bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide in plants: the history is just beginning
Authors: Beligni M. V. , Lamattina L.
Published: March 2003

Nitric oxide (NO) is a bioactive molecule that exerts a number of diverse activities in phylogenetically distant species, as well as opposing effects in related biological systems. It was firstly described in mammals as a major messenger in the cardiovascular, immune and nervous system, in which it plays regulatory, signalling, cytoprotective and cytotoxic effects (Ignarro, Annual Review of Pharmacology and Toxicology 30, 535–560, 1990; Anbar, Experientia 51, 545–550, 1995). This versatility is mainly achieved through interactions with targets via either a redox or an additive chemistry (Stamler, Cell 78, 931–936, 1994). For this reason, metal‐ and thiol‐containing proteins serve as major target sites for NO: these include signalling proteins, receptors, enzymes, transcription factors and DNA, among others. Furthermore, NO is a small, highly diffusible molecule. It rapidly crosses biological membranes and triggers various different processes in a short period of time. In this context, NO can co‐ordinate and regulate cellular functions of microsomes and organelles such as mitochondria. The ubiquity of NO reactions, as well as the finding that the biochemical and molecular mechanisms underlying many physiological processes are well conserved between diverse species, have opened the exploration of NO chemistry in different organisms. Among these, plants were not the exception. The research in plants has been focused on three main fields: (i) the search for NO or any source of NO generation; (ii) the examination of the effects of NO upon exogenous treatments; and (iii) the search for the same molecules involved in NO‐sensitive transduction pathways as in animals (e.g. cGMP, Ca2+, calmodulin). As it is evident from this review, recent progress on NO functionality in plants has been impressive. With the use of biochemistry, molecular genetics and structural biology, together with classical physiological approaches, an explosion of new discoveries will surely begin. It is certainly a good time for plant biologists.
The essentials of direct xylem pressure measurement
Authors: Wei C. , Steudle E. , Tyree M. T. , Lintilhac P. M.
Published: March 2003

This paper discusses the essentials of the oil‐filled pressure probe technique in the measurement of negative xylem pressures, focusing in particular on the technique and physics underlying our recent, successful experiment which has rekindled the debate on the validity of the Cohesion–Tension theory. We illustrate a number of general problems associated with the cell pressure probe and xylem pressure probe techniques, and propose appropriate criteria for micropipette construction. We enumerate factors dealing with the cavitation problem and suggest methods for eliminating air seeds in the system. We introduce reliable criteria for the successful measurement of xylem pressure, and emphasize the importance of the probe pressure relaxation test. Several problems regarding the controversy over the Cohesion–Tension theory are also discussed. We discuss the correlation between xylem pressure and the transpiration rate, the existence of absolute negative xylem pressure in intact plants, the most negative values of xylem pressure measured by the pressure probe, the agreement between the pressure probe and pressure bomb techniques, and the vulnerability to cavitation (tensile strength) of pressure probes.
cDNA cloning of a mechanical/abiotic stress‐inducible calmodulin‐related gene from dodder‐infected alfalfa
Authors: Borsics T. , Lados M.
Published: March 2003

Plants respond to various biotic and abiotic stresses by the specific induction of a certain set of genes. This study involved a search for transcripts induced in alfalfa (Medicago sativa L. bv Verko) upon infection with dodder (Cuscuta trifolii Bab. et Gibs). A 570 bp cDNA, designated PPRG1, was isolated by differential display. Southern blot analysis indicates that PPRG1 is a member of a new small gene family in alfalfa. PPRG1 encodes a putative 19·2 kDa calmodulin‐related protein with 59% amino acid homology to the touch‐inducible TCH2 gene isolated from Arabidopsis. The level of PPRG1 transcript shows a rapid transient increase to mechanical stimuli such as rain, touch and wounding, to osmotic and salt stresses and to external treatment with the plant hormone abscisic acid. Constantly elevated levels of PPRG1 mRNA can be observed during cold stress, whereas heat treatment does not induce PPRG1 expression. These results implicate roles for Ca2+ and PPRG1 in the transduction of signals from the environment.
Nitric oxide interferes with plant photo‐oxidative stress by detoxifying reactive oxygen species
Authors: Beligni M. V. , Lamattina L.
Published: March 2003

Oxidative stress within chloroplasts is originated due to light‐dependent O2 reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H2O2 and then, by the Fenton reaction, H2O2 may be reduced to the hydroxyl radical (OH•). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm) and S‐nitroso‐N‐acetylpenicillamine (200 µm), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.
Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh
Authors: Ramonell K. M. , Mcclure G. , Musgrave M. E.
Published: March 2003

An unforeseen side‐effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol−1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. ‘Columbia’ was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol−1) with 0·35 mmol mol−1 CO2 in N2. Ethylene in full‐sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1‐aminocyclopropane ‐1‐carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol−1 O2. At the same time, silique ACC oxidase mRNA increased three‐fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue‐specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.
Root elongation and branching is related to local hexose concentration in Arabidopsis thaliana seedlings
Authors: Freixes S. , Thibaud M‐C. , Tardieu F. , Muller B.
Published: March 2003

