Acceleration of plastoquinone pool reduction by alternative pathways precedes a decrease in photosynthetic CO2 assimilation in preheated barley leaves

Heat stress causes inhibition of photosynthetic CO₂ assimilation, affects light photosynthetic reactions and accelerates alternative pathways of plastoquinone pool reduction (APPR). We have studied all these heat-sensitive processes after preheating to a broad range of physiological temperatures (24–46°C) to explore a role of these alternative pathways during heat stress. Primarily, the effective quantum yield of PSII photochemistry was reduced (at 40°C). This PSII downregulation was accompanied by the stimulation of APPR and preceded reduction of photosynthetic CO₂ assimilation by 2°; it occurred after preheating at 42°C because of inhibition in Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) activation process. Thus, we suggest that the heat-induced stimulation of APPR is not associated with the heat-induced inhibition of Calvin cycle as it was reported for other types of stresses. A possible role of APPR in the compensation of PSII downregulation is briefly discussed.     

Hydrolytic cleavage of N6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

Homogeneous adenine deaminases (EC 3.5.4.2) from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and a putative ADA (adenosine deaminase; EC 3.5.4.4) from Arabidopsis thaliana were obtained for the first time as purified recombinant proteins by molecular cloning of the corresponding genes and their overexpression in Escherichia coli. The enzymes showed comparable molecular properties with well-known mammalian ADAs, but exhibited much lower k(cat) values. Adenine was the most favoured substrate for the yeast enzymes, whereas the plant enzyme showed only very low activities with either adenine, adenosine, AMP or ATP. Interestingly, the yeast enzymes also hydrolysed N6-substituted adenines from cytokinins, a group of plant hormones, cleaving them to inosine and the corresponding side chain amine. The hydrolytic cleavage of synthetic cytokinin 2,6-di-substituted analogues that are used in cancer therapy, such as olomoucine, roscovitine and bohemine, was subsequently shown for a reference sample of human ADA1. ADA1, however, showed a different reaction mechanism to that of the yeast enzymes, hydrolysing the compounds to an adenine derivative and a side chain alcohol. The reaction products were identified using reference compounds on HPLC coupled to UV and Q-TOF (quadrupole-time-of-flight) detectors.The ADA1 activity may constitute the debenzylation metabolic route already described for bohemine and, as a consequence, it may compromise the physiological or therapeutic effects of exogenously applied cytokinin derivatives.     

Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice

We investigated the soluble forms of the earliest activation antigen of human leukocyte CD69. This receptor is expressed at the cell surface as a type II homodimeric membrane protein. However, the elements necessary to prepare the soluble recombinant CD69 suitable for structural studies are a matter of controversy. We describe the physical, biochemical and in vivo characteristics of a highly stable soluble form of CD69 obtained by bacterial expression of an appropriate extracellular segment of this protein. Our construct has been derived from one used for CD69 crystallization by further optimization with regard to protein stability, solubility and easy crystallization under conditions promoting ligand binding. The resulting protein is stable at acidic pH and at temperatures of up to 65 degrees C, as revealed by long-term stability tests and thermal denaturation experiments. Protein NMR and crystallography confirmed the expected protein fold, and revealed additional details of the protein characteristics in solution. The soluble CD69 refolded in a form of noncovalent dimers, as revealed by gel filtration, sedimentation velocity measurements, NMR and dynamic light scattering. The soluble CD69 proved to be remarkably stable in vivo when injected into the bloodstream of experimental mice. More than 70% of the most stable CD69 proteins is preserved intact in the blood 24 h after injection, whereas the less stable CD69 variants are rapidly taken up by the liver.     

Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

Feruloylation and structure of arabinoxylan in wheat endosperm cell walls from RNAi lines with suppression of genes responsible for backbone synthesis and decoration

Arabinoxylan (AX) is the major component of the cell walls of wheat grain (70% in starchy endosperm), is an important determinant of end‐use qualities affecting food processing, use for animal feed and distilling and is a major source of dietary fibre in the human diet. AX is a heterogeneous polysaccharide composed of fractions which can be sequentially extracted by water (WE‐AX), then xylanase action (XE‐AX) leaving an unextractable (XU‐AX) fraction. We determined arabinosylation and feruloylation of AX in these fractions in both wild‐type wheat and RNAi lines with decreased AX content (TaGT43_2 RNAi, TaGT47_2 RNAi) or decreased arabinose 3‐linked to mono‐substituted xylose (TaXAT1 RNAi). We show that these fractions are characterized by the degree of feruloylation of AX, <5, 5–7 and 13–19 mg bound ferulate (g^?1 AX), and their content of diferulates (diFA), <0.3, 1–1.7 and 4–5 mg (g^?1 AX), for the WE, XE and XU fractions, respectively, in all RNAi lines and their control lines. The amount of AX and its degree of arabinosylation and feruloylation were less affected by RNAi transgenes in the XE‐AX fraction than in the WE‐AX fraction and largely unaffected in the XU‐AX fraction. As the majority of diFA is associated with the XU‐AX fraction, there was only a small effect (TaGT43_2 RNAi, TaGT47_2 RNAi) or no effect (TaXAT1 RNAi) on total diFA content. Our results are compatible with a model where, to maintain cell wall function, diFA is maintained at stable levels when other AX properties are altered.     

