bluuebunny/crossref_metadata_2025 huggingface dataset:
Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L.
Authors: Jetter R. , Schäffer S. , Riederer M.
Published: March 2003

The composition and spatial arrangement of cuticular waxes on the leaves of Prunus laurocerasus were investigated. In the wax mixture, the triterpenoids ursolic acid and oleanolic acid as well as alkanes, fatty acids, aldehydes, primary alcohols and alcohol acetates were identified. The surface extraction of upper and lower leaf surfaces yielded 280 mg m−2 and 830 mg m−2, respectively. Protocols for the mechanical removal of waxes from the outermost layers of the cuticle were devised and evaluated. With the most selective of these methods, 130 mg m−2 of cuticular waxes could be removed from the adaxial surface before a sharp, physically resistant boundary was reached. Compounds thus obtained are interpreted as ‘epicuticular waxes’ with respect to their localization in a distinct layer on the surface of the cutin matrix. The epicuticular wax film can be transferred onto glass and visualized by scanning electron microscopy. Prunus laurocerasus epicuticular waxes consisted entirely of aliphatic compounds, whereas the remaining intracuticular waxes comprised 63% of triterpenoids. The ecological relevance of this layered structure for recognition by phytotrophic fungi and herbivorous insects that probe the surface composition for sign stimuli is discussed.
Physiological and photosynthetic plasticity in the amphibious, freshwater plant,Littorella uniflora, during the transition from aquatic to dry terrestrial environments
Authors: Robe W. E. , Griffiths H.
Published: March 2003

The physiological and photosynthetic responses ofLittorella uniflora(L.) Ascherson, an amphibious macrophyte of isoetid life form, to rapid and prolonged emersion onto dry land, was studied at a reservoir. Water relations were little affected in the short term, but declining water potential and turgor pressure indicated water stress after flowering. High leaf lacunal CO2concentrations suggested continued CO2uptake from sediments. In contrast, a switch from Crassulacean acid metabolism (CAM) to C3photosynthesis was indicated by much lower levels of ΔH+(down minus dusk titratable acidity) and phosphoenolpyruvate carboxylase (PEPC) activity in new terrestrial leaves, 7–8‐fold higher activity of ribulose bisphosphate carboxylase oxygenase (Rubisco), and increased chlorophyll and soluble protein contents. Accumulated nitrate and amino acid pools were depleted, whereas storage of carbohydrates as soluble sugars, fructan and starch increased. Plant carbon and nitrogen isotope ratios (δ13C and δ15N) declined, perhaps reflecting changes in C fixation processes, N metabolism, and source C and N. In leaves of plants grown half‐emersed for an extended period, contrasting activities of PEPC and Rubisco were found in submersed and emersed portions. Overall,L. uniflorashowed considerable phenotypic plasticity, yet seemed to remain poised for re‐submersion; these characteristics could be adaptive in the unpredictable water margin habitat.
Strategies providing success in a variable habitat: II. Ecophysiology of photosynthesis of Cladophora glomerata
Authors: Ensminger I. , Hagen C. , Braune W.
Published: March 2003

Cladophora glomerata (L.) Kütz. is the dominant filamentous algae of the river Ilm, Thuringia, Germany. For most of the year it can be found at open as well as at shaded sites. Photosynthetic acclimation of C. glomerata to different light intensities was detected by chlorophyll fluorescence measurements and pigment analysis. Cladophora glomerata from highlight sites showed decreased values of efficiency of open photosystem II (Fv/Fm) as compared with C. glomerata from low‐light sites. Winter populations revealed higher Fv/Fm values than summer populations. A light‐induced decrease in efficiency of the closed photosystem II was observed at increasing irradiance intensities. The decrease was higher in C. glomerata from shaded sites compared with plants from open sites. Differences in the photosynthetic electron transport rate of different populations of C. glomerata were shown by photosynthesis–irradiance curves. Summer populations from high‐light sites yielded higher maximum electron transport rates than plants from low‐light sites, whereas winter populations exhibited significantly decreased values compared with the summer populations. Results of the analysis of photosynthetic pigments corresponded with data from chlorophyll fluorescence measurements. In addition to these long‐term acclimation effects, C. glomerata expressed its ability to cope with rapid changes in the light environment by the de‐epoxidation of violaxanthin during exposure to high light intensities.
The induction of inorganic carbon transport and external carbonic anhydrase in Chlamydomonas reinhardtii is regulated by external CO2 concentration
Authors: Bozzo G. G. , Colman B.
Published: March 2003

Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO2‐grown Chlamydomonas reinhardtii 2137 mt + cells to well‐defined dissolved inorganic carbon (Ci) limited conditions. The CCM components investigated were active HCO3− transport, active CO2 transport and extracellular carbonic anhydrase (CAext) activity. The CAext activity increased 10‐fold within 6 h of acclimation to 0·035% CO2 and there was a further slight increase over the next 18 h. The CAext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO2 and HCO3− uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO2 transport was induced prior to active HCO3− transport. Similar results were obtained during acclimation to air in darkness. The critical Ci concentrations effecting the induction of active Ci transport and CAext activity were determined by allowing cells to acclimate to various inflow CO2 concentrations in the range 0·035–0·84% at constant pH. The total Ci concentration eliciting the induction and repression of active Ci transport was higher during acclimation at pH 7·5 than at pH 5·5, but the external CO2 concentration was the same at both pHs of acclimation. The concentration of external CO2 required for the full induction and repression of Ci transport and CAext activity were 10 and 100 μM, respectively. The induction of CAext and active Ci transport are not correlated temporally, but are regulated by the same critical CO2 concentration in the medium.
Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV‐A and UV‐B radiation in developing rye primary leaves as assessed by ultraviolet‐induced chlorophyll fluorescence measurements
Authors: Burchard P. , Bilger W. , Weissenböck G.
Published: March 2003

