) was used to calibrate each assay.

Results  There were a greater number of group O individuals in the Rev F, Rev G and FC donors than in the HSC and Auto‐C donors. The mean VWF:Ag level was > 100 IU/dl, VWF:RCo activity was > 90 U/dl, the VWF:RCo/Ag ratio was close to 1, and the percentage of 11–15 mers was above 100% of RP in the 10 individual plasma units from Rev G, HSC, FC, and Auto‐C and in their respective pools. The mean percentage of multimers > 15 mers, relative to RP, was significantly less in Rev G plasmas (48 ± 17%; range 32–91%), compared with Auto‐C, HSC and FC plasmas (P = 0·0211; 0·0257; and 0·0376, respectively). The VWF:CB activity of the 30‐donation pools was 61 and 60 U/dl in Auto C and HSC, 50 U/dl in Rev F and FC, and 43 U/dl in the Rev G pool. The VWF:CB/Ag ratio was 0·54 (Auto‐C), 0·49 (HSC), 0·46 (Rev F), 0·45 (FC) and 0·37 (Rev G), compared with 0·81and 0·92 in NPPs. The percentage of VWF multimers of 11–15 mers in apheresis plasma and NPP was normal. VWF multimers > 15 mers ranged from 38 to 64% of that of RP plasma, and was 111 and 112% in NPPs.

Conclusions  The VWF:Ag, VWF:RCo activity and 11–15 mer VWF multimers were well preserved in all plasma units from each of the five apheresis procedures. The VWF:CB activity and the percentage of multimers > 15 mers in apheresis plasma was less than in normal plasma pools and differed slightly among procedures.
The Mitochondrial Carnitine Palmitoyltransferase System — From Concept to Molecular Analysis
Authors: McGarry J. Denis , Brown Nicholas F.
Published: July 2004

First conceptualized as a mechanism for the mitochondrial transport of long‐chain fatty acids in the early 1960s, the carnitine palmitoyltransferase (CPT) system has since come to be recognized as a pivotal component of fuel homeostasis. This is by virtue of the unique sensitivity of the outer membrane CPT I to the simple molecule, malonyl‐CoA. In addition, both CPT I and the inner membrane enzyme, CPT II, have proved to be loci of inherited defects, some with disastrous consequences. Early efforts using classical approaches to characterize the CPT proteins in terms of structure/function/regulatory relationships gave rise to confusion and protracted debate. By contrast, recent application of molecular biological tools has brought major enlightenment at an exponential pace. Here we review some key developments of the last 20 years that have led to our current understanding of the physiology of the CPT system, the structure of the CPT isoforms, the chromosomal localization of their respective genes, and the identification of mutations in the human population.
Cleavage Requirements for Activation of Factor V by Factor Xa
Authors: Thorelli Elisabeth , Kaufman Randal J. , Dahlbäck Björn
Published: July 2004

Coagulation factor V circulates in plasma as a single chain protein which expresses little procoagulant activity. After its activation by limited proteolysis by thrombin or factor Xa, factor Va functions as cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves human factor V at Arg709, Arg1018 and Arg1545 and factor Va is formed by the heavy and light chains, which correspond to the N‐terminal and C‐terminal fragments, respectively. Factor Xa has been shown to cleave factor V at Arg1018 and at a second undefined position close to Arg709. The factor‐Xa‐mediated cleavage at Arg1018 has been proposed to be sufficient for expression of full factor Va activity. To study the activation of factor V by factor Xa, site‐directed mutagenesis was used to convert Arg709 to Gln, Arg1018 to Ile, and Arg1545 to Gln. Constructs containing all possible combinations of native and mutated residues in these positions were expressed transiently in COS 1 cells. The various factor‐V mutants were incubated with factor Xa or thrombin. The proteolytic cleavage pattern was analyzed by Western blotting, and the specific factor‐Va activities determined in a prothrombinase assay. Control experiments using thrombin gave results which were in agreement with those on record, i.e. cleavages at both Arg709 and Arg1545 were required for expression of full factor‐Va activity, whereas the cleavage at Arg1018 enhanced the rate of cleavage at Arg1545. Factor Xa was found to cleave factor V at all three thrombin cleavage sites, i.e. at Arg709, Arg1018 and Arg1545. An additional factor‐Xa‐cleavage site was found in the light chain region at Arg1765. Cleavage at Arg1018 by factor Xa was not sufficient for expression of full factor‐Va activity. Full factor‐Va activity was only obtained after cleavage at both Arg709 and Arg1545. The factor‐Xa‐mediated cleavage at Arg709 was kinetically favourable over that at Arg1545. Factor V which was mutated at all three sites (at positions 709, 1018 and 1545) was resistant to activation by thrombin. However, treatment with factor Xa yielded an increased factor‐Va activity which was associated with the cleavage at Arg1765. Our study extends previously results on thrombin activation of factor V and elucidates the relative importance of the different cleavage sites for activation of factor V by factor Xa.
Production and Characterization of Recombinant Active Mouse Gelatinase B from Eukaryotic Cells and in vivo Effects after Intravenous Administration
Authors: Masure Stefan , Paemen Liesbet , Van Aelst Use , Fiten Pierre , Proost Paul , Billiau Alfons , Van Damme Jo , Opdenakker Ghislain
Published: July 2004

Gelatinase B is a matrix metalloproteinase involved in tissue remodelling. When mouse cells are triggered in vitro with interleukin‐1, bacterial endotoxin, virus‐mimicking double‐stranded RNA or cytokine inducers, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. Hybrid genomic DNA‐cDNA constructs under the control of two constitutive viral promoters were generated by PCR‐mediated exon amplification. In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79‐kDa protein, and expression in CHO cells resulted in an intact glycosylated 110‐kDa gelatinase B which was enzymically active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell‐culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B. Therefore, the enzyme was expressed in the yeast Pichia pastoris. Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/1. Amino‐terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin‐Sepharose yielded the expected N‐glycosylated proenzyme form (110 kDa) in addition to an amino‐terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo. After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h. In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow. Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.