Compostional variations and patterns of condont reworking in Late Devonian and early carboniferous calciturbidites (Moravia, Czech Republic)

Summary Compositional variations and grain-size properties of both carbonate constituents and conodonts as an alternative component group were used for interpreting the processes governing the deposition of upper Famennian and middle Tournaisian calciturbidites in Moravia, Czech Republic. Both the composition and grain-size properties of conodont element associations showed to be markedly dependant on facies type of their host sediment. Upper Devonian calciturbidite successions deposited on flanks of wide, Moravian-Silesian carbonate platform are composed mainly of echinoderm-and peloid-rich wacke/packstones and intraclastic float/rudstones (fine-grained calciturbidites, “normal” calciturbidites with Tab Bouma sequences, debris-flow breccias) with abundance of shelf-and shelf margin conodont taxa and epipelagic and “mesopelagic” conodonts. Upper Devonian calciturbidites deposited on slopes of volcanic sea-mounts are composed of echinoderm-and peloid-rich wacke/packstones and float/rudstones with increased proportion of intraclasts and volcanigenic lithoclasts (fine-grained calciturbidites, normal calciturbidites), yeilding abundant conodont associations with higher proportion of “mesopelagic” taxa compared to the platform-flank examples. Middle Tournaisian calciturbidite succession composed of crinoid-, peloid-, intraclast-and lithoclast-rich lime mudstones, wacke/packstones and float/rudstones (normal calciturbidites and debris-flow breccias) yielded conodont element associations rich in shelt-and shelf-margin taxa, “mesopelagic” conodonts and reworked Middle-and Upper Devonian conodonts. In general, the ratio of shelf-and shelf margin conodont taxa to “mesopelagic” taxa is distinctly lower in finegrained calciturbidites than it is in normal calciturbidites and debris-flow breccias. Grain-size properties (mean grain size and sorting) and percentage of fragmented conodont elements, too, are markedly dependant on the facies type: in fine-grained calciturbidites the values of mean grain-size and fragmentation are low and the sorting is good to very good whereas in normal calciturbidites and debris-flow breccias the values of mean grain-size and fragmentation are distinctly higher and the sorting is poorer. The interdependence of facies type and composition and grain-size properties of conodont element associations in gravity-flow deposits is explained as resultant from hydrodynamic sorting during turbidity current flow and final deposition of the bed. Compositional variations observed in our sections may thus be attributed to facies variability (coarsening-and thickening-upward trends) rather than to sea-level fluctuations (highstand shedding of carbonate platforms). On the other hand, significant enrichment in reworked conodont taxa in middle Tournaisian normal calciturbidites compared to scarcity and/or absence of such conodonts in essentially identical facies of upper Famennian age indicate sea-level to be the major control governing such compositional variations, with low relative sea-level stand in middle Tournaisian and high relative sea-level stand in upper Famennian. Thorough analysis of conodont evolution, palaeoecology and taphonomy, with emphasis on understanding the processes of deposition of their host rock, are recommended for any biostratigraphic and biofacies study to be done in carbonate sediments deposited under strong hydrodynamic regimes, such as calciturbidites, temperstites, debris-flow deposits, shelf-edge oolitic sands, tidal-channel facies etc.     

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.     

Atrial BNP endocrine function during chronic unloading of the normal canine heart

The goal of the study was to define the effect of chronic unloading of the normal heart on atrial endocrine function with a focus on brain natriuretic peptide (BNP), specifically addressing the role of load and neurohumoral stimulation. Although produced primarily by atrial myocardium in the normal heart, controversy persists with regard to load-dependent vs. neurohumoral mechanisms controlling atrial BNP synthesis and storage. We used a unique canine model of chronic unloading of the heart produced by thoracic inferior vena caval constriction (TIVCC), which also resulted in activation of plasma endothelin (ET-1), ANG II, and norepinephrine (NE), known activators of BNP synthesis, compared with sham. TIVCC was produced by banding of the inferior vena cava for 10 days (n = 6), whereas in control (n = 5) the band was not constricted (sham). In a third group (n = 7), the band was released on day 11, thus acutely reloading the heart. Chronic TIVCC decreased cardiac output and right atrial pressure with a decrease in atrial mass index consistent with atrial atrophy. Atrial BNP mRNA decreased compared with sham. Immunoelectron microscopy revealed an increase in BNP in atrial granules consistent with increased storage. Acute reloading increased cardiac filling pressures and resulted in an increase in plasma BNP. We conclude that chronic unloading of the normal heart results in atrial atrophic remodeling and in suppression of atrial BNP mRNA despite intense stimulation by ET, ANG II, and NE, underscoring the primacy of load in the control of atrial endocrine function and structure.