Epidermally located ultraviolet (UV)‐absorbing phenolic compounds, flavonoids and hydroxycinnamic acid esters (HCAs), can shield the underlying tissues in plants against harmful UV‐radiation. The relative importance of the two different classes of phenolic compounds for UV‐screening was a matter of recent debate. Using a non‐invasive method based on chlorophyll fluorescence measurements to estimate epidermal UV transmittance, the relationship between epidermal UV shielding and the content of the two different groups of secondary phenolic compounds in the epidermal layers and the underlying photosynthetic mesophyll of developing rye primary leaves grown under supplementary UV‐B radiation was investigated. From the fourth to the tenth day after sowing, epidermally located flavonoids increased in an age‐ and irradiation‐dependent manner, whereas mesophyll flavonoids and epidermal HCAs, mainly ferulic acid and p‐coumaric acid esters, were constitutively present and did not vary in their contents over the observed time period. There was an excellent correlation between epidermal UV‐A and UV‐B absorbances as assessed by chlorophyll fluorescence measurements and contents of epidermal flavonoids. However, HCAs showed an additional contribution to UV‐B shielding. In contrast, mesophyll flavonoids did not seem to play a respective role. When absorbances of the abaxial and adaxial epidermal layers were compared, it became apparent that in fully expanded primary leaves epidermal tissues from both sides were equally effective in absorption of UV‐radiation. However, the earlier and more UV‐exposed abaxial epidermis of young unrolling leaves showed a significantly higher absorption. It is shown that in early stages of development the epidermal HCAs are the dominant UV‐B protective compounds of the primary leaf. This function is increasingly replaced by the epidermal flavonoids during leaf development and acclimation. The application of chlorophyll fluorescence measurements has been proven to be a useful tool for estimating relative contents of these compounds in epidermal tissue.
Effects of photoinhibition on whole‐plant carbon gain assessed with a photosynthesis model
Authors: Werner C. , Ryel R. J. , Correia O. , Beyschlag W.
Published: March 2003

A canopy photosynthesis model was modified to assess the effect of photoinhibition on whole‐plant carbon gain. Photoinhibitory changes in maximum quantum yield of photosystem II (Fv/Fm) could be explained solely from a parameter (Lflux) calculated from the light micro‐environment of the leaves. This relationship between Fv/Fm and the intercepted cumulative light dose, integrated and equally weighted over several hours was incorporated into the model. The effect of photoinhibition on net photosynthesis was described through relationships between photoinhibition and the shaping parameters of the photosynthetic light‐response curve (quantum use efficiency, convexity, and maximum capacity). This new aspect of the model was then validated by comparing measured field data (diurnal courses of Fv/Fm) with simulation results. Sensitivity analyses revealed that the extent of photoinhibitory reduction of whole‐plant photosynthesis was strongly dependent on the structural parameters (LAI and leaf angle). Simulations for a Mediterranean evergreen oak, Quercus coccifera, under climatic conditions which cause mild photoinhibition revealed a daily loss of 7·5–8·5% of potential carbon gain in the upper sunlit canopy layers, a 3% loss in the bottom canopy, and an overall loss of 6·1%. Thus, this canopy photoinhibition model (CANO‐PI) allows the quantitative evaluation of photoinhibition effects on primary production.
Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum
Authors: Matsuda Y. , Hara T. , Colman B.
Published: March 2003

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO2 to air and related to H2CO3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K0·5[DIC]) value in high CO2‐grown cells was 1009 mmol m−3 but was reduced three‐fold by the addition of bovine carbonic anhydrase (CA), whereas in air‐grown cells K0·5[DIC] was 71 mmol m−3, irrespective of the presence of CA. The maximum rate of photosynthesis (Pmax) values varied between 300 and 500 μmol O2 mg Chl−1 h−1 regardless of growth pCO2. Bicarbonate dehydration kinetics in artificial seawater were re‐examined to evaluate the direct HCO3− uptake as a substrate for photosynthesis. The uncatalysed CO2 formation rate in artificial seawater of 31·65°/oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m−3 min−1 at 100 mmol m−3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air‐ and high CO2‐grown cells, respectively. These data indicate that even high CO2‐grown cells of P. tricornutum can take up both CO2 and HCO3− as substrates for photosynthesis and HCO3− use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO2‐grown cells to air. The development of high‐affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO2‐grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K0·5[DIC] values in acclimated cells appeared to be correlated only with [CO2(aq)] in the medium but not to HCO3−, CO32−, total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO2(aq)] in the medium.
Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble
Authors: Davoine C. , Le Deunff E. , Ledger N. , Avice J.‐C. , Billard J.‐P. , Dumas B. , Huault C.
Published: March 2003

Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H2O2 were investigated during the ageing of leaf sheaths of ryegrass (Lolium perenne L.) stubble. The accumulation of H2O2 during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca‐oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Ca‐oxalate and OxO, the production of H2O2 and Ca2+ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass.
Changes in synthesis and degradation of Rubisco and LHCII with leaf age in rice (Oryza sativa L.) growing under supplementary UV‐B radiation
Authors: Takeuchi A. , Yamaguchi T. , Hidema J. , Strid A. , Kumagai T.
Published: March 2003

The effects of supplementary ultraviolet‐B (UV‐B) radiation on the changes in synthesis and degradation of ribulose ‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and light‐harvesting chlorophyll a/b binding protein of PSII (LHCII) were examined, as well as mRNA levels for small and large subunits of Rubisco (rbcS and rbcL, respectively) and LHCII (cab) with leaf age in UV‐sensitive rice (Norin 1) and UV‐resistant rice (Sasanishiki). Both Rubisco and LHCII were actively synthesized until the leaf had fully expanded, and then decreased with increasing leaf age. Synthesis of Rubisco, but not LHCII, was significantly suppressed by UV‐B in Norin 1. The degradation of Rubisco was enhanced by UV‐B around the time of leaf maturation in the two cultivars. The levels of rbcS and rbcL were reduced by UV‐B at the early stages after leaf emergence in both cultivars. Cab transcripts were first present at high levels in the two cultivars, but drastically decreased due to UV‐B treatment immediately after leaf emergence in Norin 1. It was shown that synthesis and degradation of Rubisco and LHCII greatly changed with leaf age: Rubisco synthesis was significantly suppressed by supplementary UV‐B radiation at the transcription step during the early leaf stages. It was also suggested that the difference in UV‐B sensitivity in Rubisco synthesis between the two rice cultivars might be due to specific suppression both transcriptionally and post‐transcriptionally.
A hydrothermal time model explains the cardinal temperatures for seed germination
Authors: Alvarado V. , Bradford K. J.
Published: March 2003

Temperature (T) and water potential (y) are two primary environmental regulators of seed germination. Seeds exhibit a base or minimum T for germination (Tb), an optimum T at which germination is most rapid (To), and a maximum or ceiling T at which germination is prevented (Tc). Germination at suboptimal T can be characterized on the basis of thermal time, or the T in excess of Tb multiplied by the time to a given germination percentage (tg). Similarly, germination at reduced y can be characterized on a hydrotime basis, or tg multiplied by the y in excess of a base or threshold y that just prevents germination (yb). Within a seed population, the variation in thermal times to germination among different seed fractions (g) is based on a normal distribution of yb values among seeds (yb(g)). Germination responses across a range of suboptimal T and y can be described by a general hydrothermal time model that combines the T and y components, but this model does not account for the decrease in germination rates and percentages when T exceeds To. We report here that supra‐optimal temperatures shift the ψb(g) distribution of a potato (Solanum tuberosum L.) seed population to more positive values, explaining why both germination rates and percentages are reduced as T increases above To. A modified hydrothermal time model incorporating changes in ψb(g) at T > To describes germination timing and percentage across all T and ψ at which germination can occur and provides physiologically relevant indices of seed behaviour.
Components of quantitative resistance to powdery mildew (Erysiphe pisi) in pea (Pisum sativum)
Authors: Viljanen‐Rollinson S. L. H. , Gaunt R. E. , Frampton C. M. A. , Falloon R. E. , McNeil D. L.
Published: March 2003

Components of quantitative resistance in pea (Pisum sativum) to Erysiphe pisi, the pathogen causing powdery mildew, were investigated. Conidium germination, infection efficiency, latent period and conidium production dynamics on cv. Quantum (quantitatively resistant) were compared with those on Pania and Bolero (susceptible). There was an additional comparison in conidium germination experiments with the resistant cv. Resal. Quantitative resistance in Quantum did not affect conidium germination, but infection efficiency of conidia on this cultivar was 34% less than on the susceptible Pania. More conidia germinated on 5‐day‐old leaflets than on 15‐day‐old leaflets but the age of the plant did not affect percentage germination or infection efficiency. The length of the latent period did not differ between cultivars. Total conidium production (AUC) per unit leaflet area on Quantum was 25% less than on Pania. The maximum conidium production per day (CMAX) per unit leaflet area on Quantum was 33% less than on Pania. The time to maximum conidium production per day (TMAX) was 10% longer on Quantum than on Pania. The cv. Bolero, reported to be susceptible, also showed some degree of quantitative resistance, but this differed from that of Quantum. Total conidium production was less on Bolero than on Quantum, but the conidia on Bolero were produced sooner, and for a shorter period, than on Quantum. The stability of these responses was tested by analysing components in three different temperature regimes and testing for interactions with temperature, and with leaflet age. Temperature affected all conidium production variables. AUC per leaflet area was nearly seven times as great and CMAX nearly 15 times greater at 23°C than at 13°C. TMAX increased by 1.5 times when temperature increased from 13°C to 18°C or 23°C. Several interactions occurred and these are described.
Factors affecting the control of Pythium ultimum damping‐off of sugar beet by Pythium oligandrum
Authors: Holmes K. A. , Nayagam S. D. , Craig G. D.
Published: March 2003

