Differential oxidation of protein-tyrosine phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.     

Determinants of the nucleolar targeting of protein phosphatase-1

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.     

Distinctive architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium helveticum

The chloroplast genome has experienced many architectural changes during the evolution of chlorophyte green algae, with the class Chlorophyceae displaying the lowest degree of ancestral traits. We have previously shown that the completely sequenced chloroplast DNAs (cpDNAs) of Chamydomonas reinhardtii (Chlamydomonadales) and Scenedesmus obliquus (Sphaeropleales) are highly scrambled in gene order relative to one another. Here, we report the complete cpDNA sequence of Stigeoclonium helveticum (Chaetophorales), a member of a third chlorophycean lineage. This genome, which encodes 97 genes and contains 21 introns (including four putatively trans-spliced group II introns inserted at novel sites), is remarkably rich in derived features and extremely rearranged relative to its chlorophycean counterparts. At 223,902 bp, Stigeoclonium cpDNA is the largest chloroplast genome sequenced thus far, and in contrast to those of Chlamydomonas and Scenedesmus, features no large inverted repeat. Interestingly, the pattern of gene distribution between the DNA strands and the bias in base composition along each strand suggest that the Stigeoclonium genome replicates bidirectionally from a single origin. Unlike most known trans-spliced group II introns, those of Stigeoclonium exhibit breaks in domains I and II. By placing our comparative genome analyses in a phylogenetic framework, we inferred an evolutionary scenario of the mutational events that led to changes in genome architecture in the Chlorophyceae.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Nucleotide sequence data reported are available in the GenBank database under the accession number DQ630521.     

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.     

Nocturnal migrants do not incur higher collision risk at wind turbines than diurnally active species

Nocturnally migrating birds, particularly passerines, are known to be vulnerable to collision with man‐made structures such as buildings, towers or offshore platforms, yet information with respect to wind farms is ambiguous. We recorded bird flight intensities using radar during autumn migration at four wind farms situated within a major migration flyway in northern Germany and simultaneously conducted systematic searches for collision fatalities at the same sites. We found that migration traffic rates at rotor height estimated by radar observations were significantly higher during the night, yet strictly nocturnal migrants constituted only 8.6% of all fatalities at the wind farms. In contrast to the situation at other vertical structures, nocturnal migrants do not have a higher risk of collision with wind energy facilities than do diurnally active species, but rather appear to circumvent collision more effectively.     

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.     

Combined flash- and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms

The difference in light-emission kinetics between the Ca(2+)-triggered bioluminescent reaction of the photoprotein aequorin (AEQ) and the alkaline phosphatase (ALP)-catalyzed chemiluminescent hydrolysis of dioxetane aryl phosphate substrates was exploited for the analysis of both alleles of biallelic polymorphisms in a single microtiter well. The genotyping of the IVS-1-110 locus of the human beta-globin gene was chosen as a model. Genomic DNA, isolated from whole blood, was first subjected to polymerase chain reaction using primers flanking the polymorphic site. A single oligonucleotide-ligation reaction employing two allele-specific probes, labeled with biotin and digoxigenin, and a common probe carrying a characteristic tail was then performed. The ligation products were captured in a microtiter well through hybridization of the tail with an immobilized complementary oligonucleotide. The products were detected by adding a mixture of streptavidin-aequorin complex and antidigoxigenin-alkaline phosphatase conjugate. AEQ was measured first by adding Ca(2+) and integrating the signal for 3s followed by the addition of the substrate for ALP. The ratio of the luminescence signals obtained from ALP and AEQ gives the genotype of each sample. The coefficient of variation of the dual assay ranged from 7 to 11% for each allele. The reproducibility of the ALP/AEQ signal ratio was about 14%. The proposed assay allows for many samples to be screened in parallel in a single microtiter plate, for single-nucleotide polymorphisms.     

Cyclitol metabolism is a central feature of Burkholderia leaf symbionts

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant–bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.