A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.     

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

Synthesis of non-glycosidic 4,6'-thioether-linked disaccharides as hydrolytically stable glycomimetics

Michael addition of 1,2:3,4-di-O-isopropylidene-6-thio-alpha-D-galactose (2) to 2-propyl 6-O-acetyl-3,4-dideoxy-alpha-D-glycero-hex-3-enopyranosid-2-ulose (1) afforded, as the major diastereoisomer, 2-propyl 6-O-acetyl-3-deoxy-4-S-(6-deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranos-6-yl)-4-thio-alpha-D-threo-hexopyranosid-2-ulose (3, 91% yield). Reduction of the carbonyl group of 3, followed by O-deacetylation gave the two epimers 7 (alpha-D-lyxo) and 8 (alpha-D-xylo) in a 1:2 ratio. On removal of the protecting groups of 8 by acid hydrolysis, formation of an 1,6-anhydro bridge was observed in the 3-deoxy-4-thiohexopyranose unit (10). The free non-glycosidic thioether-linked disaccharide 3-deoxy-4-S-(6-deoxy-alpha,beta-D-galactopyranos-6-yl)-4-thio-alpha,beta-D-xylo-hexopyranose (11) was obtained by acetolysis of 10 followed by O-deacetylation. A similar sequence starting from the enone 1 and methyl 2,3,4-tri-O-benzoyl-6-thio-alpha-D-glucopyranoside (12) led successfully to 2-propyl 3-deoxy-4-S-(methyl 6-deoxy-alpha-D-glucopyranos-6-yl)-4-thio-alpha-D-lyxo-hexopyranoside (17) and its alpha-D-xylo analog (19, major product). In this synthetic route, orthogonal sets of protecting groups were employed to preserve the configuration of both reducing ends and to avoid the formation of the 1,6-anhydro ring.     

Self-association of nucleotides

The self-association of nucleosides decreases within the series adenosine>guanosine>inosine>cytidine ≈uridine. The same trend is observed for the corresponding nucleotides, though less pronounced, as the charge effect governs series like adenosine ? AMP^2?>ADP^3??ATP^4?. Protonation of adenosine considerably reduces its self-stacking tendency: this is different with ATP^4?, where a maximum is reached for H₂(ATP)^2? caused by additional ionic interactions in the [H₂(ATP)]₂ ^4? dimer. Metal ion coordination may promote self-association, e.g., of ATP^4? via (mainly) charge neutralization (Mg²⁺) and the formation of intermolecular bridges in dimeric stacks (Zn²⁺, Cd²⁺). These results allow definition of conditions with negligible self-association and thus the determination of the stability and structure of monomeric nucleotide complexes in aqueous solution, e.g., quantification of macrochelate formation in M(ATP)^2? complexes. Some biological implications of the results are indicated.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

Predator-induced morphology enhances escape locomotion in crucian carp

Fishes show a remarkable diversity of shapes which have been associated with their swimming abilities and anti-predator adaptations. The crucian carp (Carassius carassius) provides an extreme example of phenotypic plasticity in body shape which makes it a unique model organism for evaluating the relationship between body form and function in fishes. In crucian carp, a deep body is induced by the presence of pike (Esox lucius), and this results in lower vulnerability to gape-limited predators, such as pike itself. Here, we demonstrate that deep-bodied crucian carp attain higher speed, acceleration and turning rate during anti-predator responses than shallow-bodied crucian carp. Therefore, a predator-induced morphology in crucian carp enhances their escape locomotor performance. The deep-bodied carp also show higher percentage of muscle mass. Therefore, their superior performance in escape swimming may be due to a combination of higher muscle power and higher thrust.     

High-density genetic map of durum wheat × wild emmer wheat based on SSR and DArT markers

A genetic linkage map of tetraploid wheat was constructed based on a cross between durum wheat [Triticum turgidum ssp. durum (Desf.) MacKey] cultivar Langdon and wild emmer wheat [T. turgidum ssp. dicoccoides (K?rn.) Thell.] accession G18-16. One hundred and fifty-two single-seed descent derived F(6) recombinant inbred lines (RILs) were analyzed with a total of 690 loci, including 197 microsatellite and 493 DArT markers. Linkage analysis defined 14 linkage groups. Most markers were mapped to the B-genome (60%), with an average of 57 markers per chromosome and the remaining 40% mapped to the A-genome, with an average of 39 markers per chromosome. To construct a stabilized (skeleton) map, markers interfering with map stability were removed. The skeleton map consisted of 307 markers with a total length of 2,317 cM and average distance of 7.5 cM between adjacent markers. The length of individual chromosomes ranged between 112 cM for chromosome 4B to 217 cM for chromosome 3B. A fraction (30.1%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1A, 1BL, 2BS, 3B, and 4B. DArT markers showed high proportion of clustering, which may be indicative of gene-rich regions. Three hundred and fifty-two new DArT markers were mapped for the first time on the current map. This map provides a useful groundwork for further genetic analyses of important quantitative traits, positional cloning, and marker-assisted selection, as well as for genome comparative genomics and genome organization studies in wheat and other cereals.