Application of Pythium oligandrum to a soil‐based compost as a mycelial suspension (5 × 102 CFU g−1 of dry compost) and oospore alginate pellets (105 oospores/g of dry compost) controlled pre‐ and postemergence damping‐off of sugar beet caused by Pythium ultimum to a level similar to metalaxyl seed treatment. Oospore seed treatments and aqueous suspensions of oospores applied to compost failed to control disease. Problems in the use of P. oligandrum oospore inocula for the control of damping‐off were highlighted. It was shown that treatment of oospores with cellulase (20 g L−1) increased germination approximately three‐fold in comparison to untreated spores. Untreated and cellulase pretreated oospores were subsequently evaluated as seed treatments for their ability to control damping‐off of sugar beet. The highest rate of pretreated oospores (104 oospores/seed) gave levels of emergence and establishment in infested compost that were not significantly different from the uninfested controls, whereas seed treatment with untreated oospores gave no significant reduction in disease. In a trial carried out in a controlled environment to assess the effect of pH (4.5–8.0), P. oligandrum (104 cellulase pretreated oospores/seed) was shown to control pre‐ and postemergence damping‐off of sugar beet at pH 7.0 and 7.5 only.
Spatio‐temporal development of black rot (X. campestris pv. campestris) in cabbage in relation to initial inoculum levels in field plots in The Netherlands
Authors: Kocks  , Zadoks  , Ruissen
Published: March 2003

The effects of initial inoculum levels on spatio‐temporal development in black rot of cabbage from artificial sources, one per plot, were evaluated in replicated field experiments. The results support the hypothesis that black rot is a polycyclic disease. Its development was inoculum‐dependent because the progress rate of epidemics and spatial spread were both positively correlated with the strength of the source. Fast disease development was related to the number of rain days. The spread of black rot, associated with the primary gradient, at first resulted from allo‐infection, later followed by a phase during which allo‐infection was complemented by auto‐infection. Three‐dimensional maps of disease severity showed the dominance of the primary focus. Maximum distance of black rot symptoms from the source of the focus was limited to a few metres so that damage to cabbage by focal inoculum was limited to the plants close to the source. Spatio‐temporal development and initial inoculum were related. High inoculum levels in point sources resulted in faster outward spread of black rot, and differences between low and high levels were generally significant. Under the conditions of the experiments, performed during three relatively dry seasons, a single source of infection measuring 0.5 × 0.5 m was not capable of spreading disease over all the plants in a plot of 6.5 × 6.5 m. The results imply that severe disease where whole crops are infected, as observed regularly in The Netherlands, can only originate from a large number of small foci per field.
Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis
Authors: Van Zyl  , Wingfield  , Alfenas  , Crous
Published: March 2003

A single 3 kb segment of double‐stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis. These dsRNA‐containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA‐free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence‐associated traits as those of the original dsRNA‐containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis.
Genetic control of resistance to fenpropimorph in Ustilago maydis
Authors: Markoglou  , Ziogas
Published: March 2003

Fenpropimorph‐resistant mutants of Ustilago maydis were obtained at high frequency (30 × 10−6) after UV‐irradiation followed by selection on media containing fenpropimorph (50 μg mL−1). Genetic analysis of 30 such mutants resulted in the identification of two unlinked chromosomal loci, the U/fpm‐1 locus with two allelic genes (U/fpm‐1A and U/fpm‐1B) and the U/fpm‐2 locus. The mutant genes U/fpm‐1A and U/fpm‐2 are responsible for high resistance levels (Rf: 75–100 or 257–286 based on MICs or ED50s, respectively), while the U/fpm‐1B mutation gives only a small reduction (approximately 7–10‐fold) in fenpropimorph sensitivity. Cross‐resistance studies with other SBIs showed that the major gene (U/fpm‐1A and U/fpm‐2) mutants were cross‐resistant to the related compound fenpropidin (Rf: 15–20 or 53–66 based on MICs or ED50s values, respectively) and to tridemorph (Rf: 5 or 7.1–9.5 based on MICs or ED50s values, respectively), but not to the inhibitors of steps of ergosterol biosynthesis preceding the Δ14‐reductase. The minor gene (U/fpm‐1B) mutants also had low‐level resistance (approximately 5‐fold) to tridemorph and to fenpropidin, but in contrast with the major gene mutants they were 2–10 times more sensitive to the triazoles studied (triadimefon, triadimenol, propiconazole and flusilazole) and to the pyridine, pyrifenox.

