bluuebunny/crossref_metadata_2025 huggingface dataset:
Mature dendritic cells differentiated in the presence of interferon-b and interleukin-3 prime functional antigen-specific CD8+ T cells
Authors: Renneson J , Salio M , Mazouz N , Goldman M , Marchant A , Cerundolo V
Published: February 2005

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-β and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-β/IL-3 DCs) that are particularly efficient at eliciting IFN-γ and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8+ T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-β/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A26−35-specific CD8+ T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8+ T cells produced IFN-γ and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8+ T cell numbers was generally higher following priming with CD40-L than with polyinosinic–polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-β/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.
Plasminogen activator inhibitor-1 is elevated, but not essential, in the development of bleomycin-induced murine scleroderma
Authors: Matsushita M , Yamamoto T , Nishioka K
Published: February 2005

Accumulative data have demonstrated that plasminogen activator inhibitor-1 (PAI-1) plays an important role in the extracellular matrix metabolism; however, the involvement of PAI-1 in scleroderma has not been fully elucidated. In this study, we investigated the role of PAI-1 in bleomycin-induced murine scleroderma. 100 µg of bleomycin was injected subcutaneously to the back skin of C3H/HeJ mice on alternate day for 4 weeks. Histopathological findings revealed that PAI-1 was positive in macrophage-like cells and fibroblastic cells in the dermis, in parallel with the induction of dermal sclerosis. PAI-1 mRNA expression in the whole skin was up-regulated at 1 and 4 weeks. The production of active PAI-1 protein in the lesional skin was significantly increased 3 and 4 weeks after bleomycin treatment. Next, we examined whether dermal sclerosis is induced by bleomycin in PAI-1-deficient (PAI-1–/–) mice. 10 µg of bleomycin was subcutaneously injected to PAI-1–/– and wild type (WT) mice 5 days per week for 4 weeks. Histological examination revealed that dermal sclerosis was similarly induced even in PAI-1–/– as well as WT mice. Dermal thickness and collagen contents in the skin were significantly increased by bleomycin injection in both PAI-1–/– and WT mice, and the rate of increase was similar. These data suggest that PAI-1 plays an important role, possibly via TGF-β pathway activation. However, the fact that PAI-1 deficiency did not ameliorate skin sclerosis suggest that PAI-1 is not the essential factor in the development of bleomycin-induced scleroderma, and more complex biochemical effects other than PA/plasmin system are greatly suspected.
a4b1+ and a4b7+ CD4+ T cell numbers increase and CLA+ CD4+ T cell numbers decrease in systemic sclerosis
Authors: Scala E , Paganelli R , Sampogna F , Abeni D , Colonna L , De Pità O , Puddu P , Russo G
Published: February 2005

We studied the expression of adhesion molecules affecting recirculation and homing on peripheral blood CD4+ T cells of patients with systemic sclerosis (SSc), in order to evaluate whether the distribution of tissue targeted subsets could reflect the participation of internal organs or the extent of cutaneous involvement [i.e. limited cutaneous (lc) and diffuse cutaneous (dc)]. Peripheral blood mononuclear cells (PBMC) from 51 patients with SSc and 19 sex- and age-matched controls were investigated by cytofluorimetric analysis for lymphocyte subpopulations carrying the following surface molecules: CD3, CD4, CLA, α4β7 and α4β1. Standard routine biochemistry and clinical examinations were also performed in all patients. We found that both α4β1+ and α4β7+ cells within the CD4+ T cell population were significantly increased, while CLA+ CD4+ T cells were significantly reduced in SSc, compared to healthy donors. Significantly lower absolute numbers of α4β7+ cells were found in lc- compared to dc-SSc. Patients with oesophageal involvement had high numbers of α4β7+ cells, while those with nephritis also showed low levels of CLA+ cells. Lung involvement was related directly to α4β1+ cell numbers and inversely to α4β7+ CD4 cell numbers. Taken together, our findings demonstrate that distinct CD4+ T cell populations with selective homing properties show changes from normal distribution in SSc, and such changes are related to clinical expression and organ involvement in the course of the disease.
A new approach to the analysis of the human sleep/wakefulness continuum
Authors: PARDEY JAMES , ROBERTS STEPHEN , TARASSENKO LIONEL , STRADLING JOHN
Published: February 2005