Root architecture can be profoundly affected by the carbon availability in the plant. We hypothesized that this effect could be mediated by the carbon status of root cells involved in elongation and branching processes. Arabidopsis thaliana plants were grown at several photosynthetic photon flux densities (PPFD) and were supplied with various sucrose concentrations in the root medium. Hexose and sucrose concentration was estimated in individual roots in the apical growing region of the primary root and of secondary roots as well as in the zone of primordia development. Local sugar concentration was high in fast‐growing and in highly branched roots and robust relationships between root elongation rate or branching and hexose concentration (but not sucrose) were found that were common to all situations experienced. Moreover, these relationships accounted for the plant‐to‐plant variability within a treatment as well as for the variability among individual secondary roots within a plant. These results support the view that local hexose concentration integrates changes in carbon availability from several sources and acts as a signal to induce at least part of the response of the root architecture to the environment.
The use of chlorophyll fluorescence excitation spectra for the non‐destructive in situ assessment of UV‐absorbing compounds in leaves
Authors: CEROVIC Z. G. , OUNIS A. , CARTELAT A. , LATOUCHE G. , GOULAS Y. , MEYER S. , MOYA I.
Published: March 2003

In this study a method was designed to assess non‐destructively the type of UV‐screening compounds present in the leaf epidermis. The method is based on the recording and calculation of the ratio of UV‐excitation spectra of chlorophyll fluorescence (FER) from the adaxial and abaxial sides of bifacial leaves, or from older and younger segments of monocotyledonous leaves. The logarithm of this ratio (logFER) matched the absorption spectrum of the UV‐absorbers present in the leaf, as confirmed by its overlap with the absorption spectrum of the methanolic extract of the leaf or of the isolated epidermis. By using the logFER approach, it was possible to demonstrate that the concentration but not the classes of compounds present in the epidermis that are responsible for UV‐screening is affected by the side and the age of the leaves. In contrast, measurements from the leaves of seven dicots and one monocot indicated large difference in the classes of these compounds between species. Finally, it was shown that the logFER in the UV is independent of the emission wavelength, and that the method can be used for quantitative measurements. This method expands to the spectral domain the use of ChlF for the estimation of the leaf epidermal transmittance.
Characterization of a hypovirulent insertional mutant of Pyrenophora graminea and analysis of the barley defence response after inoculation
Authors: Valè  , Aragona  , Torrigiani  , Cattivelli  , Montigiani  , Stanca  , Porta‐Puglia
Published: March 2003

Pyrenophora graminea is the causal agent of barley leaf stripe. A mutant of the fungus (T2) was obtained by insertional mutagenesis from the highly virulent isolate I2 by random insertion of a plasmid that has no homology with the fungal genome. A marked reduction of disease incidence was observed in two barley cultivars susceptible to leaf stripe, Nudinka and Mirco, when inoculated with the isogenic insertional mutant T2 in comparison with P. graminea isolate I2. Southern blot and RAPD analysis were performed to confirm that the insertion had affected only a single locus and that consequently the loss of the corresponding genetic function had led to a consistent reduction of virulence. A RAPD amplification product was found to be associated with the genomic region tagged by the plasmid, perhaps therefore representing a part of the inactivated gene. The accumulation of four defence‐related transcripts was monitored in the root tips of cv. Nudinka challenged with both the virulent isolate and the isogenic hypovirulent T2. Two kinds of response were observed in rootlets inoculated with T2 when compared with the wild type: a delay in the accumulation of the mRNAs homologous to thaumatin‐like protein, thionin and peroxidase genes and, 24 h after inoculation, a higher accumulation of mRNAs homologous to β‐(1,3)‐glucanases and thionins. The relationships between the plant defence response and the virulent and hypovirulent effects, as well as the potential use of this insertional mutant in the isol\Gamma ation and characterization of the inactivated virulence gene are discussed.
Ascospores as primary inoculum for epidemics of white leaf spot (Mycosphaerella capsellae) in winter oilseed rape in the UK
Authors: Inman  , Fitt  , Todd  , Evans
Published: March 2003

