High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.     

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.     

Regulation of stretch-activated ANP secretion by chloride channels

This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl(-) channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736microl for volume load vs. 129microl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca2+-activated Cl(-) channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl(-) channel, diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl(-) channels may differently regulate stretch-activated ANP secretion.     

Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration

We made a three-dimensional (3-D) nanofibrous fibroin scaffold (NFS) with high porosity (94%) and examined its feasibility in bone regeneration. Under scanning electron microscopy, MC3T3-E1 preosteoblasts on the scaffold showed more spread on the first day after seeding compared with a 2-D scaffold. MTT assay showed significantly increased proliferation in 3-D NFS compared with 2-D NFS 7 days after seeding (P < 0.05). Western immunoblotting for activated paxillin, FAK, AKT, C-Src, and ERK1/2 antibodies showed signals from the extracellular matrix were significantly increased in 3-D NFS. Newly developed 3-D electrospun NFS may be a good candidate for use in bone regeneration. Ki and Park equally contributed in this paper.     

A novel pepper membrane-located receptor-like protein gene CaMRP1 is required for disease susceptibility, methyl jasmonate insensitivity and salt tolerance

Plant receptor proteins are involved in the signaling networks required for defense against pathogens. The novel pepper pathogen-induced gene CaMRP1 was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). This gene is predicted to encode a membrane-located receptor-like protein that has an N-terminal signal peptide and a C-terminal transmembrane helix. A CaMRP1-GFP fusion protein localized primarily to the plasma membrane of plant cells. Strong and early induction of CaMRP1 expression occurred following exposure of pepper plants to Xcv, Colletotricum coccodes, methyl jasmonate (MeJA) and wounding stress. Virus-induced gene silencing (VIGS) of CaMRP1 in pepper conferred enhanced basal resistance to Xcv infection, accompanied by induction of genes encoding basic PR1 (CaBPR1), defensin (CaDEF1) and SAR8.2 (CaSAR82A). In contrast, CaMRP1 overexpression (OX) in transgenic Arabidopsis plants resulted in increased disease susceptibility to Hyaloperonospora parasitica infection. Arabidopsis plants overexpressing CaMRP1 exhibited insensitivity to MeJA by causing reduced expression of MeJA-responsive genes. Overexpression also resulted in tolerance to NaCl and during salt stress, the expression of several abscisic acid-responsive genes was induced. Together, these results suggest that pepper CaMRP1 may belong to a new subfamily of membrane-located receptor-like proteins that regulate disease susceptibility, MeJA-insensitivity and salt tolerance.     

Optimization of Vi capsular polysaccharide production during growth of Salmonella enterica serotype Typhi Ty2 in a bioreactor

Vi capsular polysaccharide is synthesized during growth of Salmonella typhi Ty2 and is spontaneously released from the bacterial cells into the culture medium during culture. Vi production was dependent on cell growth and the greater the cell mass the greater the production of Vi. Using fed batch culture to optimize bacterial growth resulted is an increase in cell mass and consequently Vi production. The yield of Vi obtained in fed batch culture was 415 mg l⁻¹, which was over three times that, obtained in batch culture. A proportion of the Vi remained cell associated in the form of a capsule and at least part of this was released from the bacterial surface by sonication. The size of the Vi polysaccharide produced was consistently high and did not change during the different phases of bacterial growth. The synthesis of Vi was also dependent upon the media components and the fermentation conditions. The presence of high concentrations of glucose at the beginning of growth inhibited the production of Vi, particularly during the stationary phase. At a concentration of 400 mM sodium phosphate the synthesis of Vi was strongly inhibited.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.     

Differential effect of NF-kappaB activity on beta-catenin/Tcf pathway in various cancer cells

beta-Catenin/Tcf and NF-kappaB pathways play an important role in biological functions. We determined the underlying mechanisms of differential interaction between two pathways in various human cancer cell lines. NF-kappaB positively regulated beta-catenin/Tcf pathways in human glioblastoma, whereas it has an opposite effect on beta-catenin/Tcf pathways in colon, liver, and breast cancer cells. Expression of lucine zipper tumor suppressor 2 (lzts2) was positively regulated by NF-kappaB activity in colon, liver, and breast cancer cells, whereas negatively regulated in glioma cells. Downregulation of lzts2 increased the beta-catenin/Tcf promoter activity and inhibited NF-kappaB-induced modulation of the nuclear translocation of beta-catenin. These data indicate that the differential crosstalk between beta-catenin/Tcf and NF-kappaB pathway in various cancer cells is resulted from the differences in the regulation of NF-kappaB-induced lzts2 expression.     

Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2 Å; the ADP-Mg²⁺, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP-Mg²⁺-complex at 2.0 Å. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP-Mg²⁺-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP-Mg²⁺ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK.     

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.