Studies of the fitness of U. maydis mutants showed that in major gene mutants, resistance was not associated with changes in growth rate in liquid culture or pathogenicity on young maize plants. The minor gene mutation reduced significantly the growth rate in liquid culture and the pathogenicity, either in homozygous or heterozygous condition in dikaryotic mycelium.
Elimination of Citrus psorosis virus by somatic embryogenesis from stigma and style cultures
Authors: D'Onghia A. M. , Carimi F. , De Pasquale F. , Djelouah K. , Martelli G. P.
Published: March 2003

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich‐indirect‐enzyme‐linked immunosorbent assay (DASI‐ELISA) in explants and embryogenic callus, but was not detected in any of the plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.
Evaluation of sorghum germplasm used in US breeding programmes for sources of sugary disease resistance*
Authors: Dahlberg J. A. , Bandyopadhyay R. , Rooney W. L. , Odvody G. N. , Madera‐Torres P.
Published: March 2003

Ergot or sugary disease of sorghum has become an important constraint in North and South American countries that rely on F1 hybrid seeds for high productivity. The objective of this research was to determine the vulnerability of various germplasm sources and publicly bred sorghum lines to sugary disease (Claviceps africana) in the United States. Flower characteristics associated with sugary disease resistance were also studied. A‐/B‐line pairs, R‐lines, putative sources of resistance and their hybrid combinations with an A3 cytoplasmic male‐sterile source were evaluated using a disease incidence, severity, and dual‐ranking system. Trials were planted in a randomized complete block design with three replications and repeated in at least two planting dates. Planting dates and pedigrees had significant effects on overall ranking for resistance. A‐lines were most susceptible to sugary disease. R‐lines were more susceptible than B‐lines with respect to incidence and severity of the disease. Newer releases of A‐ and B‐lines were more susceptible to sugary disease than older releases. Sugary disease reaction of A‐lines was a good indicator of disease reaction of B‐lines. Tx2737, a popular R‐line, was highly susceptible to sugary disease in spite of being a good pollen shedder because the stigma emerged from glumes 2–3 days before anthesis. The combination of flower characteristics associated with resistance were least exposure time of stigma to inoculum before pollination, rapid stigma drying after pollination, and small stigma. An Ethiopian male‐fertile germplasm accession, IS 8525, had good levels of resistance. Its A3 male‐sterile hybrid had the highest level of resistance in the male‐sterile background. IS 8525 should be exploited in host‐plant resistance strategies.
Sporulation ofPyrenopeziza brassicae(light leaf spot) on oilseed rape (Brassica napus) leaves inoculated with ascospores or conidia at different temperatures and wetness durations
Authors: Karolewski Z. , Evans N. , Fitt B. D. L. , Todd A. D. , Baierl A.
Published: March 2003

In controlled environment experiments, sporulation ofPyrenopeziza brassicaewas observed on leaves of oilseed rape inoculated with ascospores or conidia at temperatures from 8 to 20°C at all leaf wetness durations from 6 to 72 h, except after 6 h leaf wetness duration at 8°C. The shortest times from inoculation to first observed sporulation (l0), for both ascospore and conidial inoculum, were 11–12 days at 16°C after 48 h wetness duration. For both ascospore and conidial inoculum (48 h wetness duration), the number of conidia produced per cm2leaf area with sporulation was seven to eight times less at 20°C than at 8, 12 or 16°C. Values of Gompertz parametersc(maximum percentage leaf area with sporulation),r(maximum rate of increase in percentage leaf area with sporulation) andl37(days from inoculation to 37% of maximum sporulation), estimated by fitting the equation to the observed data, were linearly related to values predicted by inserting temperature and wetness duration treatment values into existing equations. The observed data were fitted better by logistic equations than by Gompertz equations (which overestimated at low temperatures). For both ascospore and conidial inoculum, the latent period derived from the logistic equation (days from inoculation to 50% of maximum sporulation,l50) ofP. brassicaewas generally shortest at 16°C, and increased as temperature increased to 20°C or decreased to 8°C. Minimum numbers of spores needed to produce sporulation on leaves were ≈25 ascospores per leaf and ≈700 conidia per leaf, at 16°C after 48 h leaf wetness duration.
Allotype‐Associated Differences in Concentrations of Human IgA2
Authors: Mattila  , Seppälä  , Sarvas  , Mäkelä
Published: March 2003

Concentrations of immunoglobulin (Ig)A2 were determined in 176 Finnish blood donor sera. Their IgA, IgM, IgE, IgG1, IgG2, IgG3 and IgG4 concentrations and Gm allotypes had been determined earlier. The mean concentration of IgA2 was higher in individuals carrying the Gm(ax) allele (0.15 g/l) than in those negative for Gm(x) (0.103 g/l). The difference was statistically significant (P = 0.006). As ≥ 70% of IgA was usually IgA1, its concentration could be calculated fairly reliably by subtracting the IgA2 value from the IgA value. The mean IgA1 concentration (2.03 g/l) seemed to be independent of the Gm allotypes.
Down‐Regulation of Lymphokine Synthesis by Intravenous Gammaglobulin is Dependent Upon Accessory Cells
Authors: Skansén‐Saphir  , Andersson  , Björk  , Ekberg  , Fehniger  , Henter  , Andersson
Published: March 2003

