Sexual reproduction advances autumn leaf colours in mountain birch (Betula pubescens ssp. czerepanovii)

Autumnal change in leaf colour of deciduous trees is one of the most fascinating displays in nature. Current theories suggest that autumn leaf colours are adaptations to environmental stress. Here I report that the number of ripening female catkins altered timing of yellow autumn leaf colours in mountain birch. The tree's autumnal colour change was brought forward if the tree matured plenty of female catkins. Since yellow colour pigments in leaves are unmasked as leaf nitrogen is re-translocated, sexual reproduction may alter resource allocation at times of leaf senescence. Thus, our current view on the reasons for leaf senescence has to be re-examined, and a novel evolutionary explanation is needed for the appearance of yellow autumn leaf colours.     

Leaf water content and palisade cell size

The palisade cell sizes in leaves of Eucalyptus pauciflora were estimated in paradermal sections of cryo-fixed leaves imaged in the cryo-scanning electron microscope, as a quantity called the cell area fraction (CAF). Cell sizes were measured in detached leaves as a function of leaf water content, in intact leaves in the field during a day"s transpiration as a function of balance pressure of adjacent leaves, and on leaf disks equilibrated with air of relative humidities from 100 to 58%. Values of CAF ranged from 0.82 at saturation to approx. 0.3 in leaves dried to a relative water content (RWC) of 0.5, and in the field to approx. 0.58 at 15 bar (1.5 MPa) balance pressure. At a CAF of 0.58, the moisture content of the cell walls is in equilibrium with air at 90% relative humidity, which is the estimated relative humidity in the intercellular spaces. It is shown that at this moisture content, the cell walls could be exerting a pressure of approx. 50 bar on the cell contents.     

Herbal monoterpene alcohols inhibit propofol metabolism and prolong anesthesia time

2,6-Diisopropylphenol (Propofol) is a short-acting intravenous anesthetic that is rapidly metabolized by glucuronidation and ring hydroxylation catalyzed by cytochrome P450. The goal of this research was to determine whether dietary monoterpene alcohols (MAs) could be used to prolong the anesthetic effect of propofol by inhibiting propofol metabolism in animals. Mice were injected intraperitoneally (i.p.) with MAs (100-200) mg/kg followed by the administration of 100 mg/kg propofol 40 min later via an i.p. injection. The time of the anesthesia of each mouse was recorded. It was found that (+/-)-borneol, (-)-carveol, trans-sobrerol, and menthol significantly extended the anesthetic effect of propofol (>3 times). The concentration of propofol in the mouse blood over time (up to 180 min) also increased in mice pre-treated with (-)-borneol, (-)-carveol, and trans-sobrerol. The volume of distribution of propofol decreased in the (-)-borneol (p<0.05), pre-treated group as compared to the propofol control group. Moreover, the maximum blood concentration of propofol and the concentration of propofol in the blood as indicated by the area under the curve were significantly increased in (-)-borneol and (-)-carveol pre-treated groups. Additional evidence using rat hepatocytes showed that (-)-borneol inhibited propofol glucuronidation whereas trans-sobrerol and (-)-carveol inhibited cytochrome P450 dependent microsomal aminopyrine N-demethylation. These results suggest that (-)-borneol extends propofol-induced anesthesia by inhibiting its glucuronidation in the mouse whereas trans-sobrerol (-)-carveol extends propofol-induced anesthesia by inhibiting P450 catalyzed propofol metabolism.     

Crown development in a pioneer tree, Rhus trichocarpa, in relation to the structure and growth of individual branches

Based on an allometric reconstruction, the structure and biomass-allocation patterns of branches and current-year shoots were investigated in branches of various heights in the pioneer tree Rhus trichocarpa, to evaluate how crown development is achieved and limited in association with height. Path analysis was conducted to explore the effects of light availability, basal height and size of individual branches on branch structure and growth. Branch angle was affected by basal height, whereas branch mass was influenced primarily by light availability. This result suggests that branch structure is strongly constrained by basal height, and that trees mediate such constraints under different light environments. Previous-year leaf area and light availability showed positive effects on current-year stem mass. In contrast, branch basal height and mass negatively affected current-year stem mass. Moreover, the length of stems of a given diameter decreased with increasing branch height. Therefore the cost of biomass investment for a unit growth in length is greater for branches of larger size and at upper positions. Vertical growth rate in length decreased with increasing height. Height-dependent changes in stem allometry and angle influenced the reduction in vertical growth rate to a similar degree.     