SUMMARY The conventional approach to the analysis of human sleep uses a set of pre‐defined rules to allocate each 20 or 30‐s epoch to one of six main sleep stages. The application of these rules is performed either manually, by visual inspection of the electroencephalogram and related signals, or, more recently, by a software implementation of these rules on a computer. This article evaluates the limitations of rule‐based sleep staging and then presents a new method of sleep analysis that makes no such use of pre‐defined rules and stages, tracking instead the dynamic development of sleep on a continuous scale. The extraction of meaningful features from the electroencephalogram is first considered, and for this purpose a technique called autoregressive modelling was preferred to the more commonly‐used methods of band‐pass filtering or the fast Fourier transform. This is followed by a qualitative investigation into the dynamics of the electroencephalogram during sleep using a technique for data visualization known as a self‐organizing feature map. The insights gained using this map led to the subsequent development of a new, quantitative method of sleep analysis that utilizes the pattern recognition capabilities of an artificial neural network. The outputs from this network provide a second‐by‐second qualification of the sleep/wakefulness continuum with a resolution that far exceeds that of rule‐based sleep staging. This is demonstrated by the neural network's ability to pinpoint micro‐arousals and highlight periods of severely disturbed sleep caused by certain sleep disorders. Both these phenomena are of considerable clinical value, but neither are scored satisfactorily using rule‐based sleep staging.
Sleepiness and driving: the experience of UK car drivers
Authors: MAYCOCK G.
Published: February 2005

SUMMARY A postal questionnaire survey aimed at exploring the relationship between accidents and daytime sleepiness was sent to 9000 male drivers of which 4621 (51.3%) responded (mean age 47.7, SD 17.1). Drivers provided details of the accidents they had experienced in the last 3 y, and identified those factors, including tiredness, they thought contributed to the accident. In addition, drivers completed the Epworth scale measuring daytime sleepiness and reported whether they had felt close to falling asleep whilst driving during the past 12 mo. Analysis of the data showed that 29% of drivers had felt close to falling asleep at the wheel in the last 12 mo, the probability of which depended on Epworth score, age, occupational group, annual mileage, the proportion of time spent driving on motorways and in built‐up areas, how long the driver is prepared to drive before taking a break, and whether the driver is driving a company car o r not. Overall, about 7% of accident ‘involvements’ were associated with tiredness (representing 9‐10Y0 of accidents)‐a figure which is higher on motorways than on rural roads or built‐up roads and higher still in the early hours of the morning. Accident rates of company car drivers and for those who have felt close to falling asleep at the wheel in the last year are shown to be associated with daytime sleepiness. For example, a company car driver who has felt close to falling asleep at the wheel in the last 12 mo and who scores highly on the Epworth scale has an accident liability which is 70% higher than a similar driver who scores zero on the Epworth scale. Snoring every night increases accident liability by about 30%).
Sleep deprivation reduces the citalopram –induced inhibition of serotoninergic neuronal firing in the nucleus raphe dorsalis of the rat
Authors: PRÉVOT ELISABETH , MAUDHUIT CORINNE , LEPOUL EMMANUEL , HAMON MICHEL , ADRIEN JOüLLE
Published: February 2005

SUMMARY Sleep deprivation (SD) for one night induces mood improvement in depressed patients. However, relapse often occurs on the day after deprivation subsequently to a sleep episode. In light of the possible involvement of central serotonin (5–hydroxytryptamine, 5‐HT) neurotransmission in both depression and sleep mechanisms, we presently investigated, in the rat, the effects of SD and recovery sleep on the electrophysiological response of 5–HT neurons in the nucleus raphe dorsalis (NRD) to an acute challenge with the 5–HT reuptake blocker citalopram. In all rats, citalopram induced a dose–dependent inhibition of the firing of NRD neurons recorded under chloral hydrate anaesthesia. After SD, achieved by placing rats in a slowly rotating cylinder for 24 h, the inhibitory action of citalopram was significantly reduced (with a concomitant 53% increase in its ED5, value). After a recovery period of 4 h, a normal susceptibility of the firing to citalopram was restored. The decreased sensitivity of 5‐HT neuronal firing to the inhibitory effect of citalopram after SD probably results in an enhancement of 5‐HT neurotransmission. Such an adaptive phenomenon (similar to that reported after chronic antidepressant treatment), and its normalization after recovery sleep, parallel the mood improvement effect of SD and the subsequent relapse observed in depressed patients. These data suggest that the associated changes in 5–HT autocontrol of the firing of NRD serotoninergic neurons are relevant to the antidepressant action of SD.
Control of anthocyanin biosynthesis in flowers of Antirrhinum majus
Authors: Martin Cathie , Prescott Andy , Mackay Steve , Bartlett Jeremy , Vrijlandt Eli
Published: February 2005

