Characterization of a novel y-type high molecular weight glutenin subunit at <Emphasis Type="Italic">Glu-D1</Emphasis> locus

Glu-D1y12.K as a novel y-type subunit was found in HMW-GSs encoded at the Glu-D1 locus in the JB20, which a Korean wheat line from F₉ lines crossed by Keumkang with Glu-D1d and Chinese Spring (CS) with Glu-D1a alleles. This novel subunit shows faster electrophoretic mobility and lower molecular weight than Dy12 subunit on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result of linear ion-trap and Fourier-transform mass spectrometry (LTQ-FT-MS) based on two-dimensional electrophoresis (2-DE) showed that the Dy12.K subunit has high similarity against protein ID: P08488 (GLT3_WHEAT) as ‘Glutenin, high molecular weight subunit 12’ form UniProtKB. The gene of the Glu-1Dy12.K subunit is composed of 1962 nucleotide base pairs containing open reading frame (ORF) as 652 amino acids corresponding to about 70.1 kDa. It has four indels (36 bp insertions: two repeated 18 and 24 bp deletion: two deletions with 6?+?18 bp) and 21 SNPs compared to Glu-1Dy10 (GI: 164457872 in NCBI), and one deletion (18 bp) and three SNPs compared to Glu-1Dy12 (GI: 1036031968) by DNA markers. Consequentially, in comparison with Dy10, 13 SNPs were non-synonymous SNPs and eight SNPs were synonymous SNPs of 21 SNPs. In comparison with Dy12, only one SNP was non-synonymous SNP of three SNPs. Furthermore, the deduced peptide sequences as ‘TGQGQQ’ corresponding to ‘AACAGGACAAGGGCAACA’ are deleted only in the Dy12.K subunit.     

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

Production of Recombinant Hinnavin II/MSH Hybrid in Insect Cells

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. For the purpose of this study, a cDNA encoding hinnavin II‐α‐melanocyte stimulating hormone (hin/MSH) hybrid was chemically synthesized, annealed, and then cloned into transfer vector pBacPAK 9 for expression in Sf21 insect cells. Recombinant hin/MSH (rhin/MSH) hybrid was efficiently produced in baculovirus expression vector system (BEVS) as a hybrid peptide. The antibacterial activity of the rhin/MSH hybrid was compared to that of the recombinant hinnavin II (rhin), using inhibition zone and overlay assay. This new recombinant hybrid peptide may serve as an attractive candidate for powerful novel class of antimicrobial pharmaceuticals.     

Efficient and accurate analysis of microRNA using a specific extension sequence

Molecular Biology Reports - We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay...     

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.     

Deuteration of Escherichia coli Enzyme I alters its stability

Enzyme I^Ntr is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI^Ntr. Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4 °C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI^Ntr are evaluated.     

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).