Blood reactions in vitro in crayfish against a fungal parasite, Aphanomyces astaci

The immediate and subsequent reactions in crayfish blood upon making contact with hyphae of the crayfish plague fungus were observed in vitro. Blood cells agglomerated and clumped rapidly on the hyphae in streaming blood. Very brief contact by chance with a hypha, or with cell extensions from cells or a cell clump on the hypha, caused flowing cells to stop and adhere. Chemotaxis was not believed to be of any importance. Particles originating from hemocyte granules were specifically attached to, and “encapsulated” the distal portion of, the hypha (even without the presence of the cells themselves), where their content of polyphenol oxidase became activated. These processes may be connected with the defense mechanism in crayfish. Within a few hours the process of melanization could be observed on the hyphal surface. Both the enzyme(s) and the substrate(s) for the melanization reaction seem to originate from the blood cells. Dihydroxyphenylalanine (DOPA) could replace the natural substrate and greatly enhanced the process. The melanization in whole blood was much heavier in a crayfish resistant to the disease than in a susceptible one.     

Two-dimensional high-resolution electrophoresis of elastin-derived peptides

Degradation of human aortic elastin in vivo yields a restricted number of differentially sized and charged peptides. Elastin-derived peptides (EDP) can be analyzed by two-dimensional electrophoresis after their extraction from human abdominal aortic tissue by 0.2 M sodium chloride. The peptides were separated according to charge by acetic acid-urea-PAGE and then according to molecular mass by SDS-PAGE. The identity of these peptides as EDP was continued by immunoprecipitation and Western blots. The two-dimensional electrophoretic system can resolve desmosine-like cross-linked EDP of the similar molecular configuration but differing in the number of positively charged amino acid residues. The new separation technique of EDP has the capacity to identify defects in desmosine-like cross-links and may be useful in characterizing abberations in elastin structures.     

Antagonism against fomes annosus. Comparison between different test methods in vitro and in vivo

The antagonism of Trichoderma harzianum, Scytalidium album and an unidentified fungus (isolated from Monotropa hypopitys) against the root rot fungus Fomes annosus was studied in vitro and in vivo. Antibiosis was demonstrated on malt extract agar and on powdered wood. Competition between F. annosus and its antagonists was studied in stem discs and in living stems of Norway spruce. Effective antibiotics on agar and sawdust did not always bring about effective antagonism against F. annosus in stem discs or living trees. Factors such as growth rate of the antagonists in wood and their ability to invade the substrate during competition may influence the effectiveness of F. annosus suppression. The results from studies on antagonism in stem discs agreed with the results from tests in living trees. The frequency of penetrations through the wood cell walls was drastically lowered by the presence of the antagonists but was not closely correlated with the abundance of F. annosus.     

Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interaction

A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 °C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.     

Allocation of carbon to fine root compounds and their residence times in a boreal forest depend on root size class and season

Fine roots play a key role in the forest carbon balance, but their carbon dynamics remain largely unknown. We pulse labelled 50 m(2) patches of young boreal forest by exposure to (13)CO(2) in early and late summer. Labelled photosynthates were traced into carbon compounds of < 1 and 1-3 mm diameter roots (fine roots), and into bulk tissue of these and first-order roots (root tips). Root tips were the most strongly labelled size class. Carbon allocation to all size classes was higher in late than in early summer; mean residence times (MRTs) in starch increased from 4 to 11 months. In structural compounds, MRTs were 0.8 yr in tips and 1.8 yr in fine roots. The MRT of carbon in sugars was in the range of days. Functional differences within the fine root population were indicated by carbon allocation patterns and residence times. Pronounced allocation of recent carbon and higher turnover rates in tips are associated with their role in nutrient and water acquisition. In fine roots, longer MRTs but high allocation to sugars and starch reflect their role in structural support and storage. Accounting for heterogeneity in carbon residence times will improve and most probably reduce the estimates of fine root production.     

