bluuebunny/crossref_metadata_2025_split huggingface dataset:
The C‐terminal tail of Arabidopsis 14‐3‐3ω functions as an autoinhibitor and may contain a tenth α‐helix
Authors: Shen Wei , Clark A. Clay , Huber Steven C. 
Published: May 2003

SummaryThe eukaryotic regulatory protein 14‐3‐3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me2+) and some polyamines interact with the loop 8 region of the 14‐3‐3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C‐terminal regions of the 14‐3‐3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C‐terminal tail of the Arabidopsis 14‐3‐3ω isoform and evaluated its contributions to the inhibition of pNR. Nested C‐terminal truncations of the recombinant 14‐3‐3ω protein revealed that the removal of the C‐terminal tail renders the protein partially Mg2+‐independent in both pNR binding and inhibition of activity, suggesting that the C‐terminus functions as an autoinhibitor. The C‐terminus of 14‐3‐3ω appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys‐247. C‐terminal truncation of 14‐3‐3ω at Thr‐255 increased its interaction with antibodies to the C‐terminus of 14‐3‐3ω in non‐denaturing conditions, but not in denaturing conditions, suggesting that the C‐terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C‐terminal peptide, from Trp‐234 to Lys‐249, revealed that the C‐terminal tail might contain a tenth α‐helix, in agreement with the in silico predictions. The function of the putative tenth α‐helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14‐3‐3 protein is restricted by the C‐terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.
Identification and expression of a soybean nodule‐enhanced PEP‐carboxylase kinase gene (NE‐PpcK) that shows striking up‐/down‐regulation in vivo
Authors: Xu Wenxin , Zhou You , Chollet Raymond 
Published: May 2003

SummaryVarious isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post‐translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC‐kinase (PpcK), a novel, ‘minimal but functional’ Ser/Thr kinase. To date, this plant‐specific kinase has been investigated in molecular terms exclusively in non‐leguminous plants, such as Crassulacean‐acid‐metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up‐regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady‐state abundance of this former, nodule‐enhanced transcript (NE‐PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up‐/down‐regulation of NE‐PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation‐state of PEPC, and the abundance of a previously identified, nodule‐enhanced transcript (GmPEPC7) that encodes the target enzyme (NE‐Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK‐Ppc‘expression‐partners’ in plants, exemplified by NE‐PpcK and NE‐Ppc in the soybean nodule.
Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4‐dichloroaniline
Authors: Loutre Caroline , Dixon David P. , Brazier Melissa , Slater Malcolm , Cole David J. , Edwards Robert 
Published: May 2003

SummaryThe pollutant 3,4‐dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N‐β‐d‐glucopyranosyl‐DCA exported into the medium. The N‐glucosyltransferase (N‐GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5‐trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N‐GT and O‐GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4‐hydroxyphenylpyruvic acid. Both O‐GT and N‐GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.
The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation
Authors: Amor Besma Ben , Shaw Sidney L. , Oldroyd Giles E. D. , Maillet Fabienne , Penmetsa R. Varma , Cook Douglas , Long Sharon R. , Dénarié Jean , Gough Clare 
Published: May 2003

SummaryEstablishment of the Rhizobium–legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation‐defective (Nod−) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor‐activated signal transduction pathway. Characterisation of a new M. truncatula Nod− mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod− mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild‐type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor‐induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild‐type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.
Nanolitre‐scale assays to determine the activities of enzymes in individual plant cells
Authors: Roy Stuart J. , Cuin Tracey A. , Leigh Roger A. 
Published: May 2003

SummaryThere are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single‐cell sampling to extract 10–100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre‐scale fluorescence‐based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole‐leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successfully to Arabidopsis and, as in barley, revealed cell‐specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single‐cell level.
Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non‐dehiscent anthers
Authors: Steiner‐Lange Sabine , Unte Ulrike S. , Eckstein Luca , Yang Caiyun , Wilson Zoe A. , Schmelzer Elmon , Dekker Koen , Saedler Heinz 
Published: May 2003