The intensity and pattern of anthocyanin biosynthesis in Antirrhinum flowers is controlled by several genes. We have isolated six cDNA clones encoding enzymes in the pathway committed to flavonoid biosynthesis and used these to assay how the regulatory genes that modify colour pattern affect the expression of bio‐synthetic genes. The biosynthetic genes of the later part of the pathway appear to be co‐ordinately regulated by two genes, Delila (Del), and Eluta (El), while the early steps (which also lead to flavone synthesis) are controlled differently. This division of control is not the same as control of anthocyanin biosynthesis by the regulatory genes R(S) and C1 in maize aleurone, and may result from the adaptive significance of different flavonoids in flowers and seeds, reflecting their attractiveness to insects and mammals respectively. EI and del are probably involved in transcriptional control and both genes appear to be able to repress expression of some biosynthetic genes and activate expression of others.
The impact of decreased Rubisco on photosynthesis, growth, allocation and storage in tobacco plants which have been transformed with antisense rbcS
Authors: Quick W.P. , Schurr U. , Fichtner K. , Schulze E.‐D. , Rodermel S.R. , Bogorad L. , Stitt M.
Published: February 2005

Transgenic tobacco plants tranformed with antisense to rbcS to decrease expression of ribulose‐1,5–bisphosphate carboxylase‐oxygenase (Rubisco) have been used to investigate (a) whether Rubisco is limiting for photosynthesis and plant growth and (b) whether biomass allocation and storage of carbohydrate and nitrogen are regulated in response to decreased rate of photosynthesis.

The rate of photosynthesis (measured in growth conditions) and plant growth were not strongly inhibited until almost half of the Rubisco was removed. When Rubisco was decreased further there was a large decrease of photosynthesis and plant growth. When photosynthesis decreased in the ‘antisense’ plants there was an increase in the shoot/root ratio and the specific leaf area. As a result, the leaf area ratio (leaf area per g plant dry weight) increased 3–4–fold. This shows that tobacco compensates for decreased photosynthesis by maximizing leaf area. The decrease of photosynthesis also resulted in lower starch and free hexose in the leaf, but the volume of the diurnal starch turnover was largely maintained. This indicates that partitioning to starch is regulated to decrease non‐productive accumulation of starch, but still maintain a pool of transient starch for export during the night. The decrease of photosynthesis was also accompanied by a large increase of the nitrogen/ carbon balance, due to a large accumulation of nitrate in the leaf. This shows that assimilation of nitrate is inhibited in response to low availability of photo‐synthate.
Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus
Authors: Luo Da , Coen Enrico S. , Doyle Sandra , Carpenter Rosemary
Published: February 2005

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild‐type olive gene is required for the production of dark‐green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (mV‐600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600‐700 bp region containing many palindromic copies of a motif sequence, possibly required in c/sfor transposition. The somatic excision of Tam4 in niV‐600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.
Transgenic tobacco plants expressing yeast‐derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions
Authors: Sonnewald Uwe , Brauer Monika , von Schaewen Antje , Stitt Mark , Willmitzer Lothar
Published: February 2005

In higher plants sucrose plays a central role with respect to both short‐term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast‐derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast‐derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N‐terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein.

Transgenic tobacco plants expressing the yeast‐derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild‐type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light‐green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.
Characterization and localization of laccase forms in stem and cell cultures of sycamore
Authors: Driouich Azeddine , Lainé Anne‐Catherine , Vian Brigitte , Faye Loic
Published: February 2005

A laccase‐type polyphenoloxidase (EC 1.10.3.2.), abundantly secreted by suspension‐cultured sycamore (Acer pseudoplatanus) cells was purified to homogeneity. This laccase form is a glycoprotein (molecular weight 110000) with high mannose and complex glycans. The polypeptide moiety has a molecular weight of 66 000, indicating that the glycoprotein is 40% carbohydrate. Laccase is abundantly present in both the cell wall and the culture medium of suspension‐cultured sycamore cells, but it is not detected in the cytoplasm, indicating that this large protein is efficiently secreted by the cells. Polyclonal rabbit antiserum was raised against the deglycosylated protein and was used to probe extracts of sycamore stem tissues. A second laccase form (molecular weight 56 000), antigenically related to laccase from cell cultures, is abundant in the epidermis of sycamore stems. In addition, this 56 kDa laccase form co‐localizes with lignin precursors on tissue prints from sycamore stems. A polypeptide (molecular weight 50 000‐56 000), antigenically related to sycamore laccase, was also immunodetected in most plant organs previously described in the literature as polyphenoloxidase‐rich.
Comparison of homologous recombination frequencies in somatic cells of petunia and tobacco suggest two distinct recombination pathways
Authors: Engels Peter , Meyer Peter
Published: February 2005

