Combined application of a laser ablation-ICP-MS assay for screening and ESI-FTICR-MS for identification of a Cd-binding protein in Spinacia oleracea L. after exposure to Cd

We have studied the binding of the toxic element Cd to plant proteins and have used for this purpose spinach (Spinacia oleracea L.) plants treated with 50 μM Cd(II) as a model system. Laser ablation ICP-MS has been applied for the screening of Cd-binding proteins after separation by native anodal polyacrylamide gel electrophoresis (AN-PAGE) and electroblotting onto membranes. The main Cd-carrying protein band was isolated and investigated by nano-electrospray ionization-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry after tryptic digestion. By this procedure, the main Cd-binding protein was identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The latter enzyme has been discussed in the literature to be affected in its activity by oxidative stress induced by Cd. However, in this paper it is demonstrated for the first time that RuBisCO directly binds Cd and thus may be directly altered by this toxic element. A commercially available protein standard was used to verify direct binding of Cd(II) to the protein, even without metabolisation. The resulting metal-protein complex was shown to be stable enough to survive AN-PAGE separation and electroblotting. By the use of size exclusion chromatography coupled with ICP-MS it was demonstrated that the RuBisCO protein standard shows similar metal binding properties to Cd. Furthermore, essential elements such as Mn(II), Fe(II) and Cu(II), which are known to possibly replace the RuBisCO activator Mg(II), were investigated in addition to Zn(II). Again, similar binding properties in comparison to the plant protein were observed.     

Poor trail making test performance is directly associated with altered dual task prioritization in the elderly--baseline results from the TREND study

Background

Deterioration of executive functions in the elderly has been associated with impairments in walking performance. This may be caused by limited cognitive flexibility and working memory, but could also be caused by altered prioritization of simultaneously performed tasks. To disentangle these options we investigated the associations between Trail Making Test performance—which specifically measures cognitive flexibility and working memory—and dual task costs, a measure of prioritization.

Methodology and Principal Findings

Out of the TREND study (Tuebinger evaluation of Risk factors for Early detection of Neurodegenerative Disorders), 686 neurodegeneratively healthy, non-demented elderly aged 50 to 80 years were classified according to their Trail Making Test performance (delta TMT; TMT-B minus TMT-A). The subjects performed 20 m walks with habitual and maximum speed. Dual tasking performance was tested with walking at maximum speed, in combination with checking boxes on a clipboard, and subtracting serial 7 s at maximum speeds. As expected, the poor TMT group performed worse when subtracting serial 7 s under single and dual task conditions, and they walked more slowly when simultaneously subtracting serial 7 s, compared to the good TMT performers. In the walking when subtracting serial 7 s condition but not in the other 3 conditions, dual task costs were higher in the poor TMT performers (median 20%; range −6 to 58%) compared to the good performers (17%; −16 to 43%; p<0.001). To the contrary, the proportion of the poor TMT performance group that made calculation errors under the dual tasking situation was lower than under the single task situation, but higher in the good TMT performance group (poor performers, −1.6%; good performers, +3%; p = 0.035).

Conclusion

Under most challenging conditions, the elderly with poor TMT performance prioritize the cognitive task at the expense of walking velocity. This indicates that poor cognitive flexibility and working memory are directly associated with altered prioritization.     

Munich information center for protein sequences plant genome resources: a framework for integrative and comparative analyses 1(W)

With several plant genomes sequenced, the power of comparative genome analysis can now be applied. However, genome-scale cross-species analyses are limited by the effort for data integration. To develop an integrated cross-species plant genome resource, we maintain comprehensive databases for model plant genomes, including Arabidopsis (Arabidopsis thaliana), maize (Zea mays), Medicago truncatula, and rice (Oryza sativa). Integration of data and resources is emphasized, both in house as well as with external partners and databases. Manual curation and state-of-the-art bioinformatic analysis are combined to achieve quality data. Easy access to the data is provided through Web interfaces and visualization tools, bulk downloads, and Web services for application-level access. This allows a consistent view of the model plant genomes for comparative and evolutionary studies, the transfer of knowledge between species, and the integration with functional genomics data.     

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

pIQPNNI: parallel reconstruction of large maximum likelihood phylogenies

SUMMARY: IQPNNI is a program to infer maximum-likelihood phylogenetic trees from DNA or protein data with a large number of sequences. We present an improved and MPI-parallel implementation showing very good scaling and speed-up behavior.     

Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.     

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.