In the UK, conidia of Pseudocercosporella capsellae, the anamorph of Mycosphaerella capsellae, were observed on white leaf spot lesions on leaves throughout the growing season. Ascomata were not observed on lesions on either green or senescent leaves, although stromatic knots and spermogonia were occasionally seen in summer. However, spermogonia and protoascomata were produced in white leaf spot pod and stem lesions in early summer. Protoascomata continued to mature after harvest in these lesions on the debris. Mature ascomata subsequently developed by early autumn, but were exhausted by early January and did not overwinter. A diurnal periodicity in numbers of air‐borne M. capsellae ascospores discharged from infected debris was observed with a Burkard spore sampler, with greatest numbers of ascospores collected near the middle of the day; the records also suggested that ascospores were released in response to wetting by dew or rain. Studies of natural white leaf spot epidemics in winter oilseed rape provided evidence that air‐borne ascospores are the primary inoculum for initiating epidemics in the autumn in the UK. White leaf spot disease gradients over 100 m across a winter oilseed rape crop at Rothamsted were fitted by both negative exponential and inverse power‐law models, with gradient slopes suggesting the deposition of air‐borne spores dispersed from a single local source of inoculum. In comparison, no obvious white leaf spot gradients were observed over 250 m in a severely diseased crop near North Petherton, Somerset, suggesting that the air‐borne spores were dispersed from a number of more distant sources in the area. Both patterns of disease were unlikely to have been initiated by P. capsellae conidia, which are dispersed only very short distances by rain‐splash. However, once epidemics have been initiated by air‐borne ascospores in the autumn, subsequent disease spread within an infected crop is dependent only on splash‐dispersed conidia. A revised disease cycle of the pathogen is proposed.
Wintering of the biotrophic fungus Puccinia lagenophorae within the annual plant Senecio vulgaris: implications for biological weed control
Authors: Frantzen  , Müller‐Schärer
Published: March 2003

Epidemics of the obligate biotrophic fungus Puccinia lagenophorae might be used to control populations of the annual plant, groundsel, Senecio vulgaris. Insight into the mechanisms of survival of P. lagenophorae over winter may help to explain the number of inoculum sources, and their strength (assessed by number and size of pustules), present in an S. vulgaris population in spring, indicating the probability and rate of progress of a subsequent epidemic. Results of the study indicated survival of the rust as mycelium within the host over winter. Survival outside the host is unlikely, because aecidiospores lost their capacity to germinate over winter and teliospores have not been reported to be infectious. Survival of S. vulgaris plants over winter was reduced by rust infection in autumn. The mortality of S. vulgaris was 30–100% depending on the date of infection. All plants infected early in autumn died but those infected late in autumn were more likely to survive. In turn, poor survival of the host impacted on the survival of P. lagenophorae over winter. Consequently, the results of the study suggest that no inoculum sources, or only a few weak ones, are present in vulgaris populations in spring. This suggestion was supported by observations of an S. vulgaris population at a ruderal site. Therefore, research on biological weed control should focus on increasing the negative impact of P. lagenophorae on S. vulgaris populations while augmenting the probability of survival of the rust over winter to start new epidemics in spring.
Phytotoxity of Fusarium subglutinans culture filtrates on in vitro plantlets and calli of resistant and susceptible pineapple (Ananas comosus)
Authors: Hidalgo  , Santos  , Tussel  , Pires de Matos  , Cabral  , Arzola  , Pérez
Published: March 2003

Various concentrations of culture filtrates from Fusarium subglutinans were tested for their effect on in vitro multiplication of plantlets and regeneration from calli of pineapple. The cultivars tested were the resistant Perolera and Spring and the susceptible Perola and Smooth Cayenne. The culture filtrate reduced shoot formation of in vitro plantlets and regeneration of plants from calli derived from the susceptible cultivars but plantlets and calli from resistant plants showed resistance. These results show the potential for culture filtrates of F. subglutinans to provide a method of in vitro screening of pineapple for resistance to the pathogen.
Genetic diversity in Botrytis cinerea populations on vegetable crops in greenhouses in south‐eastern Spain
Authors: Alfonso C. , Raposo R. , Melgarejo P.
Published: March 2003

Seventy‐nine random amplified polymorphic DNA (RAPD) markers, of which 46 were polymorphic and 33 monomorphic, were used to determine the level of genetic diversity and to study the structure of populations of Botrytis cinerea in Almería, Spain. Sixty‐five isolates were collected in the first survey (December 1992, at the beginning of the epidemic) from 23 commercial greenhouses of common winter crops in two regions, Levante and Poniente. In the second survey, conducted three months later at the end of the epidemic, 75 isolates were collected from 16 greenhouses in Poniente. The analysis of population structure revealed that genetic diversity within subpopulations sampled in the two surveys accounted for 98% of the total genetic diversity, while genetic diversity between subpopulations represented only 2% of the total diversity. The relative magnitude of gene differentiation between subpopulations, GST, averaged 0.017. In the first survey, partition of genetic diversity into hierarchical components by greenhouses within regions showed that the within‐greenhouse component of diversity was 93%, the between‐greenhouses, within‐region component was 6%, and the between‐regions component was 1%. Similar results were obtained when comparisons were made between greenhouses in the second survey or between populations of different countries: genetic diversity within subpopulations accounted for 95 and 96%, respectively, of the total genetic diversity. In all comparisons, values of GST were higher than 0.01, ranging from 0.011 to 0.055. Analysis of likelihood ratio chi‐square (G2) statistics under the null hypothesis that frequencies of markers are similar between subpopulations showed that different markers contributed to the differentiation between subpopulations in each comparison. The relative importance of several evolutionary forces in populations of B. cinerea is discussed, together with implications for the management of grey mould.
Activation of systemic disease resistance in pea by an avirulent bacterium or a benzothiadiazole, but not by a fungal leaf spot pathogen
Authors: Dann E. K. , Deverall B. J.
Published: March 2003

