bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryBasic 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

SummaryA 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

SummaryWe 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.
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.
Cellular Processing Limits the Heterologous Expression of Secretory Component in Mammalian Cells
Authors: Cottet Sandra , Corthésy Blaise 
Published: July 2004

Recombinant vaccinia‐virus‐based expression systems are very popular for the overproduction of proteins in mammalian cell lines. Both the double virus T7/vaccinia hybrid system and the single recombinant strategy based on the p11 K late promoter were evaluated for their ability to govern expression and secretion of recombinant human secretory component (SC), a glycoprotein associated with IgA in mucosal secretions. We report here that, while the T7 promoter is transcriptionally 3.4‐fold more active than the p11 K promoter, no difference in levels of secreted recombinant human SC is observed using either vaccinia system to infect CV‐1 cells. High transcription, and thus translation levels, lead to saturation of early processing steps involved in protein export. Both systems exhibit transient accumulation of comparable amount of recombinant human SC in the endoplasmic reticulum and/or the cis Golgi network, as demonstrated by immunofluorescence and endoglycosidase H (EndoH) sensitivities. Exposure of infected cells to tunicamycin results in similar inhibition of recombinant human SC export, further arguing that N‐linked glycosylation is necessary for proper folding and subsequent secretion. Moreover, pulse‐chase experiments indicate that newly synthesized recombinant human SC is not completely processed in a mature glycoprotein and that a portion of overexpressed SC might be degraded before it can be secreted. Recombinant human SC behaves identically to native SC in terms of kinetics of secretion and IgA‐binding capacity.Our results indicate that optimization of expression systems should not only rely on the design of effective vectors, but also on the identification and clearance of the cellular bottlenecks associated with maturation of the secreted proteins.
Subcellular and Tissue Distribution, Partial Purification, and Sequencing of Calmodulin‐Stimulated Ca2+‐Transporting ATPases from Barley (Hordeum Vulgare L.) and Tobacco (Nicotiana Tabacum)
Authors: Dainese Paola , James Peter , Baldan Barbara , Carafoli Ernesto 
Published: July 2004

The subcellular distribution of plasma‐membrane‐type Ca2+‐transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner‐membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)‐enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+‐transporting ATPase (molecular mass ≈ 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116‐kDa Ca2+‐transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue‐specific manner: the PM pump was dominant in leaves, while the inner‐membrane pump was expressed at a higher level in the roots.For the purification of calmodulin‐binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116kDa was partially sequenced.