SummaryA male sterile mutant with a defect in anther dehiscence was identified in an Arabidopsis thaliana population mutagenized with the Zea mays transposon En‐1/Spm. Mutants produce viable pollen that can fertilize when released mechanically from the anthers. Mutant stamens are of normal size and shape, but lack cell wall fortifications in the endothecial cell layer of the anther, which are required for the dehiscence process. The mutant phenotype was shown to be caused by a transposon insertion in AtMYB26, disrupting the putative DNA‐binding domain of this R2R3‐type MYB transcription factor. RT‐PCR revealed that expression of AtMYB26 is restricted to inflorescences. Sterility was shown to be stable under several environmental conditions. The high stability of the sterile phenotype, together with the fact that pollen is functional, makes AtMYB26 and its orthologs a valuable tool for manipulating male fertility in higher plants.
Comparative mapping between potato (Solanum tuberosum) and Arabidopsis thaliana reveals structurally conserved domains and ancient duplications in the potato genome
Authors: Gebhardt Christiane , Walkemeier Birgit , Henselewski Heike , Barakat Abdelali , Delseny Michel , Stüber Kurt 
Published: May 2003

SummaryA genetic map of potato (Solanum tuberosum) was constructed based on 293 restriction fragment length polymorphism (RFLP) markers including 31 EST markers of Arabidopsis. The in silico comparison of all marker sequences with the Arabidopsis genomic sequence resulted in 189 markers that detected in Arabidopsis 787 loci with sequence conservation. Based on conserved linkage between groups of at least three different markers on the genetic map of potato and the physical map of Arabidopsis, 90 putative syntenic blocks were identified covering 41% of the potato genetic map and 50% of the Arabidopsis physical map. The existence and distribution of syntenic blocks suggested a higher degree of structural conservation in some parts of the potato genome when compared to others. Syntenic blocks were redundant: most potato syntenic blocks were related to several Arabidopsis genome segments and vice versa. Some duplicated potato syntenic blocks correlated well with ancient segmental duplications in Arabidopsis. Syntenic relationships between different genomic segments of potato and the same segment of the Arabidopsis genome indicated that potato genome evolution included ancient intra‐ and interchromosomal duplications. The partial genome coveridge and the redundancy of syntenic blocks limits the use of synteny for functional comparisons between the crop species potato and the model plant Arabidopsis.
Look‐Back of Anti‐HCV ELISA‐Positive, HCV‐RNA PCR‐Negative Donors and Recipients of Their Blood Products
Authors: Vrielink H. , Reésink H.W. , Zaaijer H. L. , Scholten E. , Kremer L. C. M. , Cuypers H. T. M. , Lelie P. N. , van Oers M. H. J. , van der Poel C. L. 
Published: May 2003

Abstract Background and objectives: To establish the infectivity of anti‐HCV ELISA‐positive, but cDNA‐PCR‐negative blood components transfused before the introduction of routine anti‐HCV blood donor screening, we enrolled recipients of such blood products in a look‐back programme. Materials and methods: The blood components were donated by (A) RIBA™‐2‐indeterminate and cDNA‐PCR‐negative donors, and (B) RIBA‐2 and cDNA‐PCR‐negative donors. The look‐back comprised 214 blood products from group A donors and 278 from group B. Results: Of 211 recipients of group A components, 66 (31.3%) were available for testing. All other recipients could not be traced, had died, or refused collaboration. Of these 66, 3 patients had independent risk factors for HCV infection and were excluded. All remaining 63 recipients were anti‐HCV ELISA‐negative. Of 274 recipients of group B components, 84 (30.7%) were available for testing. All others could not be traced, had died, or refused collaboration. Of these 84, six patients had an independent risk factor for HCV infection and were excluded. All remaining 78 recipients were anti‐HCV ELISA‐negative. None of the recipients of blood products from previous donations of anti‐HCV ELISA‐positive, cDNA‐PCR‐negative, and RIBA‐2‐indeterminate or negative donors were HCV‐infected. Conclusions: Donors and patients with such reactivities in anti‐HCV ELISA, RIBA‐2, and cDNA‐PCR can be assured that they are not infected with HCV. The donors involved can re‐enter the donor pool, provided that future donations are anti‐HCV ELISA‐negative.
Typing of Hepatitis C Virus Antibody with Specific Peptides in Seropositive Blood Donors and Comparison with Genotyping of Viral RNA
Authors: León Pilar , López José A. , Elola Consuelo , Quan Stella , Echevarría José M. 
Published: May 2003

