A dynamic leaf gas‐exchange strategy is conserved in woody plants under changing ambient CO2: evidence from carbon isotope discrimination in paleo and CO2 enrichment studies

Rising atmospheric [CO₂], c_a, is expected to affect stomatal regulation of leaf gas‐exchange of woody plants, thus influencing energy fluxes as well as carbon (C), water, and nutrient cycling of forests. Researchers have proposed various strategies for stomatal regulation of leaf gas‐exchange that include maintaining a constant leaf internal [CO₂], c_i, a constant drawdown in CO₂ (c_a ? c_i), and a constant c_i/c_a. These strategies can result in drastically different consequences for leaf gas‐exchange. The accuracy of Earth systems models depends in part on assumptions about generalizable patterns in leaf gas‐exchange responses to varying c_a. The concept of optimal stomatal behavior, exemplified by woody plants shifting along a continuum of these strategies, provides a unifying framework for understanding leaf gas‐exchange responses to c_a. To assess leaf gas‐exchange regulation strategies, we analyzed patterns in c_i inferred from studies reporting C stable isotope ratios (δ¹³C) or photosynthetic discrimination (?) in woody angiosperms and gymnosperms that grew across a range of c_a spanning at least 100 ppm. Our results suggest that much of the c_a‐induced changes in c_i/c_a occurred across c_a spanning 200 to 400 ppm. These patterns imply that c_a ? c_i will eventually approach a constant level at high c_a because assimilation rates will reach a maximum and stomatal conductance of each species should be constrained to some minimum level. These analyses are not consistent with canalization toward any single strategy, particularly maintaining a constant c_i. Rather, the results are consistent with the existence of a broadly conserved pattern of stomatal optimization in woody angiosperms and gymnosperms. This results in trees being profligate water users at low c_a, when additional water loss is small for each unit of C gain, and increasingly water‐conservative at high c_a, when photosystems are saturated and water loss is large for each unit C gain.     

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

ATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake.     

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.     

Regulation of middle cerebral artery blood velocity during recovery from dynamic exercise in humans.

We sought to examine the regulation of cerebral blood flow during 10 min of recovery from mild, moderate, and heavy cycling exercise by measuring middle cerebral artery blood velocity (MCA V). Transfer function analyses between changes in arterial blood pressure and MCA V were used to assess the frequency components of dynamic cerebral autoregulation (CA). After mild and moderate exercise, the decreases in mean arterial pressure (MAP) and mean MCA V (MCA Vm) were small. However, following heavy exercise, MAP was rapidly and markedly reduced, whereas MCA Vm decreased slowly (-23 +/- 4 mmHg and -4 +/- 1 cm/s after 1 min for MAP and MCA Vm, respectively; means +/- SE). Importantly, for each workload, the normalized low-frequency transfer function gain between MAP and MCA Vm remained unchanged from rest to exercise and during recovery, indicating a maintained dynamic CA. Similar results were found for the systolic blood pressure and systolic MCA V relationship. In contrast, the normalized low-frequency transfer function gain between diastolic blood pressure and diastolic MCA V (MCA Vd) increased from rest to exercise and remained elevated in the recovery period (P < 0.05). However, MCA Vd was quite stable on the cessation of exercise. These findings suggest that MCA V is well maintained following mild to heavy dynamic exercise. However, the increased transfer function gain between diastolic blood pressure and MCA Vd suggests that dynamic CA becomes less effective in response to rapid decreases in blood pressure during the initial 10 min of recovery from dynamic exercise.     

Cytochemical localization of glycogen in Chlamydia trachomatis inclusions.

The origin and distribution of glycogen in inclusions of Chlamydia trachomatis were demonstrated with silver proteinate stain for electron microscopy. Glycogen particles were detected in all developmental stages of C. trachomatis, as well as free in the inclusions. Intrachlamydial glycogen was most common in elementary bodies but was also detected in intermediate forms and reticulate bodies (RBs). Abnormal divisions and breakdown of cytoplasmic membranes were common in RBs. Cytoplasmic contents, including glycogen particles, were released into the inclusions after rupture of the outer membranes of abnormal RBs and intermediate forms. From these observations, we conclude that glycogen in inclusions of C. trachomatis originates in the organisms themselves.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1