The below-ground perspective of forest plants: soil provides mainly organic nitrogen for plants and mycorrhizal fungi

? Nitrogen (N) availability has a major impact on a wide range of biogeochemical processes in terrestrial ecosystems. Changes in N availability modify the capacity of plants to sequester carbon (C), but despite the crucial importance for our understanding of terrestrial ecosystems, the relative contribution of different N forms to plant N nutrition in the field is not known. Until now, reliably assessing the highly dynamic pool of plant-available N in soil microsites was virtually impossible, because of the lack of adequate sampling techniques. ? For the first time we have applied a novel microdialysis technique for disturbance-free monitoring of diffusive fluxes of inorganic and organic N in 15 contrasting boreal forest soils in situ. ? We found that amino acids accounted for 80% of the soil N supply, while ammonium and nitrate contributed only 10% each. In contrast to common soil extractions, microdialysis revealed that the majority of amino acids are available for plant and mycorrhizal uptake. ? Our results suggest that the N supply of boreal forest soils is dominated by organic N as a major component of plant-available N and thus as a regulator of growth and C sequestration.     

Antimicrobial peptides in the first line defence of human colon mucosa

Antimicrobial peptides and proteins are effector molecules in the protection of epithelial surfaces. We have evaluated the presence of antimicrobial peptides/proteins that can participate in human colonic defence against microbes. A peptide/protein extract of normal human colon mucosa was found to be active against Gram-positive bacteria, Gram-negative bacteria, and fungi. Four polypeptides with antimicrobial activity were isolated from this material and they were identified by N-terminal amino acid sequence analysis as ubiquicidin, histone H2B, eosinophil cationic protein, and phospholipase A(2) (PLA(2)). Using immunodetection and mass spectrometry, LL-37, HNP1-3, and HBD-1 were also identified. Combined, these results indicate that the colon mucosa is protected by a complex mixture of polypeptides, able to kill invading microbes and working in synergy as a barrier against bacterial invasion.     

A GC-MS method for determination of amino acid uptake by plants

In this study, we present a rapid, robust and sensitive method for quantification of plant amino acid uptake using universally (U) (¹³C, ¹⁵N)-labelled amino acids and gas chromatography-mass spectrometry (GC-MS). Amino acids were analysed as their tert -butyldimethylsilyl (tBDMS) derivatives and displayed detection limits in the range 10–100 fmol on column, depending on the amino acid. The technique allows for simultaneous detection and quantification of both unlabelled and isotopically labelled species of amino acids. This makes simple quantification of plant amino acid uptake from an isotopically labelled source possible. The analytical variation was low, concerning total amino acid concentrations (relative standard deviation, rsd , less than 5.3%) as well as enrichment of U-¹³C, ¹⁵N-labelled glycine (Gly), arginine (Arg) and glutamic acid (Glu) ( rsd <2.1%). An application of the GC-MS method was conducted on non-mycorrhizal Pinus sylvestris roots supplied with U-¹³C, ¹⁵N-labelled amino acids. Intact, labelled amino acids were traced in root extracts. This provided conclusive evidence of plant root uptake of intact amino acids. Uptake rates of the three amino acids Gly, Glu and Arg in the range 0.5–37.9 μmol g⁻¹ dry weight h⁻¹ were recorded. These rates are comparable with those recorded in earlier studies of amino acid uptake, using other methods, as well as uptake rates measured for nitrate and ammonium.     

A conditional marker gene allowing both positive and negative selection in plants

Selectable markers enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage, respectively. We present a marker gene, dao1, encoding D-amino acid oxidase (DAAO, EC 1.4.3.3) that can be used for either positive or negative selection, depending on the substrate. DAAO catalyzes the oxidative deamination of a range of D-amino acids. Selection is based on differences in the toxicity of different D-amino acids and their metabolites to plants. Thus, D-alanine and D-serine are toxic to plants, but are metabolized by DAAO into nontoxic products, whereas D-isoleucine and D-valine have low toxicity, but are metabolized by DAAO into the toxic keto acids 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. Hence, both positive and negative selection is possible with the same marker gene. The marker has been successfully established in Arabidopsis thaliana, and proven to be versatile, rapidly yielding unambiguous results, and allowing selection immediately after germination.