Inoculation of first expanded leaves of pea seedlings with an avirulent strain of Pseudomonas syringae pv. pisi, or treatment with sprays of a benzothiadiazole (20 or 100 μg a.i. mL−1), decreased the susceptibility of subsequent leaves 7 or 14 days later to challenge inoculation with Mycosphaerella pinodes. Inoculation of first leaves with a virulent strain of P. syringae pv. pisi or with M. pinodes did not decrease the susceptibility of plants to M. pinodes. Treatments effective in decreasing susceptibility to M. pinodes were similarly active against Uromyces viciae‐fabae and virulent P. syringae pv. pisi. Effective treatments also enhanced the activities of the enzymes β‐1,3‐glucanase and chitinase in untreated upper leaves 6 days later. Ineffective treatments for decreased susceptibility had no effect on the activity of the enzymes. None of the treatments enhanced peroxidase activities. The results are discussed in relation to the reported signalling effects of the benzothiadiazole and in relation to a suggested high activity of the avirulent P. syringae pv. pisi strain and inactivity of M. pinodes in enhancing natural signalling.
Biocontrol of the pathogen Phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci
Authors: Vigo C. , Norman J. R. , Hooker J. E.
Published: March 2003

The impact of colonization by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on tomato root necrosis caused by the soil‐borne pathogen Phytophthora parasitica was investigated. Studies in situ permitted infection loci to be identified and the effects of the AMF on numbers to be elucidated. Effects were significant and, 7 and 16 days after inoculation with zoospores of the pathogen, roots of plants colonized by the AMF had 39% and 30%, respectively, fewer infection loci than those that were not. Concurrent studies of the rate of spread of necrosis within roots showed no changes caused by the AMF. At harvest, 26 days following inoculation with the pathogen, 61% of roots of noncolonized plants were necrotic compared with only 31% in AMF‐colonized plants. It is concluded that effects on numbers of infection loci are one mechanism via which AMF achieve biocontrol of this pathogen in tomato. Measures of the effects of the AMF on root system architecture suggest that no significant changes occur and are thus not the reasons for the reduction in infection loci. The implications of these data for agricultural practice and biocontrol research are discussed.
Characterization of Ascochyta isolates and susceptibility of pea cultivars to the ascochyta disease complex in Alberta
Authors: Wang H. , Hwang S. F. , Chang K. F. , Turnbull G. D. , Howard R. J.
Published: March 2003

The relative virulence of 109 Ascochyta isolates collected from pea fields in Alberta from 1996 to 1998 were evaluated on 10‐day‐old seedlings by the excised leaf‐assay technique. Twenty‐eight isolates were avirulent, while the others produced lesions of various sizes on pea leaves. DNA samples from 86 isolates were amplified by the RAPD technique using PCR with single primers. One dominant genotype of Ascochyta pisi was identified throughout Alberta, but variations in virulence were not clearly differentiated by the RAPD technique. Five Ascochyta isolates, four virulent and one avirulent, were used to assess the susceptibility of 20 field pea cultivars available in Alberta, including 13 yellow types and seven green types. Based on symptom development, the yellow‐type cultivars Swing, Eiffel and Delta, and the green‐type cultivar Orb, were the most susceptible. Of yellow‐type cultivars, Voyageur, Carneval and Montana were most resistant to Ascochyta infection.
Modelling the progress of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in relation to leaf wetness and temperature
Authors: Papastamati K. , Welham S. J. , Fitt B. D. L. , Gladders P.
Published: March 2003

A compartmental model was developed to describe the progress with time of light leaf spot (Pyrenopeziza brassicae) on leaves of winter oilseed rape (Brassica napus) during the autumn in the UK. Differential equations described the transition between the four compartments: healthy susceptible leaves, infected symptomless leaves, sporulating symptomless leaves and leaves with necrotic light leaf spot lesions, respectively. The model was fitted to data on the progress of light leaf spot on winter oilseed rape at a single site during the autumn of the 1990–1991 season. Model parameters were used to describe rates of leaf appearance, leaf death, infection by airborne ascospores (primary inoculum) and infection by splash‐dispersed conidiospores (secondary inoculum). Infection was dependent on sufficient leaf wetness duration. The model also included delay terms for the latent period between infection and sporulation and the incubation period between infection and the appearance of necrotic light leaf spot lesions. This modified SEIR model formulation gave a reasonable fit to the experimental data. Sensitivity analysis showed that varying the parameter accounting for the rate of infection by ascospores affected the magnitude of the curves after the start of the epidemic, whilst including a parameter for conidiospore infection improved the fit to the data. Use of ascospore counts from different sites and different years showed variation in spore release patterns sufficient to affect model predictions.
Development of apothecia of the eyespot pathogen Tapesia on cereal crop stubble residue in England
Authors: Dyer P. S. , Bateman G. L. , Wood H. M.
Published: March 2003