Arbuscular mycorrhizal fungi associated with Populus-Salix stands in a semiarid riparian ecosystem

This study examined the activity, species richness, and species composition of the arbuscular mycorrhizal fungal (AMF) community of Populus-Salix stands on the Verde River (Arizona, USA), quantified patterns of AMF richness and colonization along complex floodplain gradients, and identified environmental variables responsible for structuring the AMF community. Samples from 61 Populus-Salix stands were analyzed for AMF and herbaceous composition, AMF colonization, gravimetric soil moisture, soil texture, per cent organic matter, pH, and concentrations of nitrate, bicarbonate phosphorus and exchangeable potassium. AMF species richness declined with stand age and distance from and elevation above the channel and was positively related to perennial species cover and richness and gravimetric soil moisture. Distance from and elevation above the active channel, forest age, annual species cover, perennial species richness, and exchangeable potassium concentration all played a role in structuring the AMF community in this riparian area. Most AMF species were found across a wide range of soil conditions, but a subset of species tended to occur more often in hydric areas. This group of riparian affiliate AMF species includes several not previously encountered in the surrounding Sonoran desert.     

Foliar pH as a new plant trait: can it explain variation in foliar chemistry and carbon cycling processes among subarctic plant species and types?

Plant traits have become popular as predictors of interspecific variation in important ecosystem properties and processes. Here we introduce foliar pH as a possible new plant trait, and tested whether (1) green leaf pH or leaf litter pH correlates with biochemical and structural foliar traits that are linked to biogeochemical cycling; (2) there is consistent variation in green leaf pH or leaf litter pH among plant types as defined by nutrient uptake mode and higher taxonomy; (3) green leaf pH can predict a significant proportion of variation in leaf digestibility among plant species and types; (4) leaf litter pH can predict a significant proportion of variation in leaf litter decomposability among plant species and types. We found some evidence in support of all four hypotheses for a wide range of species in a subarctic flora, although cryptogams (fern allies and a moss) tended to weaken the patterns by showing relatively poor leaf digestibility or litter decomposability at a given pH. Among seed plant species, green leaf pH itself explained only up to a third of the interspecific variation in leaf digestibility and leaf litter up to a quarter of the interspecific variation in leaf litter decomposability. However, foliar pH substantially improved the power of foliar lignin and/or cellulose concentrations as predictors of these processes when added to regression models as a second variable. When species were aggregated into plant types as defined by higher taxonomy and nutrient uptake mode, green-specific leaf area was a more powerful predictor of digestibility or decomposability than any of the biochemical traits including pH. The usefulness of foliar pH as a new predictive trait, whether or not in combination with other traits, remains to be tested across more plant species, types and biomes, and also in relation to other plant or ecosystem traits and processes.     

Up-regulation of dopamine D(2)L mRNA levels in the ventral tegmental area and dorsal striatum of amphetamine-sensitized C57BL/6 mice: role of Ca(v)1.3 L-type Ca(2+) channels

Dopamine D(2) long (D(2)L) and D(2) short (D(2)S) isoforms of the D(2) receptor play an important role in psychostimulant-induced neuronal adaptations. In this study, we used quantitative real-time PCR to specifically amplify these two splice variants to examine their mRNA expression in the dorsal striatum (dStr), nucleus accumbens (NAc) and the ventral tegmental area (VTA) of amphetamine-sensitized C57BL/6 mice. We found a significant increase in D(2)L mRNA in the VTA and dStr of amphetamine-treated mice that positively correlated with the sensitized locomotor response. We also found a significant increase in D(2)S mRNA in the VTA. We further examined the role of the Ca(v)1.3 subtype of L-type Ca(2+) channels in up-regulation of D(2)L and D(2)S mRNA in the VTA. Amphetamine-pretreated Ca(v)1.3 wild-type (Ca(v)1.3(+/+)) mice exhibited sensitized behavior and a significant increase in D(2)L and D(2)S mRNA compared with saline-pretreated mice Amphetamine-pretreated homozygous Ca(v)1.3 knockout (Ca(v)1.3(-/-)) mice did not exhibit sensitized behavior. There was a significant increase in D(2)S mRNA, but not D(2)L mRNA. In conclusion, our results find that amphetamine increases D(2)L mRNA expression in the dStr and the VTA, an adaptation that correlates with expression of sensitized behavior and dependence on Ca(v)1.3 Ca(2+) channels.     

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.     

Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima

Studies of the structural basis of protein thermostability have produced a confusing picture. Small sets of proteins have been analyzed from a variety of thermophilic species, suggesting different structural features as responsible for protein thermostability. Taking advantage of the recent advances in structural genomics, we have compiled a relatively large protein structure dataset, which was constructed very carefully and selectively; that is, the dataset contains only experimentally determined structures of proteins from one specific organism, the hyperthermophilic bacterium Thermotoga maritima, and those of close homologs from mesophilic bacteria. In contrast to the conclusions of previous studies, our analyses show that oligomerization order, hydrogen bonds, and secondary structure play minor roles in adaptation to hyperthermophily in bacteria. On the other hand, the data exhibit very significant increases in the density of salt-bridges and in compactness for proteins from T.maritima. The latter effect can be measured by contact order or solvent accessibility, and network analysis shows a specific increase in highly connected residues in this thermophile. These features account for changes in 96% of the protein pairs studied. Our results provide a clear picture of protein thermostability in one species, and a framework for future studies of thermal adaptation.     

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.