Homologous recombination in plants was studied using an extrachromosomal recombination assay in which intermolecular homologous recombination between two complementary plasmids restored a selectable marker gene. Several vectors containing an insertion into or deletions within the coding region of the neomycin phosphotransferase (NPT‐II) gene were designed. Plasmids were introduced, in pairwise combinations, into protoplasts and homologous recombination events were measured by counting the number of NPT‐ll‐resistant colonies. A 10‐fold increase in recombination frequency was observed in Petunia hybrids RL01 compared to Nicotiana tabacum SRI. This difference occurred when one or both of the co‐transferred recombination plasmids was offered in a circular form. Apart from such specific differences between two cultivars from different species, a two‐to fivefold increase in recombination frequencies was observed when the genomic TBS (transformation booster sequence) fragment from P. hybrids was added onto one of the transferred plasmids. TBS‐specific stimulation of recombination was observed in Petunia RL01. These data suggest that two different recombination pathways may be present in plants.
Behaviour of the maize transposable element Ac in Arabidopsis thaliana
Authors: Dean Caroline , Sjodin Christina , Page Tania , Jones Jonathan , Lister Clare
Published: February 2005

The somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T‐DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re‐integrated in different positions in the genome.
Parallel pathways for intracellular Ca2+ release from the vacuole of higher plants
Authors: Johannes Eva , Brosnan James M. , Sanders Dale
Published: February 2005

A novel pathway for intracellular Ca2+ release via a voltage‐gated channel has been diered in the tonoplast of Beta vulgaris (sugar beet) tap roots. The channel is characterized electrophysiologically in isolated vacuoles and radiometrically in tonoplast‐enriched microsomes. Single channel properties were studied in excised membrane patches. With 5‐20 mM Ca2+ as a charge carrier on the vacuolar side, the unitary current saturates to a maximal value of 0.59 ± 0.05 pA as membrane voltage approaches +30 to +50 mV. The maximal slope conductance at non‐saturating voltages is 12.45 ± 1.06 pS. Open‐state probability increases markedly with positive‐going voltage changes in the physiological range. Channel activity is also increased by vacuolar, but not by cytosolic Ca2+. The lanthanide Gd3+ is alone among a large range of Ca2+ channel antagonists in acting as an effective inhibitor of the channel, primarily as a result of a dramatic effect on the open‐state probability. We conclude that this voltage‐gated channel could constitute an alternative, inositol (1,4,5)‐trisphosphate‐independent pathway for vacuolar Ca2+ release during signal transduction in plants.
In‐situ localization of chalcone synthase mRNA in pea root nodule development
Authors: Yang Wei‐Cai , Cramers Hayo C.J. Canter , Hogendijk Peter , Katinakis Panagiotis , Wijffelman Carel A. , Franssen Henk , Van Kammen Albert , Bisseling Ton
Published: February 2005

In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it is hypothesized that the Rhizobium Nod factor induces cell division in the root cortex by stimulating the production of flavonoids that function as auxin transport inhibitors. In nodules CHS mRNA is predominantly present in a region at the apex of the nodule consisting of meristematic and cortical cells. These cells are not infected by Rhizobium. Therefore it is postulated that CHS plays a role in nodule development rather than in a defence response. In roots CHS mRNA is located at a similar position as in nodules, suggesting that CHS has the same function in both root and nodule development.

When nodules are formed by mutants of Rhizobium leguminosarum bv. viciae that are unable to secrete β(1‐2) glucan and to synthesize the O‐antigen containing LPS I, CHS genes are also expressed in regions of the nodule that are infected by Rhizobium. It is postulated that the impaired development of nodules formed by these mutants is due to an induction of a plant defence response.
Characterization of a shoot‐specific, GA3‐ and ABA‐Regulated gene from tomato
Authors: Shi Lifang , Gast Robert T. , Gopalraj Manjula , Olszewski Neil E.
Published: February 2005

A tomato (Lycopersicon esculentum) gene (GAST1) that encodes an RNA whose abundance increases > 20‐fold in shoots of the GA‐deficient gib 1 mutant following spraying with GA3 has been characterized. An increase in GAST1 RNA levels is detectable 2 h after treatment and levels continue to increase for at least an additional 10 h. Between 12 and 24 h following treatment, the amount of GAST1 RNA begins to decline and at 48 h the level is nearly equivalent to that of water‐treated control plants. Nuclear runoff analysis indicates that 8 h after treatment with GA3, transcription of the GAST1 gene has increased only threefold, suggesting that GA acts both transcriptionally and post‐transcriptionally. ABA partially inhibits the GA‐mediated increase in GAST1 RNA abundance while ethephon, kinetin, and 2, 4‐D have little effect. GAST1 RNA is detectable in untreated leaves, stems, petioles and flowers, but not in roots. The GAST1 gene encodes a 0.7 kb transcript. The sequence of the GAST1 cDNA and genomic clones indicates that the gene is interrupted by three introns and potentially encodes a 112 amino acid protein of unknown function.
A functionally active protein import complex from
Authors: Soil Jürgen , Waegemann Karin
Published: February 2005