We have investigated one mechanism by which pooled human IgG preparations for intravenous use (IVIg) selectively down‐regulates lymphokine synthesis. Effects of IVIg on cytokine production were quantified at a cellular level by using an immunocytochemical staining technique. Pure T‐lymphocyte preparations (from the peripheral blood of healthy adults) were separated by the use of magnetic beads and were then used in parallel experiments with unfractionated mononuclear cells (MNC). Cell activation was induced either by a combination of the protein kinase C activator, phorbol 12‐myristate 13‐acetate (PMA), and the calcium ionophore, ionomycin, or by direct ligation of the T‐cell receptor, using immobilized anti‐CD3 monoclonal antibody (MoAb). Cells were cultured in the presence or absence of IVIg and subsequently harvested and stained for the following cytokines: interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ), tumour necrosis factor‐β (TNF‐β) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Assessment of the frequencies of positively stained cells was performed by manual microscopy and by computerized image analysis. Activation by PMA/ionomycin or by immobilized anti‐CD3 MoAb induced substantial lymphokine production in both MNC and in purified T cells. Addition of IVIg led to a diminished synthesis of all of the T‐cell products studied in unfractionated MNC preparations, whereas production was maintained or occasionally increased in the purified T‐cell preparations. These findings indicate that the immunomodulatory effect by IVIg on T‐cell activation and lymphokine production was dependent on accessory cells.
The Effects of Leflunomide and Cyclosporin A on Rejection of Cardiac Allografts in the Rat
Authors: Östraat  , Qi  , Tufveson  , Hedlund  , Ekberg
Published: March 2003

Leflunomide is a new low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate‐dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and cyclosporin A (CsA) are different, we postulated a synergistic effect of the two drugs and tested graft survival following leflunomide administration alone or in combination with CsA in a rat cardiac transplantation model. Low‐ and high‐responder rat strain combinations were used in parallel and the experiments were performed both with and without challenge with Linomide, an immunomodulator which promotes graft rejection in this model. In the low‐responder rat strain combination (Piebald Virol Glaxo graft to Dark Agouti recipient; PVG to DA), graft survival appeared to be a dichotomous variable, being characterized by tolerance or early rejection. Leflunomide (10 or 5 mg/kg) given for 10 days induced tolerance and CsA did likewise; the addition of Linomide abolished the immunosuppressive effect of leflunomide but not that of CsA. In the high‐responder combination (DA to PVG), no tolerance was seen and graft survival was moderately prolonged both after leflunomide and after CsA treatment; the addition of Linomide to CsA or to leflunomide (5 mg/kg) abolished the immunosuppressive effect of the drugs. However, when CsA–Linomide or leflunomide–Linomide were supplemented with the second immunosuppressive drug, leflunomide or CsA respectively, graft survival was significantly prolonged (P < 0.001 in both cases). This suggests leflunomide and CsA have additive potential.
Endogenous Interleukin‐4 Does Not Suppress the Resistance Against a Primary or a Secondary Listeria monocytogenes Infection in Mice
Authors: Samsom  , Annema  , Langermans  , Van Furth
Published: March 2003

Interleukin‐4 (IL‐4), a cytokine produced by T‐helper 2 (Th2) cells, can inhibit the development of T‐helper 1 (Th1) cells, which results in a decreased release of cytokines by the latter. As interferon‐γ (IFN‐γ), produced by Th1 cells, is involved in the resistance against a Listeria monocytogenes infection, the role of endogenously formed IL‐4 during a Listeria infection in mice was investigated. Neutralization of endogenously formed cytokines by subcutaneously injected alginate‐encapsulated monoclonal antibody (MoAb)‐forming cells results in high antibody titres in the circulation over a long time period. The aim of the present study was to re‐evaluate the effect of neutralization of IL‐4 during a primary Listeria infection and to investigate the role of IL‐4 during a secondary infection in mice using encapsulated MoAb‐forming cells. During the course of a primary infection in mice given anti‐IL‐4 antibody‐forming cells (anti‐IL‐4‐FC), the number of Listeria found in the liver and spleen was comparable to that found in control mice given anti‐β‐galactosidase antibody‐forming cells (anti‐β‐gal‐FC). Activation of macrophages measured by inhibition of Toxoplasma gondii proliferation and the release of reactive nitrogen intermediates (RNI) was not affected by anti‐IL‐4‐FC treatment during infection. Furthermore, during a secondary L. monocytogenes infection the number of bacteria in the liver and spleen of anti‐IL‐4‐treated immune mice was comparable to anti‐β‐gal‐FC‐treated, control, immune mice. The concentration of IFN‐γ in plasma of anti‐IL‐4‐treated immune mice was similar to that of control immune mice. Taken together, these findings demonstrate that neutralization of endogenously formed IL‐4 does not affect resistance to a primary or a secondary L. monocytogenes infection in mice.
Induction of Gut Mucosal Immune Responses: Importance of Genetic Background and Th1/Th2 Cross‐Regulation
Authors: Kjerrulf  , Grdic  , Kopf  , Lycke
Published: March 2003

The reciprocal regulation of T‐helper cell (Th) subsets is widely documented in various animal models of infectious diseases. In this study IFN‐γ/IL‐4 double knockout (DKO) mice were used to analyse the role of Th subsets in mucosal immune responses. We found that the DKO mice had normal IgA differentiation but impaired induction of specific gut mucosal antibody responses after oral immunization using cholera toxin adjuvant. Both Th1 and Th2 responses were reduced compared with wild‐type mice. Despite the absence of both IFN‐γ and IL‐4 in the DKO mice the overall results were similar to previous observations in IFN‐γ receptor‐knockout (IFN‐γR−/−) mice and did not suggest a strict cross‐regulation of the two Th subsets in the gut mucosa. To further examine the role of IFN‐γ in mucosal immunity we compared two different mouse strains lacking IFN‐γ, i.e. IFN‐γ−/− (C57BL/6) and IFN‐γR−/− mice (129/Sv). We found that IFN‐γR−/− mice exhibited reduced mucosal antibody responses and decreased Th1 and Th2 activity after oral immunization, while IFN‐γ−/− mice had intact antibody responses and increased Th2 responses. Thus, genetic differences were found to critically affect the development of a specific gut mucosal immune response. An enhanced Th2 activity in the Peyer's patches following oral immunization was associated with an ability to mount strong intestinal IgA immunity.
Immunological Effects of a Hyperinsulinaemic Euglycaemic Insulin Clamp in Healthy Males
Authors: Kappel  , Dela  , Barington  , Galbo  , Pedersen
Published: March 2003