Abstract Background and objectives: Serotyping of antibody to hepatitis C virus (anti‐HCV) with specific peptides has been developed as an alternate method for typing HCV infections. The method does not require a prior amplification of viral RNA sequences from the sample. Identification of the viral genotype may be relevant for prognosis and clinical management. Materials and methods: We used a previously described HCV serotyping assay (RIBA™ HCV Serotype SIA kit, Chiron Corp.) to investigate the serotype in 191 samples from blood donors selected for anti‐HCV patterns (positive and indeterminate), ALT levels, and the presence or absence of viral RNA. The serotypes were compared with the genotypes obtained from typing the 5′‐noncoding region of the viral RNA in 82 viremic samples. Results: We were able to obtain the viral serotype in 85% (114/134) of samples positive for anti‐HCV but in only 3.5% (2/57) of the indeterminates. Lack of anti‐NS4 in the sample was significantly associated with both untypable results and the presence of HCV serotypes other than serotype 1. The overall correlation with genotyping was 78% (64/82), rising to 95.5% (64/67) if only samples that could be both genotyped and serotyped were considered. The assay was easy to perform, gave reactivity patterns easy to interpret, and performed with high proficiency on anti‐HCV‐positive samples lacking detectable levels of viral RNA. Conclusions: This is a practical and useful method for typing HCV infections in the clinical setting. The poor ability of the Core peptides to give the serotype in samples lacking anti‐NS4 and the lack of specific peptides to recognize HCV types other than 1, 2, and 3 are, however, some aspects of the method that need improvement in the future.
Transmission of GB Virus C by Blood Transfusions during Heart Surgery
Authors: Shimizu Masaru , Osada Koji , Okamoto Hiroaki 
Published: May 2003

Abstract Background and objectives: Hepatitis‐causing viral agents other than those designated A to E are being reported. Their epidemiology, modes of transmission, and infectivity are not yet clear, although they may be transmitted by transfusion. Materials and methods: Thirty five patients underwent open‐heart surgery, receiving an average of 10.2±10.8 (range 1 to 35) units of blood. The patients were investigated postoperatively for the RNA of the putative non‐A‐to‐E hepatitis virus designated as GB virus C (GBV‐C). Results: Four patients (12%) acquired GBV‐C RNA in the serum within 2 to 4 weeks after the operation. GBV‐C RNA was detected in at least one of the blood units received by three patients, all of whose units were available for testing, with a sequence in the nonstructural 3 region identical to that from the corresponding patient. Three patients developed elevated alanine aminotransferase levels which persisted for two weeks or longer in two of them. Conclusions: GBV‐C is transmissible by transfusion and can induce elevated transaminase levels.
Does Anti‐HBc Reactivity Reflect ‘Lifestyle’ Risk in North London Blood Donors?
Authors: Moore M. C. , Godfrey G. S. , Hewitt P. E. , Barbara J. A. J. 
Published: May 2003

Abstract Background and objectives: To determine whether antibody to hepatitis B core (anti‐HBc) reactivity, as a marker of hepatitis B virus infection, reflects a ‘lifestyle’ risk in donors in North London, we have obtained detailed histories from donors with various manifestations of HBV infection. Materials and methods: Interviews based on a standardised questionnaire were conducted with 70 donors confirmed to be anti‐HBc reactive. Results: Comparison with controls (known to be anti‐HBc‐negative) and donors reactive for anti‐hepatitis‐C virus showed that current donor strategies are effective; in our area, reactivity for anti‐HBc is not associated with a lifestyle risk for those donors who are unreactive in current mandatory screening tests. Conclusions: Blood from donors positive for both anti‐HBc and anti‐HBs should be considered suitable for transfusion purposes. This is consistent with the practice of accepting blood from naturally immune donors for preparation of hepatitis B immunoglobulin.
Epidemiology and Prevention of Transfusion‐Associated Human Immunodeficiency Virus Transmission in Sub‐Saharan Africa
Authors: McFarland William , Mvere David , Shandera Wayne , Reingold Arthur 
Published: May 2003