Isolated outer chloroplast envelope membranes were solubilized by digitonin and separated on linear sucrose density gradients. A membrane complex was recovered from the gradients and exhibited characteristics of a protein import apparatus, i.e. the interaction of the complex with the precursor polypeptides depends on the presence of a transit sequence, ATP and protease‐sensitive components. Furthermore, trans‐location intermediates detected in the organellar system are also found after interaction of the precursor polypeptide with the isolated import complex.
Cold‐plaque screening: a simple technique for the isolation of low abundance, differentially expressed transcripts from conventional cDNA libraries
Authors: Hodge Rachel , Paul Wyatt , Draper John , Scott Rod
Published: February 2005

We report here a simple method for obtaining organ‐or tissue‐specific clones from a cDNA library which are represented at low or medium abundance in the mRNA pool. An anther cDNA library has been screened for anther‐specific clones using a new technique we have called cold‐plaque screening. Using cold‐plaque screening we have confirmed the prediction that a large proportion (40%) of unique transcripts in the anther are organ‐specific but that the majority are of low abundance and therefore not identified by conventional differential screening.
Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5
Authors: Bramley Peter , Teulieres Chantal , Blain Ian , Bird Colin , Schuch Wolfgang
Published: February 2005

Transgenic tomato plants expressing antisense RNA to a ripening‐related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell‐free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene.

Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.
Bean homologs of the mammalian glucose‐regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum
Authors: D'Amico Leone , Valsasina Barbara , Daminati M. Gloria , Fabbrini M. Serena , Nitti Gianpaolo , Bollini Roberto , Ceriotti Aldo , Vitale Alessandro
Published: February 2005

Treatment of developing bean cotyledons with the inhibitor of N ‐glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse‐chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N‐terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose‐regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glyco‐proteins phaseolin, phytohemagglutinin or α‐amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin‐treated tissue, the GRP78 homolog was always co‐precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.
Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics
Authors: Handberg Kurt , Stougaard Jens
Published: February 2005

In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.
PEPC‐mediated carbon fixation in transmitting tract cells reflects style–pollen tube interactions
Authors: Jansen Marcel A.K. , Sessa Guido , Malkin Shmuel , Fluhr Robert
Published: February 2005

Styles nurture and guide pollen tubes to the ovules. The styles of Nicotiana tabacum, a C3 plant, contain a concentric strand of transmitting tract cells replete with well‐developed chloroplasts. It is shown that the chloroplasts have normal ultrastructure and electron transport ability. However, they were found to be devoid of Rubisco, the key enzyme responsible for carbon fixation in C3 plants. Nevertheless, non‐invasive fluorescence techniques showed a light‐driven photosynthetic flux. Carbon fixation via phosphoenol pyruvate carboxylase (PEPC) into malate was demonstrated, the latter accumulating during stylar development. Characterization of stylar PEPC in vitro and in vivo revealed apparent Km values consistent with bicarbonate as a rate limiting factor for photosynthetic flux. Presumably, in the closed confines of the intact style, respired CO2 is the source of carbonate. Enhanced photosynthetic flux was detected following pollination, suggesting utilization of the additional respired bicarbonate and underlining metabolic interactions between the style and the elongating pollen tube.
RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour
Authors: Barzen E. , Mechelke W. , Ritter E. , Seitzer J. F. , Salamini F.
Published: February 2005

An RFLP linkage map for the nine chromosomes of sugar beet (Beta vulgaris L. ssp. vulgaris var. altissima Doell) was constructed by using a segregating population from a cross between two plants which were heterozygous for several agronomically interesting characters. One hundred and eleven RFLP loci have been mapped to nine linkage groups using 92 genomic markers. The current RFLP map covers a total length of 540 cM. Evidence for the existence of a major gene for rhizomania resistance (Rr1) is given, together with its map position on linkage group IV in the interval between loci GS44 and GS28a. The presence of an RFLP fragment at the GS3d locus is, until now, the best molecular marker for rhizomania‐resistant genotypes in segregating populations of sugar beet; GS3d is linked to Rr1 with 6.7 cM. The gene MM, controlling the polygerm/monogerm seed type, has been mapped on linkage group IX in a distal position at 4.2 cM from the locus GS7. The gene R controlling the hypocotyl colour maps to linkage group VII and does not recombine with the RFLP locus GS42. The inheritance of a group of RFLP loci revealed the possible presence of a translocation in the population used to establish the map. The data presented are discussed in relation to the possibility of using RFLP markers in sugar beet breeding.
Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment
Authors: Langridge Peter , Brettschneider Reinhold , Lazzeri Paul , Lörz Horst
Published: February 2005