The purpose of this study was to determine the in‐vivo and in‐vitro effects of insulin, at physiological and supraphysiological concentrations, on the human immune system. Ten healthy young men went through a sequential two‐step hyperinsulinaemic euglycaemic clamp. Plasma insulin concentrations were increased from baseline (9.0 μU/ml) to 49.1 μU/ml after 1 h of insulin infusion (step I) and to 1281 μU/ml (step II) after 2 h of infusion. As control experiments infusions of isotonic saline were performed. The unstimulated natural killer (NK) cell activity among blood mononuclear cells (BMNC) increased in response to supraphysiological plasma insulin levels (baseline versus step II: 20.6 ± 11.3 versus 27.8 ± 14.4%). The percentages of the CD16+ NK cells did not change, indicating an enhanced cytotoxic capability per individual NK cell. Insulin also slightly increased the activity of NK cells in vitro. A decline at step II in the concentrations of monocytes (0.29 ± 0.09 versus 0.12 ± 0.03 × 109/L), lymphocytes (1.57 ± 0.46 versus 1.22 ± 0.25 × 109/L), and CD16+ (24.2 ± 17.5 versus 16.7 ± 11.2 × 107/L), CD14+ (20.9 ± 10.8 versus 8.6 ± 3.9 × 107/L), HLA‐DR+ (37.2 ± 22.1 versus 19.2 ± 10.7 × 107/L) and CD45RO+ (91.6 ± 33.4 versus 61.7 ± 6.4 × 107/L) cells as well as in the percentages of CD14+ cells (11.2 ± 4.7 versus 6.4 ± 2.3%) and CD14+/HLA‐DR+ monocytes (9.7 ± 3.9 versus 4.8 ± 2.8%) were observed. No changes were found at step I. Hyperinsulinaemia did not change the percentages of the CD3+, CD4+, CD8+, CD19+, CD56+, CD11a+, CD45RO+ and CD45RA+ cells, the numbers of circulating immunoglobulin (Ig)G‐, IgA‐ and IgM‐ secreting cells, or the proliferative responses of BMNC to phytohaemagglutinin, purified derivative of tuberculin or interleukin (IL)‐2. Hyperinsulinaemia did not change the in‐vitro sensibility to insulin. In conclusion, supraphysiological insulin levels increased the activity of the individual NK cells, but decreased the numbers of NK cells, lymphocytes and activated monocytes. The findings are presumably of minor clinical relevance but may indicate an insulin‐induced immune activation.
Binding of Properdin to Solid‐Phase Immune Complexes: Critical Role of the Classical Activation Pathway of Complement
Authors: Junker  , Baatrup  , Svehag  , Wang  , Holmström  , Sturfelt  , Sjöholm
Published: March 2003

The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme‐linked immunosorbent assay using solid‐phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake was hardly detectable. Alternative pathway‐mediated deposition of C3 with slow kinetics was demonstrated in C2‐deficient serum and in NHS depleted of C1q, factor D and properdin (C1qDP‐depleted serum) after reconstitution with factor D and properdin. Efficient uptake of properdin required a functional classical pathway, in the presence of which C3 and properdin were rapidly deposited onto the IC. Judging from findings in C3‐deficient serum, factor I‐deficient serum, and C1qDPB‐depleted serum, the uptake of properdin was strictly C3‐dependent, and did not require the presence of factors B and D. Thus, C3b fixed to IC was the principal ligand for properdin in the assay. The findings could have biological implications relating to complement‐mediated modification of immune complexes in disease.
Catabolism of Lipid‐Free Recombinant Apolipoprotein Serum Amyloid A by Mouse Macrophages In Vitro Results in Removal of the Amyloid Fibril‐Forming Amino Terminus
Authors: Elliott‐Bryant  , Liang  , Sipe  , Cathcart
Published: March 2003

Serum amyloid A fibrils are formed when the normally rapid catabolism of the acute‐phase reactant apolipoprotein serum amyloid A (apoSAA) is incomplete; thus amyloidosis may be viewed as a condition of dysregulated proteolysis. There is evidence that apoSAA is dissociated from plasma high‐density lipoprotein (HDL) prior to fibril formation. The objective of this study was to investigate degradation of lipid‐free apoSAA by tissue macrophages derived from amyloid‐susceptible CBA/J mice in vitro. Peritoneal macrophages derived from untreated (normal) mice converted apoSAA (12 kDa) to a single 4 kDa C‐terminal peptide while splenic macrophages converted apoSAA to 10, 7 and 4 kDa C‐terminal peptides and a 4 kDa peptide that lacked the C‐ and N‐terminal regions. Similar patterns of proteolysis occurred when peritoneal and splenic macrophages from amyloidotic CBA/J mice were used. Conditioned medium prepared from peritoneal, but not splenic macrophages, degraded apoSAA. Specific sites of cleavage indicated activity of cathepsin G‐ and elastase‐like neutral proteases. The data indicate that lipid‐free apoSAA can be degraded by secreted or cell‐associated neutral proteases that are generated by macrophages to yield peptides that lack fibrillogenic potential.
Acute, Lethal Graft‐Versus‐Host Disease in an F1‐Hybrid Model using Grafts from Parental‐Strain, T‐Cell Receptor‐δ Gene Knockout Donors
Authors: Ellison  , Gartner
Published: March 2003