A reservoir of infection of Tapesia yallundae may exist after harvest in bases of cereal stems due to the presence of apothecia capable of discharging infective ascospores. Apothecia of T. yallundae developed in a seasonal pattern on winter barley inoculated with the pathogen, with maximum numbers of apothecia produced on stubble 5–7 months after harvest. A similar pattern of development was observed on infected winter wheat. However, the peak in numbers of mature apothecia was observed 2 months later than in winter barley. Apothecia capable of discharging ascospores were present for up to 6 months on stubble. Apothecia of T. acuformis were not detected on spring or winter barley, or spring or winter wheat stubble, despite inoculation of growing crops with isolates of compatible mating type.
Changes in populations of the eyespot fungiTapesia yallundaeandT. acuformisunder different fungicide regimes in successive crops of winter wheat, 1984–2000
Authors: Bierman S. M. , Fitt B. D. L. , Van Den Bosch F. , Bateman G. L. , Jenkyn J. F. , Welham S. J.
Published: March 2003

Effects of regular treatments with the fungicides carbendazim and prochloraz applied to whole plots divided into subplots with different initial population mixtures of carbendazim‐sensitive or carbendazim‐resistantTapesia yallundaeorT. acuformiswere studied in successive crops of winter wheat from 1984/85 to 1999/2000. In unsprayed and carbendazim‐sprayed whole plots, a stable coexistence of about 50% each ofT. yallundaeandT. acuformisdeveloped within five seasons, but in whole plots sprayed with prochloraz or prochloraz plus carbendazim, the proportion ofT. acuformisincreased to > 80%. A discrete time difference equation model was derived from knowledge of the biology of eyespot and competition theory to describe the population changes. The model was fitted to the data from treatments where coexistence occurred [subplots in unsprayed (1985–92) and carbendazim‐sprayed (1985–89) whole plots], using nonlinear least squares regression. The optimized value of the resource overlap coefficient was small, suggesting niche differences between the two species. Populations were nearly 100% carbendazim‐resistant in carbendazim‐sprayed whole plots by July 1985 (one season) and in whole plots sprayed with prochloraz plus carbendazim by July 1986 (two seasons). In prochloraz‐sprayed whole plots, the proportion of carbendazim‐resistant isolates decreased more rapidly than in unsprayed whole plots in the 1980s, but by July 1992 a shift in populations in unsprayed and prochloraz‐sprayed whole plots towards predominantly carbendazim‐resistant strains had occurred.
Production of monoclonal antibodies against synthetic peptides of the N‐terminal region of Potato virus Y coat protein and their use in PVY strain differentiation
Authors: Ounouna H. , Kerlan C. , Lafaye P. , Loukili M. J. , ElGaaied A.
Published: March 2003

Antibodies were prepared against two synthetic peptides, P19 and P11, derived from the coat protein N‐terminal region of two pepper isolates of Potato virus Y from Tunisia (PVY‐P21 and PVY‐P2, respectively). The peptides were selected by comparing the predicted amino acid sequences of three pepper and four potato PVY isolates on the basis of their polymorphism and hydrophilicity. Sera with high titres were only obtained against P19. Three MAbs, raised in response to P19, reacted with the homologous virus (PVY‐P21) in TAS‐ELISA. When tested against a broad range of PVY isolates and related viruses, MAb 3C5 proved to be PVY species specific, whereas MAbs 8A4 and 1D6 reacted specifically with standard isolates of PVYO, PVYC and PVYN‐W strains, but not with other PVY isolates. Consequently, epitope(s) recognized by 8A4 and 1D6 MAbs may be specific to a PVY group comprising all serologically PVYnon–N isolates. Surprisingly, and unlike isolate PVY‐P21, many Tunisian field pepper isolates did not carry this epitope(s), thus revealing serological heterogeneity within the PVY pepper group. As PVY is one of the most economically important plant pathogens in a range of crops, including pepper, these MAbs will provide a useful tool for practical diagnosis and strain identification of PVY.
Thick root of cucumber: other susceptible plants and the effect of pH
Authors: Van Der Gaag D. J. , Paternotte P. , Hamelink R.
Published: March 2003