It has been proposed that transgenic plants of cereals can be generated by inoculating florets with Agrobacterium at or near anthesis. This procedure is shown to lead to the production of embryos of wheat and barley with enhanced resistance to antibiotic selection. It has also been possible to recover plants of wheat, barley and maize that gave positive hybridization signals with probes produced from within the T‐DNA of the Agrobacterium vector. However, no evidence was found for transmission of the bands detected by hybridization in the progeny of the putative transgenic plants nor could enzyme activity associated with the resistance genes be found in plant extracts. Furthermore, undigested genomic DNA from the plants that were positive when probed with the T‐DNA, showed hybridization to bands smaller than the genomic DNA. It is suggested that the apparent transformation is an artifact of the procedure and does not reflect transformation of the plant nuclear genome.
Isolation of a maize bZIP protein subfamily: candidates for the ocs‐element transcription factor
Authors: Foley Rhonda C. , Grossman Carol , Ellis Jeff G. , Llewellyn Danny J. , Dennis Elizabeth S. , Peacock W. James , Singh Karam B.
Published: February 2005

Ocs‐elements, a family of 20 bp DNA sequences, are components of a number of promoters active in plants. In the maize BMS cell line the dominant ocs‐element binding activity is the ocs‐element transcription factor complex called OTF. The isolation of cDNA clones from a BMS cDNA expression library for two bZIP (basic region‐leucine zipper) proteins that bind the ocs‐element sequence and are good candidates for forming at least part of OTF is described. The two ocs‐element binding proteins, called OBF3.1 and OBF3.2, are closely related, with the OBF3.1 protein sharing 95.8% amino acid homology with part of the OBF3.2 protein although there were significant differences in the 3′ untranslated regions. Genomic Southern blot analysis revealed a small gene family with a minimum of two OBF3 loci mapping to chromosomes 3L 105 and 8L075. The OBF3.1 protein shared considerable homology with the wheat HBP1b protein (80% amino acid identity) and to a lesser extent with the tobacco TGA1a protein. OBF3.1 like HBP1b was able to bind well to the Hex sequence but poorly to G‐box/ABRE sequences. Interestingly, OBF3.1 bound eightfold more efficiently to an ocs‐element sequence than TGA1a, raising the possibility that OBF3.1 and TGA1a may be distinct members of an OBF3/TGA subfamily.
Construction of integrated genetic linkage maps by means of a new computer package: Join Map
Authors: Stam Piet
Published: February 2005

A computerized procedure to construct integrated genetic maps is presented. The computer program (Join Map) can handle raw data from F2s, backcrosses and recombinant inbred lines, as well as listed pair‐wise recombination frequencies. The procedure is useful for combining linkage data that have been collected in different experiments; the result is a mathematical alignment of the distinct genetic maps. Data from single experiments can be dealt with as well. In view of the fast growing amount of linkage information for molecular markers, which is often being generated by different research groups, integrated maps provide useful information on the map position of genes and DNA markers.

The procedure performs a sequential build‐up of the map and, at each step, a numerical search for the best fitting order of markers. Weighted least squares is used for the estimation of map distances.
Substrate‐dependent negative selection in plants using a bacterial cytosine deaminase gene
Authors: Stougaard Jens
Published: February 2005

The inability of many higher eukaryotes to convert 5‐fluorocytosine to cytotoxic 5‐fluorouracil presents the possibility of using the bacterial cytosine deaminase codA gene for negative selection. In transformed plant callus, expression of codA results in cell death on 5‐fluorocytosine. In transgenic tobacco and Lotus japonicus plants the substrate‐dependent negative marker segregates as a single dominant gene, and on 5‐fluorocytosine CodA+ seedlings stop growing at the early seedling stage. Positive selection of CodA+ tobacco on the pyrimidine biosynthetic inhibitor N‐(phosphonacetyl)‐l‐aspartate was obtained, by pyrimidine salvage from external cytosine. Activity of cytosine deaminase was determined by conversion of labelled cytosine to uracil followed by separation in thin layer chromatography. The codA marker therefore provides substrate‐dependent negative and positive selection, together with cytosine deaminase reporter activity.
Differential expression of the rol A plant oncogene and its effect on tobacco development
Authors: Carneiro Mauro , Vilaine Françoise
Published: February 2005

