The effect of past changes in inter‐annual temperature variability on tree distribution limits

Aim The northern limits of temperate broadleaved species in Fennoscanndia are controlled by their requirements for summer warmth for successful regeneration and growth as well as by the detrimental effects of winter cold on plant tissue. However, occurrences of meteorological conditions with detrimental effects on individual species are rare events rather than a reflection of average conditions. We explore the effect of changes in inter‐annual temperature variability on the abundances of the tree species Tilia cordata, Quercus robur and Ulmus glabra near their distribution limits using a process‐based model of ecosystem dynamics. Location A site in central Sweden and a site in southern Finland were used as examples for the ecotone between boreal and temperate forests in Fennoscandia. The Finnish site was selected because of the availability of varve‐thickness data. Methods The dynamic vegetation model LPJ‐GUESS was run with four scenarios of inter‐annual temperature forcing for the last 10,000 years. In one scenario the variability in the thickness of summer and winter varves from the annually laminated lake in Finland was used as a proxy for past inter‐annual temperature variability. Two scenarios were devised to explore systematically the effect of stepwise changes in the variance and shape parameter of a probability distribution. All variability scenarios were run both with and without the long‐term trend in Holocene temperature change predicted by an atmospheric general circulation model. Results Directional changes in inter‐annual temperature variability have significant effects on simulated tree distribution limits through time. Variations in inter‐annual temperature variability alone are shown to alter vegetation composition by magnitudes similar to the magnitude of changes driven by variation in mean temperatures. Main conclusions The varve data indicate that inter‐annual climate variability has changed in the past. The model results show that past changes in species abundance can be explained by changes in the inter‐annual variability of climate parameters as well as by mean climate. Because inter‐annual climatic variability is predicted to change in the future, this component of climate change should be taken into account both when making projections of future plant distributions and when interpreting vegetation history.     

Structure of type I antifreeze protein and mutants in supercooled water

Many organisms are able to survive subzero temperatures at which bodily fluids would normally be expected to freeze. These organisms have adapted to these lower temperatures by synthesizing antifreeze proteins (AFPs), capable of binding to ice, which make further growth of ice energetically unfavorable. To date, the structures of five AFPs have been determined, and they show considerable sequence and structural diversity. The type I AFP reveals a single 37-residue alpha-helical structure. We have studied the behavior of wild-type type I AFP and two "inactive" mutants (Ala17Leu and Thr13Ser/Thr24Ser) in normal and supercooled solutions of H(2)O and deuterium oxide (D(2)O) to see if the structure at temperatures below the equilibrium freezing point is different from the structure observed at above freezing temperatures. Analysis of 1D (1)H- and (13)C-NMR spectra illustrate that all three proteins remain folded as the temperature is lowered and even seem to become more alpha-helical as evidenced by (13)C(alpha)-NMR chemical shift changes. Furthermore, (13)C-T(2) NMR relaxation measurements demonstrate that the rotational correlation times of all three proteins behave in a predictable manner under all temperatures and conditions studied. These data have important implications for the structure of the AFP bound to ice as well as the mechanisms for ice-binding and protein oligomerization.     

Alternative roles for putative ice-binding residues in type I antifreeze protein

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.     

E2a/Pbx1 induces the rapid proliferation of stem cell factor-dependent murine pro-T cells that cause acute T-lymphoid or myeloid leukemias in mice

Oncoprotein E2a/Pbx1 is produced by the t(1;19) chromosomal translocation of human pre-B acute lymphoblastic leukemia. E2a/Pbx1 blocks differentiation of primary myeloid progenitors but, paradoxically, induces apoptosis in established pre-B-cell lines, and no transforming function of E2a/Pbx1 has been reported in cultured lymphoid progenitors. Here, we demonstrate that E2a/Pbx1 induces immortal proliferation of stem cell factor (SCF)-dependent pro-T thymocytes by a mechanism dependent upon both its transactivation and DNA-binding functions. E2a-Pbx1 cooperated with cytokines or activated signaling oncoproteins to induce cell division, as inactivation of conditional E2a/Pbx1 in either factor-dependent pro-T cells or pro-T cells made factor independent by expression of Bcr/Abl resulted in pro-T-cell quiescence, while reactivation of E2a/Pbx1 restored cell division. Infusion of E2a/Pbx1 pro-T cells in mice caused T lymphoblastic leukemia and, unexpectedly, acute myeloid leukemia. The acute lymphoblastic leukemia did not evidence further maturation, suggesting that E2a/Pbx1 establishes an early block in pro-T-cell development that cannot be overcome by marrow or thymic microenvironments. In an E2a/Pbx1 pro-T thymocyte clone that induced only pro-T acute lymphoblastic leukemia, coexpression of Bcr/Abl expanded its leukemic phenotype to include acute myeloid leukemia, suggesting that unique functions of cooperating signaling oncoproteins can influence the lymphoid versus myeloid character of E2a/Pbx1 leukemia and may cooperate with E2a/Pbx1 to dictate the pre-B-cell phenotype of human leukemia containing t(1;19).     

Assessment of potential (inhalation and dermal) and actual exposure to acetamiprid by greenhouse applicators using liquid chromatography-tandem mass spectrometry

New analytical methods based on liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) have been developed and validated for assessing the exposure of greenhouse workers to acetamiprid. Both ambient (potential inhalation and dermal exposure) and internal dose (biological monitoring of urine samples) measurements were carried out. Potential inhalation exposure was assessed using Chromosorb 102 cartridges connected to air personal samplers. Potential dermal exposure was estimated by using whole body dosimetry. The measurement of actual exposure was done by analyzing the parent compound in urine samples of the applicators, after a solid-phase extraction (SPE) step. The methods showed a good accuracy (72-92%), precision (2-13%) and lower limits (few microg l(-1)). The validated approaches have been applied to assess potential and actual exposure of agricultural workers spraying acetamiprid in greenhouses. The results shown the need to wear personal protective equipment (suits) in order to reduce the absorbed dose of acetamiprid.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

VADAR: a web server for quantitative evaluation of protein structure quality

VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.     

Interaction of a peptide from the receptor-binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: changes in backbone dynamics induced by binding

The C-terminal receptor-binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N NMR relaxation experiments of the (15)N-labeled recombinant PAK pilin peptide in complex with an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus, were performed in order to probe for changes in the mobilities and dynamics of the peptide backbone as a result of antibody binding. The major results of these studies are as follows: binding of Fab leads to the preferential ordering of the first turn over the second turn in each isomer, binding of Fab partially stabilizes peptide backbone regions undergoing slow (microsecond to millisecond) exchange-related motions, and binding of Fab leads to a greater loss in backbone conformational entropy at pH 7.2 versus pH 4.5. The biological implications of these results will be discussed in relation to the role that fast and slow backbone motions play in PAK pilin peptide immunogenicity and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.     

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.