Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria

Dioxins invade the body mainly through the diet, and produce toxicity through the transformation of aryl hydrocarbon receptor (AhR). An inhibitor of the transformation should therefore protect against the toxicity and ideally be part of the diet. We examined flavonoids ubiquitously expressed in plant foods as one of the best candidates, and found that the subclasses flavones and flavonols suppressed antagonistically the transformation of AhR induced by 1 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin, without exhibiting agonistic effects that transform AhR. The antagonistic IC(50) values ranged from 0.14 to 10 microM, close to the physiological levels in human.     

Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

Summary The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[d-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[d-Dab-Gly-Trp-d-Leu] (II) (DNS=dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a β-turn at residues 2 and 3.     

Elimination of bean seed-borne bacteria by thermotherapy and meristem culture

Plant Cell, Tissue and Organ Culture (PCTOC) -     

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.