Abstract Background and objectives: Compared to industrialised nations, countries in sub‐Saharan Africa experience a greater amount of transfusion‐associated HIV transmission due to high rates of transfusion in some groups of patients, a higher incidence and prevalence of HIV infection in donor populations, a lack of HIV antibody screening in some areas, and a higher residual risk of contamination in blood supplies despite antibody screening. Materials and methods: Epidemiologic review. Results: Epidemiologic evidence supports the effectiveness of three relatively inexpensive strategies to prevent transfusion‐associated HIV transmission in sub‐Saharan Africa: HIV antibody screening, avoidance of unnecessary use of blood products, and exclusion of donors at high risk of infection. Such prevention strategies have not been universally implemented. Conclusions: International aid to establish and maintain HIV antibody screening programmes, implementation of sound criteria for transfusion, and the search for HIV risk factors to use as donor exclusion criteria must be expanded in the region.
Comparison between Intravenous and Subcutaneous Recombinant Human Erythropoietin (Epoetin Alfa) Administration in Presurgical Autologous Blood Donation in Anemic Rheumatoid Arthritis Patients Undergoing Major Orthopedic Surgery
Authors: Mercuriali F. , Inghilleri G. , Biffi E. , Colotti M. T. , Vinci A. , Sinigaglia L. , Gualtieri G. 
Published: May 2003

Abstract Background and objectives: Intravenous (i.v.) Recombinant erythropoietin (Epoetin alfa) is effective in allowing autologous blood donation in patients unable to donate because of anemia. We undertook this open pilot study in order to asses whether a low subcutaneous (s.c.) dose of Epoetin alfa would prove as effective and well tolerated as the higher i.v. dose. Such a move would also decrease costs. Materials and methods: A total Epoetin alfa s.c. dose of 800 IU/kg was compared with a total i.v. dose of 1,800 IU/kg. Twenty‐two rheumatoid arthritis patients, unable to donate because of hemoglobin (Hb) <11 g/dl, received 300 IU/kg of IV Epoetin alfa twice weekly for 3 weeks (11 patients), or 100 IU/kg of s.c. Epoetin alfa twice weekly for 3 weeks plus an i.v. bolus of 200 IU/kg of Epoetin alfa at the first visit (11 patients). At each visit, all patients received 100 mg of i.v. iron saccharate and when the hematocrit (hct) 34%, 350 ml of autologous blood (AB) were collected. Results: No significant differences were observed between the 2 groups of treated patients in terms of units of AB collected (2.6±0.6 vs. 2.5±0.5 units for i.v. and s.c. groups, respectively), ml of RBC produced during the study period (291±99 vs. 337±65 ml for the i.v. and s.c. groups, respectively), or in the degree of reduced exposure to allogeneic blood in comparison with the control group. Conclusions: Lower dose of Epoetin alfa (reduced by 56%), supplemented by i.v. iron, is as effective and well tolerated as higher doses administered i.v., supplemented by i.v. iron.
Evaluation of a New Prestorage Leukoreduction Filter for Red Blood Cell Units
Authors: AuBuchon J. P. , Elfath M. D. , Popovsky M. A. , Stromberg R. R. , Pickard C. , Herschel L. , Whitley P. , McNeil D. , Arnold N. , O'Connor J. L. 
Published: May 2003

Abstract Background and objectives: Prestorage leukoreduction offers a variety of potential benefits and is becoming more commonly practiced. The LeukoNet™ prestorage leukoreduction filtration system is intended for leukoreduction of red blood cells and uses a vent to allow automatic drainage of red cells from the filter. Materials and methods: We studied the functional characteristics and the in‐vivo and in‐vitro properties of leukoreduced AS‐1 Red Blood Cells prepared with this new system. Units of AS‐1 Red Blood Cells were filtered at 4°C through the LeukoNet™ filter 24–48 h after collection and stored under usual conditions for 42 days. Residual leukocytes were enumerated using a Nageotte chamber or with a polymerase chain reaction (PCR) technique. In the clinical trial (phase one), 21 donors had units stored with and without leukoreduction for 42 days; biochemical assays were done before and after storage, and 51Cr/99mTc red cell recovery studies at the end of the storage period. Results: Leukocyte content after filtration was 3.2±2.6×104/unit (n = 21), and all units had <1×105 leukocytes (median: 3.8×104). In‐vivo paired studies showed no difference in 24‐hour recovery (control: 82.1±5.8%; test: 82.9±6.0%). Hemolysis was halved with leukoreduction (0.59±0.30 vs. 0.29±0.11%; p<0.05), and glucose consumption was reduced by 5% compared to control units (p = <0.05). Other biochemical parameters showed no differences. In the practical trial (phase two), filtration time was 41±23 min. With a residual leukocyte content of 6.6±4.9×104/unit and 14±3% red cell loss (n = 84). Six additional units underwent leukocyte enumeration by PCR and had 2.6±1.1×104 residual leukocytes. Conclusions: Under the conditions studied, the LeukoNet™ leukoreduction filtration system produces about 4–5 log10 leukocyte content reduction.