Cell-cell junctions are an integral part of epithelia and are often disrupted in cancer cells during epithelial-to-mesenchymal transition (EMT), which is a main driver of metastatic spread. We show here that Metastasis suppressor-1 (Mtss1; Missing in Metastasis, MIM), a member of the IMD-family of proteins, inhibits cell-cell junction disassembly in wound healing or HGF-induced scatter assays by enhancing cell-cell junction strength. Mtss1 not only makes cells more resistant to cell-cell junction disassembly, but also accelerates the kinetics of adherens junction assembly. Mtss1 drives enhanced junction formation specifically by elevating Rac-GTP. Lastly, we show that Mtss1 depletion reduces recruitment of F-actin at cell-cell junctions. We thus propose that Mtss1 promotes Rac1 activation and actin recruitment driving junction maintenance. We suggest that the observed loss of Mtss1 in cancers may compromise junction stability and thus promote EMT and metastasis.     

Mapping avian distributions: the evolution of bird atlases

Capsule An increasing proportion of atlases now map patterns of abundance but they are still a minority even though they require no more input of time or fieldworkers.

Aims To examine quantitatively the evolution of bird atlas methods, from their inception to the present day, to document the most frequently used methods and to quantify temporal changes in them, and so identify broad patterns that might be of use in the planning and interpretation of future atlases.

Methods A database of over 400 atlases was compiled, and a number of variables extracted from each. Temporal trends within, and relationships between, these variables were analysed.

Results Atlases have become significantly reduced in scale over time, covering smaller areas in shorter periods of fieldwork, but at higher spatial resolutions and with increasing numbers of observers per unit area. The number of participating fieldworkers and the size of the region being covered together explain over 70% of variation between atlases in spatial resolution. The number of atlases that have mapped abundance or relative abundance, rather than simply occurrence (presence/absence) or breeding status, has increased significantly over time but remains relatively small. However, such atlases were no more expensive in terms of length of the fieldwork or preparation periods or the number of observers deployed per unit area. There is evidence of a sharp decline in the number of new bird atlases being initiated.

Conclusions There have been significant changes in the way atlas surveys are undertaken, but no standardized method has evolved. This leads to flexibility that allows atlas surveys to be undertaken over areas varying by six orders of magnitude using numbers of observers that vary by five orders of magnitude.     

cDNA cloning of human oxysterol-binding protein and localization of the gene to human chromosome 11 and mouse chromosome 19

Cellular cholesterol metabolism is regulated primarily through sterol-mediated feedback suppression of the activity of the low-density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway. We previously described the cloning of a rabbit cDNA for the oxysterol-binding protein (OSBP), a cytosolic protein of 809 amino acids that may participate in these regulatory events. We now use the rabbit OSBP cDNA to clone the human OSBP cDNA and 5' genomic region. Comparison of the human and rabbit OSBP sequences revealed a remarkably high degree of conservation. The cDNA sequence in the coding region showed 94% identity between the two species, and the predicted amino acid sequence showed 98% identity. The human cDNA was used to determine the chromosomal localization of the OSBP gene by Southern blot hybridization to panels of somatic cell hybrid clones containing subsets of human or mouse chromosomes and by RFLP analysis of recombinant inbred mouse strains. The OSBP locus mapped to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. Along with previously mapped genes including Ly-1 and CD20, OSBP defines a new conserved syntenic group on the long arm of chromosome 11 in the human and the proximal end of chromosome 19 in the mouse.     

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

The biosynthetic pathway of the type B lantibiotic actagardine (formerly gardimycin), produced by Actinoplanes garbadinensis ATCC31049, has been cloned, sequenced and annotated. The gene cluster contains the gene garA that encodes the actagardine prepropeptide, a modification gene garM , involved in the dehydration and cyclization of the prepeptide, several putative transporter and regulatory genes as well as a novel luciferase-like monooxygenase gene designated garO . Expression of these genes in Streptomyces lividans resulted in the production of ala(0)-actagardine while deletion of the garA gene from A. garbadinensis generated a strain incapable of producing actagardine. Actagardine production was successfully restored however, by the delivery of the plasmid pAGvarX. This plasmid contains an engineered cassette of the actagardine encoding gene garA and offers an alternative route to generating extensive libraries of actagardine variants. Using this plasmid, an alanine scanning library has been constructed and the mutants analysed. Further modifications include the removal of the novel garO gene from A. garbadinensis . Deletion of this gene resulted in the production of deoxy variants of actagardine, demonstrating that the formation of the sulfoxide group is enzyme catalysed and not a spontaneous chemical modification as previously believed.