The rolA gene not only provokes the abnormal traits of wrinkled leaves, shortened internodes, and deficient root growth in tobacco, but also induction of roots in leaf disks in hormone‐free medium. The rolA mRNA is most abundant in the stem of transgenic tobacco, accumulating at fivefold and 50‐fold lower levels in the leaf and the root, respectively. Promoter deletion mutants were constructed and the rolA‐induced abnormal traits were exploited to identify promoter regulatory regions. The removal of specific promoter domains provokes the appearance of new patterns of rolA transcripts in leaves, stems and roots. These new patterns are correlated with the disappearance of specific abnormal trait, thereby identifying three functional domains. Domain A includes a mosaic motif composed of a putative GT‐2 binding site and DUE‐NDE elements. This domain positively regulates rolA gene expression in the leaf and negatively regulates it in the stem and root. Domains B and C, which contain sequences homologous to the OCS enhancer and to ASF‐1 binding sites, respectively, generally activate the rolA gene expression in all tissues. The possible function of these elements on the regulation of the rolA gene is discussed.
Promoter analysis of a light‐regulated gene encoding hydroxypyruvate reductase, an enzyme of the photorespiratory glycolate pathway
Authors: Sloan James S. , Schwartz Brian W. , Becker Wayne M.
Published: February 2005

Hydroxypyruvate reductase (HPR) is a peroxisomal enzyme that catalyzes the NADH‐dependent reduction of hydroxypyruvate to glycerate in the photorespiratory glycolate pathway. HPR gene expression in cucumber seedlings is organ‐specific and light‐regulated. A 5.7 kb fragment of cucumber genomic DNA containing the entire HPR‐encoding gene, hpr‐A, plus 1069 bp OF 5′ flanking sequence was introduced into tobacco, as was a construct consisting of the upstream region (positions ‐ 1069 to +43) of the hpr‐A gene fused to the ß‐glucoronidase (GUS) reporter gene. Both constructs were expressed in the transgenic plants in an organspecific and light‐dependent manner. A deletion analysis of the 5’flanking sequence of the hpr‐A gene was conducted in transgenic tobacco. A construct in which the 5′‐flanking region had been deleted to within 299 bases of the transcription start site was sufficient to confer a high level of light‐regulated expression on the hpr‐A gene in the transgenic plants. However, deletion to position ‐218 resulted in a substantial reduction in expression. The region between positions ‐299 and ‐218 contains sequences resembling the 1 box and G box elements that are associated with other light‐regulated genes.
Evaluation of home allergen sampling devices
Authors: Sercombe J. K. , Liu‐Brennan D. , Garcia M. L. , Tovey E. R.
Published: February 2005

Background:  Simple, inexpensive methods of sampling from allergen reservoirs are necessary for large‐scale studies or low‐cost householder‐operated allergen measurement.

Methods:  We tested two commercial devices: the Indoor Biotechnologies Mitest Dust Collector and the Dräger Bio‐Check Allergen Control; two devices of our own design: the Electrostatic Cloth Sampler (ECS) and the Press Tape Sampler (PTS); and a Vacuum Sampler as used in many allergen studies (our Reference Method). Devices were used to collect dust mite allergen samples from 16 domestic carpets. Results were examined for correlations between the sampling methods.

Results:  With mite allergen concentration expressed as μg/g, the Mitest, the ECS and the PTS correlated with the Reference Method but not with each other. When mite allergen concentration was expressed as μg/m2 the Mitest and the ECS correlated with the Reference Method but the PTS did not. In the high allergen conditions of this study, the Dräger Bio‐Check did not relate to any methods.

Conclusions:  The Mitest Dust Collector, the ECS and the PTS show performance consistent with the Reference Method. Many techniques can be used to collect dust mite allergen samples. More investigation is needed to prove any method as superior for estimating allergen exposure.
Secretoneurin is released into human airways by topical histamine but not capsaicin
Authors: Korsgren M. , Fischer‐Colbrie R. , Andersson M. , Coman W. B. , Mackay‐Sim A. , Persson C. G. , Sundler F. , Wallwork B. , Greiff L.
Published: February 2005

Background:  The neuropeptide secretoneurin, with potential relevance to leukocyte trafficking, is present in nerves of the nasal mucosa in allergic rhinitis and may be released in response to allergen and histamine exposure. There is no information on the occurrence and mechanisms of release of secretoneurin in healthy human airways.