Thick root is a relatively new disorder of cucumber grown in artificial substrates. Plants of cucumber, tomato, sweet pepper, lupin, anthurium, Cucurbita ficifolia, C. maxima and two lines from crosses between C. maxima and C. moschata were grown in thick root disease (TRD)‐infested nutrient solutions containing the TRD agent. Plants from each species or line, except anthurium, developed TRD symptoms. Growth of diseased plants, except those of the line C. maxima ×C. moschata RS841, was significantly reduced compared with the nondiseased controls. Two weeks after infestation, by adding TRD‐affected nutrient solution to fresh nutrient solution, the presence of the infective TRD agent was shown in each of the nutrient solutions in which the plants had been grown, including anthurium. No infective TRD agent could be shown in nutrient solution that had not been in contact with roots of living plants. The minimum pH for thick root formation was shown to be between 5·0 and 5·5. Nutrient solutions with pH values ranging from 4·0 to 6·5 and infested with the TRD agent lost their infectivity within 8 days. The infective period decreased with decreasing pH (pH 4·0–6·5) and a pH effect on the infectivity of a nutrient solution was shown within 1 h of infestation. The data show that the risk of TRD can be reduced by decreasing the pH of the nutrient solution.
Alterations of Protein Tyrosine Phosphorylation in T Cells of Immunocompromised Patients
Authors: Müller  , Patzke  , Bonmann  , Kiehl  , Koch
Published: March 2003

Activation of the T‐cell receptor (TCR) results in recruitment of tyrosine kinases and changes of tyrosine phosphorylation levels. We quantitatively analyzed protein phosphorylation of resting and TCR stimulated T cells for healthy donors and immunocompromised patients using two‐colour flow cytometry. Stimulation of T cells of healthy persons by OKT3 antibody led to a biphasic increase of phosphotyrosine levels with the first peak after 15 s and the absolute maximum occurring after 3–5 min. Levels remained high up to 30 min and returned to baseline levels afterwards. Compared to healthy blood donors, the phosphotyrosine baseline levels were 20–30% increased in patients after bone marrow transplantation (BMT). Using OKT3 to stimulate T cells of BMT patients led to strong increases in phosphotyrosine levels comparable to those of controls. In contrast, the response of T cells of human immunodeficiency virus‐infected patients with acquired immune deficiency syndrome was severely impaired (P = 0.01). In conclusion, this flow cytometric methodology enables analyses of changes in cellular phosphotyrosine levels following TCR stimulation. The increased baseline levels in BMT patients and the observed unresponsiveness of T cells in AIDS patients could be of interest for the study of predictors of graft‐versus‐host reactivity and the clinical analysis of immune functions in AIDS patients, respectively.
Ten‐Year Follow‐Up Study of Allergen‐Specific Immunoglobulin E and Immunoglobulin G4, Soluble Interleukin‐2 Receptor, Interleukin‐4, Soluble Intercellular Adhesion Molecule‐1 and Soluble Vascular Cell Adhesion Molecule‐1 in Serum of Patients on Immunotherapy for Perennial Allergic Rhinitis
Authors: Ohashi  , Nakai  , Tanaka  , Kakinoki  , Washio  , Kato  , Masamoto  , Sakamoto  , Yamada
Published: March 2003

Recent double‐blind placebo‐controlled trials for perennial allergic rhinitis have all clearly shown the efficacy of immunotherapy. Although several mechanisms for the clinical efficacy of immunotherapy have been proposed, the exact mechanisms related to the clinical effect still remain unclear. Since immunotherapy is a form of systemic treatment and its clinical benefit is likely to be, at least in part, a consequence of its systemic effects on different phases of immunological events, our study focused exclusively on several immunological parameters in serum. A total of 47 patients with perennial allergic rhinitis due to Dermatophagoides farinae enrolled in this prospective study. Venous blood was collected for determination of specific immunoglobulin (Ig)E, specific IgG4, soluble interleukin‐2 receptor (IL‐2R), interleukin‐4 (IL‐4), soluble intercellular adhesion molecule‐1 (ICAM‐1) and soluble vascular cell adhesion molecule‐1 (VCAM‐1), six times from 20 untreated patients and 27 patients on immunotherapy, at enrolment, and 1, 2, 3, 5, and 10 years after enrolment. No specific IgE, IgG4, soluble IL‐2R, IL‐4 and soluble ICAM‐1 levels changed significantly for a span of 10 years in the untreated patients. By contrast, immunotherapy affected serum levels of specific IgE, specific IgG4, soluble IL‐2R, IL‐4 and soluble ICAM‐1, but not of soluble VCAM‐1. The rates of increase in specific IgG4 and the rates of decrease in soluble IL‐2R were correlated with the rates of decrease in symptom scores during the first 3 years, but not 5 and 10 years after the course of immunotherapy. On the other hand, the rates of decrease in specific IgE, IL‐4 and soluble ICAM‐1 were significantly correlated with the rates of decrease in symptom scores at 5 and 10 years, but not during the first 3 years. Each immunological modulation by immunotherapy was likely to be involved in the working mechanism related to clinical efficacy at different phases of immunotherapy.
Isoforms of Murine and Human Serum Amyloid P Component
Authors: Nybo  , Hackler  , Kold  , Holm Nielsen  , Steinmetz  , Svehag
Published: March 2003

Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain‐dependent isoform pattern with up to seven bands (pI 5.1–5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not affect their number. When the acute‐phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H/HeN mice showed one more acidic isoform and an increase in the concentration of the most basic isoform. C57BL/6 J mice exhibited two to three new isoforms during the acute‐phase response. This appears to be the first demonstration of the physiological existence of SAP isoforms. In contrast, demonstration of isoforms of human SAP required the presence of urea and higher SAP concentrations. IEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7–5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase treatment caused a shift of the isoforms, but no reduction in isoform number. Two‐dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.
Infective Dose Modulates the Balance between Th1‐ and Th2‐Regulated Immune Responses during Blood‐Stage Malaria Infection
Authors: TAYLOR‐ROBINSON  , PHILLIPS
Published: March 2003

Plasmodium chabaudi infection of mice provides an excellent model for examining acquired immunity to the blood‐borne stage of malaria infection. CD4+ T‐cell receptor (TCR) αβ‐bearing T lymphocytes play a critical role in mediating protection, ascribed to both T helper (Th) 1 and Th2 subsets. One factor that may influence the Th1/Th2 cell balance is infective dose. In this study, we found that the size of the infective dose of P. chabaudi, and thus the level of antigen presented to the immune system, correlated with the balance of responder CD4+ T‐cell phenotypes. Increasing the infective dose in a resistant mouse strain enhanced the Th1 cytokine (interferon‐γ; IFN‐γ) response and reduced the Th2 cytokine (interleukin‐4; IL‐4) response. In contrast, increasing the infective dose in a susceptible mouse strain led to a prominent and accelerated up‐regulation of IL‐4 production. These data show that the dose of antigen can significantly affect the balance between Th1‐ and Th2‐mediated immune functions during infection of the mammalian host with blood‐stage malaria parasites. This demonstration that parasite numbers may modulate CD4+ T‐cell regulation has novel implications for the successful implementation of antimalarial vaccination and chemotherapeutic strategies.
Mapping of Antigenic Determinants of Purified, Lipid‐Free Human Serum Amyloid A Proteins
Authors: MALLE  , HERZ  , ARTL  , IBOVNIK  , ANDREAE  , SATTLER
Published: March 2003

Serum amyloid A (SAA) is the major apolipoprotein of high‐density lipoproteins (HDL) present during the acute‐phase reaction. To map specific epitopes on purified, lipid‐free SAA, sequence‐specific antibodies raised against synthetic peptides corresponding to amino acid residues 1–17, 14–30, 27–44, 40–63, 59–72, 68–84, 79–94 and 89–104 of human SAA1 were studied. Using the indirect sandwich dissociation‐enhanced lanthanide fluorescence immunoassay, antibodies raised against epitopes comprising residues 1–17, 14–30, 40–63 and 79–94 failed to recognize the corresponding domains on isolated human SAA1/SAA2 or a mixture of both isoforms, indicating that these epitopes are masked, apparently because of specific folding and/or self‐aggregation (dimerization). The accessible antigenic determinants of isolated SAA are epitopes comprising residues 31–39, 64–78 and 95–104. The present findings indicate that: (i) the same epitopes are exposed, irrespective whether SAA is HDL‐associated or in its lipid‐free form and that (ii) monomeric and dimeric SAA co‐exist to a similar extent in the lipid‐free form, irrespective of whether conditions are non‐denaturating, denaturating, acidic or basic. From our studies it is proposed that isolated, purified SAA may serve as a reliable standard for quantification of HDL‐associated SAA and for mimicking the interaction of acute‐phase HDL particles with peripheral tissues in vitro.
Distribution of Alloreactivity Amongst the CD45 Isoforms of Circulating CD4 and CD8 T Lymphocytes
Authors: O'brien  , Kemeny
Published: March 2003

The expression of different isoforms of the CD45 surface molecule allows lymphocytes to be divided into two nonoverlapping categories, CD45RA and CD45RO. Previous studies of CD4 T cells have shown that responses to soluble antigens are present predominantly in the RO subset and to mitogens in the RA, alloreactivity being present in both subsets. Responses of CD8 T cells have not been investigated in such detail, nor have responses been compared to CD4 cells. Here we report the alloreactive responses of both CD45RA and RO subsets amongst both CD4 and CD8 T lymphocytes. Following isolation of CD4 and CD8 cells with immunomagnetic beads, CD45 subsets were separated by negative depletion using specific monoclonal antibodies. CD45RA populations were greater than 97% pure and CD45RO cells greater than 91%. One‐way primary mixed lymphocyte reactions were established using the purified responder cells with irradiated allogeneic peripheral blood mononuclear cells as stimulators; experiments were all repeated at least three times. In assays of CD4+ RA and RO subsets, reactivity was present in both isoforms, being consistently, but not significantly, greater amongst the RO subset. With CD8+ T cells, reactivity was also present in both isoforms, but was significantly greater in the CD45RA subset, with mean proliferation 2.5–3‐fold that of the CD45RO cells (P < 0.05).
Diabetes Induction in C57BL/6 Mice Reconstituted with Lymphocytes of Nonobese Diabetic <−> C57BL/6 Mouse Embryo Aggregation Chimeras
Authors: Colucci  , Bergman  , Lejon  , Holmberg
Published: March 2003