γδT cells have been implicated in the pathogenesis of acute graft‐versus‐host disease (GVHD). We therefore performed experiments to determine whether mortality from GVHD is reduced in C57BL/6 × DBA/2 F1‐hybrid (BDF1‐hybrid) mice when parental strain, T‐cell receptor‐δ (TCRδ) knockout (KO) donors are used. We compared mortality, weight loss, interferon‐γ (IFN‐γ) production and cytotoxic activity in recipients of either wild‐type or TCRδ KO grafts. In both groups there was significant weight loss and an identical level of mortality. Elevated IFN‐γ levels were present in both groups, but recipients of TCRδ KO grafts produced twice as much as recipients of wild‐type grafts. Elevated natural killer (NK) and NK‐like activity was also seen in both. These results demonstrate that TCRδ KO grafts can induce GVHD as severe as that seen in recipients of wild‐type grafts, a finding that is at odds with studies demonstrating reduced mortality when γδT cells are purged from donor mice. We suggest that the inconsistency may lie in the higher levels of IFN‐γ seen with TCRδ KO grafts and that the protection afforded by the absence of γδT cells in the graft is overwhelmed by the higher levels of IFN‐γ.
How does Cytopathicity Affect the Production of Neutralizing Antibody?
Authors: Keşmir  , De Boer
Published: March 2003

Cytopathic viruses evoke an earlier neutralizing antibody (nAb) response than noncytopathic viruses do. This was previously explained by the elimination of infected B cells by the cytotoxic T cells (CTLs), which predominate during infections with noncytopathic viruses. Using a simple mathematical model we provide a much simpler explanation for this difference in the kinetics of neutralizing antibody production. The analysis of the model shows that the delay in nAb production during infections with noncytopathic viruses is a simple consequence of the cytopathic effect alone: noncytopathic viruses infect a larger fraction of nAb‐producing B cells and as a result nAb response is delayed. Extending the model with CTLs, we find that a major effect of CTLs is to limit the antigenic stimulus of the nAb‐producing B cells. Thus, by reducing the proliferation rate of nAb‐producing B cells, CTLs further delay the production of neutralizing antibodies.
Heat Denaturation of Egg‐White Proteins Abrogates the Induction of Oral Tolerance of Specific Th2 Immune Responses in Mice
Authors: PENG  , CHANG  , TSAI  , SU  , SHEN  , CHANG
Published: March 2003

Human foods are usually prepared by cooking. Boiling of chicken egg‐white (EW) led to decreased allergenicity, and abrogated intestinal uptake of immunoreactive ovalbumin (OVA) when fed to mice. Therefore, the effects of oral administration of boiled EW were examined further in BALB/c mice. Specific IgE, IgG1 and IgG antibody responses were suppressed by raw EW, but not by EW boiled for 5 or 60 min, fed prior to sensitization with 10 μg OVA or 1 μg DNP–OVA in alum. Similar results were obtained when mice were sensitized with 10 μg conalbumin, ovomucoid or lysozyme in alum. BALB/c spleen cell proliferation and secretion of Th2 cytokines IL‐4 and IL‐5 during in vitro stimulation with OVA were also suppressed by feeding raw EW, but not by boiled EW. Although heat denaturation of proteins can minimize allergenicity, the present results suggest that over‐cooking of proteins may affect their intestinal antigen processing and thus prevent the induction of oral tolerance.
Suppression of Immunoglobulin Secretion by Soluble Annexin II
Authors: AARLI  , MATRE
Published: March 2003

Annexin II (AII) belongs to a family of glycoproteins that bind negatively charged phospholipids in the presence of calcium. The annexins exert various biological functions. We have previously shown that soluble AII suppresses mitogen‐induced lymphoproliferation in vitro. In this study we address the question of whether soluble AII may also affect immunoglobulin secretion. Mononuclear cells from peripheral blood were stimulated with pokeweed mitogen in vitro and immunoglobulin‐secreting cells were quantified using an ELISPOT assay. Retroplacental serum and soluble AII significantly inhibited secretion of IgG and IgM when added at concentrations that did not affect lymphoproliferation or cell viability. The inhibitory effect was dose‐ and time dependent. Significant suppression was observed when soluble AII was added at concentrations of 0.1 and 0.01 μg/ml. The strongest inhibition was observed when soluble AII or retroplacental serum was added initially. The data demonstrate that soluble AII can suppress immunoglobulin secretion in vitro. AII seems to be a potent immunosuppressive substance. The presence of high levels of soluble AII in retroplacental serum may indicate a possible immunomodulatory role in normal pregnancy.