Methods:  The presence of secretoneurin in nasal biopsies and its release in response to nasal capsaicin and histamine challenges were examined. Symptoms and lavage fluid levels of fucose were recorded as markers of effects in part produced by neural activity. Bronchial histamine challenges followed by sputum induction and analysis of secretoneurin were also carried out.

Results:  Nerves displaying secretoneurin immunoreactivity abounded in the nasal mucosa. Nasal capsaicin challenge produced local pain (P < 0.05) and increased the levels of fucose (P < 0.05), but failed to affect the levels of secretoneurin. Nasal histamine challenge produced symptoms (P < 0.05) and increased the mucosal output of secretoneurin (P < 0.05) and fucose (P < 0.05). Bronchial histamine challenge increased the sputum levels of secretoneurin (P < 0.05).

Conclusions:  We conclude that secretoneurin is present in healthy human airways and that histamine evokes its release in both nasal and bronchial mucosae. The present observations support the possibility that secretoneurin is involved in histamine‐dependent responses of the human airway mucosa.
Exhaled nitric oxide in a population‐based study of asthma and allergy in schoolchildren
Authors: Nordvall S. L. , Janson C. , Kalm‐Stephens P. , Foucard T. , Torén K. , Alving K.
Published: February 2005

Exhaled nitric oxide (NO) reflects inflammation in the lower airways and is well adapted for use in children. The aims of this study were to investigate the distribution of the fraction of expired NO (FENO) in school children and to compare FENO and spirometry in relation to the International Study of Asthma and Allergies in Childhood questionnaire. The study was performed in 959 randomly selected 13–14‐year‐old school children in Uppsala, Sweden. Exhaled NO was measured at an inhalation rate of 0.1 l/s (FENO0.1) and a spirometric test was performed and data from these measurements were related to questionnaire data. Exhaled NO was measured according to American Thoracic Society recommendations, except the use of a mouth wash and an exhalation flow rate of 0.1 l/s. The distribution of the mean FENO0.1 values was skewed, with a preponderance of very low levels and a widespread tail of values ranging up to 102 parts per billion (ppb). Boys exhibited significantly higher mean FENO0.1 values than girls, 5.2 (4.7–5.7) vs 4.4 (4.0–4.8) ppb (geometric mean and 95% CI), P < 0.01). Children who reported wheezing in the last year had higher FENO0.1 values than children that had not, 8.5 (7.1–10.2) vs 4.3 (4.0–4.6) ppb, P < 0.001). The same association was found to most symptoms indicating hay fever and eczema. In contrast to this, only weak or inconsistent associations were found between asthma and spirometric indices. Exhaled NO levels were found to be independently related to male gender, wheeze and rhinoconjuctivitis but not to current eczema. In conclusion, exhaled NO was closely associated with reported asthma and allergy symptoms whereas spirometric indices such as percent predicted forced expiratory volume in 1 s were not. As most asthma cases in a population are mild, the findings suggest that exhaled NO is a sensitive marker of asthma and allergy.
Determinants of increased exhaled nitric oxide in patients with suspected asthma
Authors: Malmberg L. P. , Turpeinen H. , Rytilä P. , Sarna S. , Haahtela T.
Published: February 2005

Exhaled nitric oxide (FENO) has been proposed as a marker of asthmatic inflammation, but it is unclear whether FENO in clinical use selects patients primarily according to their atopic or asthmatic status. The aim of this study was to investigate the determinants of increased FENO in patients with suspected asthma, by means of multinomial logistic regression analysis. The FENO of 132 patients referred because of symptoms suggestive of asthma were studied, and the explanatory factors tested included atopy according to prick skin tests, clinical asthma according to lung function tests, sputum eosinophilia and bronchial hyperresponsiveness (BHR). Slightly elevated FENO levels were significantly explained only by sputum eosinophilia (OR: 3.7; 95% CI: 1.1–13.1; P = 0.04), but for high levels of FENO (≥3 SD of predicted), clinical asthma (OR: 16.3; 95% CI: 5.4–49.7; P < 0.0001) and sputum eosinophilia (OR: 12.0; 95% CI: 4.1–35.0; P > 0.0001) were the characteristics with the highest prediction, followed by atopy and BHR. A significant interaction between asthma and atopy was observed relating to the effect on high FENO, but further analyses stratified by atopy showed significant associations between asthma and high FENO both in atopic and nonatopic patients. We conclude that in patients with symptoms suggesting asthma, slightly elevated and high levels of FENO are associated with sputum eosinophilia, whereas asthma is significantly associated only with high levels of FENO, irrespective of atopy. The results suggest that FENO is primarily a marker of airway eosinophilia, and that only high values of FENO may be useful to identify patients with atopic or nonatopic asthma.