A novel splicing variant encoding putative catalytic α subunit of maize protein kinase CK2

A cDNA highly homologous to the known catalytic α subunit of protein kinase CK2 was cloned from maize ( Zea mays ). It was designated ZmCK 2α-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2α-4 and the previously identified ZmCK2α-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2α-4 has three potential translation initiation sites. The three putative variants of ZmCK2α-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2αs, the obtained GST:ZmCK2α-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2β. However, GST:ZmCK2α-4 did phosphorylate casein in the presence of a large excess of the β subunit. The activity of ZmCK2α-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2α-4 and the activation loop responsible for constitutive catalytic activity of CK2α. Preliminary results suggest that ZmCK2α-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s).     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

Risk alleles within a gene desert at the 9p21 locus constitute the most prevalent genetic determinant of cardiovascular disease. Previous research has demonstrated that 9p21 risk variants influence gene expression in vascular tissues, yet the biological mechanisms by which this would mediate atherosclerosis merits further investigation. To investigate possible influences of this locus on other tissues, we explored expression patterns of 9p21-regulated genes in a panel of multiple human tissues and found that the tumor suppressor CDKN2B was highly expressed in subcutaneous adipose tissue (SAT). CDKN2B expression was regulated by obesity status, and this effect was stronger in carriers of 9p21 risk alleles. Covariation between expression of CDKN2B and genes implemented in adipogenesis was consistent with an inhibitory effect of CDKN2B on SAT proliferation. Moreover, studies of postprandial triacylglycerol clearance indicated that CDKN2B is involved in down-regulation of SAT fatty acid trafficking. CDKN2B expression in SAT correlated with indicators of ectopic fat accumulation, including markers of hepatic steatosis. Among genes regulated by 9p21 risk variants, CDKN2B appears to play a significant role in the regulation of SAT expandability, which is a strong determinant of lipotoxicity and therefore might contribute to the development of atherosclerosis.     

Using a phylogenetic approach to selection of target plants in drug discovery of acetylcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae

We present phylogenetic analyses of 32 taxa of Amaryllidaceae tribe Galantheae, 6 taxa of other Eurasian genera of Amaryllidaceae and Phaedranassa dubia as outgroup in order to provide a phylogenetic framework for selection of candidate plants for lead discovery in relation to Alzheimer’s disease. We used DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid matK and trnL-F regions. Phylogenetic analyses using maximum parsimony and Bayesian inference strongly support a monophyletic tribe Galantheae in a narrow sense, including only Acis, Galanthus and Leucojum. Infrageneric relationships of Galanthus only partly support previous classifications. Alkaloid profiles and inhibition of acetylcholinesterase (AChE) were investigated for 18 taxa using gas chromatography–mass spectrometry (GC–MS) and an assay measuring inhibition of AChE activity. AChE inhibitory activity was found in all investigated clades and was correlated with alkaloid profiles of the plants. Lowest IC₅₀ values were expressed by extracts containing either galanthamine or lycorine type compounds. Evaluation of available chemistry and activity data in a phylogenetic framework could be used to select target species for further investigation.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Identification of various substrate-binding proteins of the hyperthermophylic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

Proteins from the extracellular medium of Aeropyrum pernix K1 were separated by two-dimensional electrophoresis and identified using mass spectrometry. Six different substrate-binding proteins (SBPs) from the ATP-binding cassette (ABC) transporter family were identified: (1) ABC transporter SBP (Q9YC61); (2) Branched-chain amino-acid ABC transporter, branched-chain amino-acid-binding protein (Q9YDJ6); (3) Oligopeptide ABC transporter, oligopeptide-binding protein (Q9YBL5); (4) Probable ABC transporter SBP (Q9Y9N4); (5) ABC transporter SBP (Q9YBG7); (6) ABC transporter SBP (Q9YFD7). Based on their orthology, division into the following classes was predicted: (1) multiple sugar-transport system SBPs; (2) peptide/nickel-transport system SBPs; and (3) branched-chain amino-acid-transport system SBPs. Further bioinformatic analyses showed that the identified SBPs differ in motif and in transmembrane-domain and signal-peptide organisation. Additionally, for all of these SBPs, sequence homology was found for archaeal proteins, and homologous proteins in bacteria were also found for the ABC transporter SBP Q9YBG7 and the ABC transporter SBP Q9YFD7. This is the first study, where different ABC SBPs from the extracellular medium of A. pernix have been identified using the combined methodology of two-dimensional electrophoresis and mass spectrometry.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

The quest for Y-chromosomal markers - methodological strategies for mammalian non-model organisms

Tracing maternal and paternal lineages independently to explore breeding systems and dispersal strategies in natural populations has been high on the wish-list of evolutionary biologists. As males are the heterogametic sex in mammals, such sex-specific patterns can be indirectly observed when Y chromosome polymorphism is combined with mitochondrial sequence information. Over the past decade, Y-chromosomal markers applied to human populations have revealed remarkable differences in the demographic history and behaviour between the sexes. However, with a few exceptions, genetic data tracing the paternal line are lacking in most other mammalian species. This deficit can be attributed to the difficulty of developing Y-specific genetic markers in non-model organisms and the general low levels of polymorphisms observed on the Y chromosome. Here, we present an overview of the currently employed strategies for developing paternal markers in mammals. Moreover, we review the practical feasibility and requirements of various methodological strategies and highlight their future prospects when combined with new molecular techniques such as next generation sequencing.