To determine whether the genetic background of the insulin‐producing β cells of the pancreas contributes to autoimmune diabetes susceptibility, we have used a model of the disease based on tranferring spleen cells from nonobese diabetic (NOD) <−> C57BL/6 (B6) embryo aggregation (EA) chimeras into B6 and NOD irradiated mice. Insulitis and diabetes could be induced into both B6 and NOD hosts, albeit with low incidence. Cyclophosphamide (CY) treatment, known to accelerate diabetes in prediabetic NOD mice, was found to increase diabetes incidence up to 50–60% in both B6 and NOD mice reconstituted with chimeric splenocytes, while diabetes did not occur in CY‐treated B6 mice reconstituted with B6 splenocytes. We conclude that the genetic make‐up of the target organ does not affect the final stage of the pathogenesis of insulin‐dependent diabetes mellitus.
CD40 Expression by Human Bronchial Epithelial Cells
Authors: GORMAND  , BRIERE  , PEYROL  , RACCURT  , DURAND  , AÏT‐YAHIA  , LEBECQUE  , BANCHEREAU  , PACHECO
Published: March 2003

CD40 is a 50‐kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40 L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40 L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40 L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)‐γ was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)‐α was the best cytokine able to up‐regulate CD40 in SKLU1 cells. A combination of IFN‐γ and TNF‐α was slightly more effective than the cytokine alone at up‐regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell‐surface‐antigen expression on either cell line. The production of cytokines such as interleukin (IL)‐6 and granulocyte macrophage‐colony stimulating factor (GM‐CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL‐6 and GM‐CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF‐α‐induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.
Preparation of IgG Subclass Allotypes from Polyclonal IgG
Authors: OXELIUS
Published: March 2003

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(‐n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion‐exchange and protein A–Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(‐n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(‐n) molecules is of special interest as no genetic marker has been found to identify this allotype.
Cytokine Profile in Coeliac Disease
Authors: LAHAT  , SHAPIRO  , KARBAN  , GERSTEIN  , KINARTY  , LERNER
Published: March 2003

Coeliac disease (CD), an inflammatory enteropathy, is believed to be caused by immune sensitivity to ingested gluten. T‐cell activation appears to be implicated in the disease although little is known regarding the role of T‐cell subsets, Th1/Th2, and the cytokines they secrete. Reverse transcription‐polymerase chain reaction was used to examine the mRNA expression of a wide profile of cytokines in intestinal and peripheral samples taken from active and inactive CD paediatric patients. Differential mRNA expression was observed for cytokines, between CD patients and controls, in both compartments. The percentage of samples expressing interleukin (IL)‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐β, IL‐10, IL‐1β, TNF‐α and transforming growth factor (TGF)‐β mRNA from active CD patients was higher than from controls. A prominent finding was the expression of both Th1 (IFN‐γ, IL‐2) and Th2 (IL‐4, IL‐10)‐associated cytokine transcripts in the same biopsies and peripheral blood cells from patients with active CD implying activation of Th0 cells. The expression of IL‐2 and IL‐4 mRNA was not observed in peripheral blood samples from inactive CD patients associating them with disease activity. These results are important to the understanding of the inflammatory process in CD while cytokine levels may prove to be relevant markers of disease activity.
Interferon‐γ‐ and Tumour Necrosis Factor‐α‐Producing Cells in Humans who are Immune to Cutaneous Leishmaniasis
Authors: KEMP  , THEANDER  , HVIID  , GARFAR  , KHARAZMI  , KEMP
Published: March 2003

Individuals infected with Leishmania major usually acquire immunity to cutaneous leishmaniasis. In this study we have investigated peripheral blood mononuclear cells (PBMC) stimulated by Leishmania antigens in two groups of Sudanese individuals, one with a history of cutaneous leishmaniasis and one living in an area without the disease. The production of interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 was investigated in culture supernatants, and the cellular sources of IFN‐γ and TNF‐α were identified. Cells from individuals with a history of cutaneous leishmaniasis produced significantly higher levels of IFN‐γ and TNF‐α than cells from individuals without a history of the disease. Similar levels of IL‐10 were found in the two groups. Flow cytometric analysis revealed high numbers of CD3+ cells producing IFN‐γ and TNF‐α, and only a few CD3+ cells containing IL‐10, in the PBMC cultures from the individuals with a history of cutaneous leishmaniasis. Interferon‐γ and TNF‐α were predominantly produced by CD4+ T cells rather than CD8+ T cells. The results suggest that cellular immunity against cutaneous leishmaniasis is mediated predominantly through antigen‐specific CD4+ T cells in individuals with a history of cutaneous leishmaniasis.