Solution structure of reduced Clostridium pasteurianum rubredoxin

The solution structure of reduced Clostridium pasteurianum rubredoxin (MW 6100) is reported here. The protein is highly paramagnetic, with iron(II) being in the S=2 spin state. The Hβ protons of the ligating cysteines are barely observed, and not specifically assigned. Seventy-six percent of the protons have been assigned and 1267 NOESY peaks (of which 1037 are meaningful) have been observed. Nonselective T ₁ measurements have been measured by recording four nonselective 180°-τ-NOESY at different τ values, and fitting the intensity recoveries to an exponential recovery. Thirty-six metal-proton upper and lower distance constraints have been obtained from the above measurements. The use of such constraints is assessed with respect to spin delocalization on the sulfur donor atoms. The solution structure obtained with the program DYANA has been refined through restrained energy minimization. A final family of 20 conformers is obtained with no distance violations larger than 0.24?Å, and RMSD values to the mean structure of 0.58 and 1.03?Å for backbone and all heavy atoms, respectively (measured on residues 3–53). The structure is compared to the X-ray structure of the oxidized and of the zinc substituted protein, and to the available structures of other rubredoxins. In particular, the comparison with the crystal structure and the solution structure of the Zn derivative of the highly thermostable Pyrococcus furiosus rubredoxin suggested that the relatively low thermal stability of the clostridial rubredoxin may be tentatively ascribed to the loosening of its secondary structure elements. This research is a further achievement at the frontier of solution structure determinations of paramagnetic proteins.     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol     

An unusual homostyle inHedyotis caerulea (Rubiaceae)

Hedyotis caerulea, a spring flowering herb widely distributed in eastern North America, has distylous flowers that differ by a number of morphological and physiological traits. The presence of a strong incompatibility system is indicated by the fact that intramorph crosses or self-pollinations produced little or no seed and intermorph crosses produced copious seed. An unusual homostyle was located that had most floral characters intermediate between pin and thrum flowers, although its pollen size was that of pin. The homostyle was only moderately self-compatible, and its pollen did not behave as pin pollen on thrum stigmas. It appears that, despite their intermediate position and morphology, the homostyle stigmas were fundamentally pin. Although the exceptional rarity of the homostyle suggests that it is an inadaptive failure, workers should watch for its further occurrence since additional study of it may provide insights into the genetic and physiological basis of heterostyly in theRubiaceae.     

Cardiac copper,magnesium, and zinc in recent and old myocardial infarction

X-ray fluorescence spectrometry and atomic absorption spectrometry were used in a quantitative study of zinc, copper, and magnesium in 71 postmortal human hearts. Samples were obtained from individuals who had demonstrated no previous clinical or subsequent pathological findings of myocardial infarction and from victims of a recent or an old infarction. A significant difference (p<0.001) in the elemental levels was observed between the noninfarct and the recent infarct groups. The noninfarct group had higher cardiac levels of all three elements. However, the difference in elemental concentrations between the noninfarct and the old-infarct groups was not significant. Cardiac levels of zinc (p<0.001) and copper (p<0.01) were significantly greater in the old-infarct group than in the recent-infarct group. Magnesium levels were higher in the recent-and-old-infarct group than in the recent infarct group (p<0.01). It is possible that the elements are redistributed during myocardial infarction, and that uptake of these elements (from the serum pool) by the heart may be important in maintaining myocardial integrity and function.     

Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

Effects of cover soil stockpiling on plant community development following reclamation of oil sands sites in Alberta

Stockpiling of cover soil can influence vegetation development following reclamation. Cover soil, comprising the upper 15–30 cm of the surface material on sites scheduled for mining, is commonly salvaged prior to mining and used directly or stockpiled for various lengths of time until it is needed. Salvaging and stockpiling causes physical, chemical, and biological changes in cover soils. In particular, stockpiling reduces the availability and vigor of vegetative propagules and seed, and can lead to increases in the abundance of some weedy species. This study uses data from monitoring plots to assess how stockpiling of cover soil impacts plant community development on reclaimed oil sands mine sites in northern Alberta. Development of plant communities differed distinctly between directly placed and stockpiled cover soil treatments even 18 years after reclamation. Direct placement of cover soil resulted in higher percent cover, species richness, and diversity. Nonmetric multidimensional scaling and multiresponse permutation procedure revealed compositional differentiation between the treatments. Indicator species analysis showed that direct placement treatment was dominated by perennial species while grasses and annual forb species dominated sites where stockpiled soil was used. Results indicate that stockpiling leads to slower vegetation recovery while direct placement of cover soil supports more rapid succession (from ruderal and annual communities to perennial communities). In addition, direct placement may be less costly than stockpiling. However, scheduling of salvage and placement remains a challenge.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Low genetic diversity and fragmentation effects in a wind-pollinated tree, Polylepis multijuga Plige (Rosaceae) in the high Andes

Fragmentation is predicted to increase inbreeding depression and lower the evolutionary potential of organisms by disrupting dispersal. Trees may be more resilient to fragmentation effects due to potential long-distance dispersal mechanisms that genetically connect fragments. Polylepis woodlands in the high Andes are highly fragmented and are currently the focus of reforestation and conservation efforts. Polylepis multijuga Plige (Rosaceae) is a threatened, endemic tree species in the northern Andes of Peru. Samples were collected from 371 adult trees in nine forest fragments separated by 0.5–80 km and genotyped at amplified fragment length polymorphism loci (AFLP) and chloroplast intergenic regions to determine the connectedness of fragments and their suitability for collecting seed for restoration efforts. P. multijuga is wind-pollinated and dispersed; however, genetic diversity in P. multijuga was about half that reported for other wind-pollinated species. Genetic spatial autocorrelation and patterns of chloroplast and AFLP diversity suggest seed dispersal is very limited and that wind dispersed pollen does not effectively connect all fragments. Conservation of this species will require reforestation efforts and possibly augmentation of some fragments to increase their genetic diversity. Collecting seed from multiple large fragments and from individuals separated by at least 25 m within fragments would maximize the genetic diversity of seed collections for reforestation or augmentation. Future studies of this and other Polylepis species should determine how complex topography may affect wind mediated dispersal between fragments and patterns of genetic diversity.