Identification of the <Emphasis Type="Italic">Vibrio vulnificus ahpC</Emphasis>l gene and its influence on survival under oxidative stress and virulence

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H₂O₂. In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H₂O₂ and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H₂O₂ in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.     

Monitoring of acaricide resistance of Tetranychus urticae (Acari: Tetranychidae) from Korean apple orchards

Tetranychus urticae populations were collected from ten commercial apple orchards and their susceptibilities to 12 acaricides were tested using a leaf disc bioassay. The resistance of each T. urticae population was reported as the LC₅₀ value, the resistance ratio (RR) and the slope of the probit–concentration regression. Cross resistances of T. urticae populations were estimated using the Spearman's rank correlation coefficient. Most local populations showed low resistance levels (RR ≤ 10). Development of resistance to METI and pyrethroid acaricides differed among local populations. The highest RR value (154.6) was found in the Uiseong population to tebufenpyrad. The Geochang population was highly resistant, especially to METI and pyrethroid acaricides. T. urticae populations collected from Suwon, Chungju, Yeongju and Geochang showed moderate resistance (10 < RR ≤ 40) to more than two acaricides. Resistance ratios to abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin were low (RR ≤ 10) in all populations. The LC₅₀ values of abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin ranged from 0.06 to 0.2 mg/l, from 0.67 to 3.38 mg/l, from 10.12 to 40.85 mg/l and from 0.47 to 3.01 mg/l, respectively. We discuss possible cross-resistance to acaricides using Spearman's rank correlation coefficient.     

Proteomic characterization of the sulfur-reducing hyperthermophilic archaeon Thermococcus onnurineus NA1 by 2-DE/MS–MS

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, was isolated from a deep-sea hydrothermal vent area in Papua New Guinea. The strain requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides, amino acids and sugars. Recently, genome sequencing of Thermococcus onnurineus NA1 was completed. In this study, 2-DE/MS–MS analysis of the cytosolic proteome was performed to elucidate the metabolic characterization of Thermococcus onnurineus NA1 at the protein level. Among the 1,136 visualized protein spots, 110 proteins were identified. Enzymes related to metabolic pathways of amino acids utilization, glycolysis, pyruvate conversion, ATP synthesis, and protein synthesis were identified as abundant proteins, highlighting the fact that these are major metabolic pathways in Thermococcus onnurineus NA1. Interestingly, multiple spots of phosphoenolpyruvate synthetase and elongation factor Tu were found on 2D gels generated by truncation at the N-terminus, implicating the cellular regulatory mechanism of this key enzyme by protease degradation. In addition to the proteins involved in metabolic systems, we also identified various proteases and stress-related proteins. The proteomic characterization of abundantly induced proteins using 2-DE/MS–MS enables a better understanding of Thermococcus onnurineus NA1 metabolism.     

Arabidopsis thaliana PRP40s are RNA polymerase II C-terminal domain-associating proteins

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions as a scaffold for RNA processing machineries that recognize differentially phosphorylated conserved (YSPTSPS)_n repeats. Evidence indicates that proteins that regulate the phosphorylation status of the CTD are determinants of growth, development, and stress responses of plants; however, little is known about the mechanisms that translate the CTD phosphoarray into physiological outputs. We report the bioinformatic identification of a family of three phospho-CTD-associated proteins (PCAPs) in Arabidopsis and the characterization of the AtPRP40 (Arabidopsis thaliana PRE-mRNA-PROCESSING PROTEIN 40) family as PCAPs. AtPRP40s-CTD/CTD-PO₄ interactions were confirmed using the yeast two-hybrid assay and far-Western blotting. WW domains at the N-terminus of AtPRP40b mediate the AtPRP40b-CTD/CTD-PO₄ interaction. Although AtPRP40s interact with both phosphorylated and unphosphorylated CTD in vitro, there is a strong preference for the phosphorylated form in Arabidopsis cell extract. AtPRP40s are ubiquitously expressed and localize to the nucleus. These results establish that AtPRP40s are specific PCAPs, which is consistent with the predicted function of the AtPRP40 family in pre-mRNA splicing.     

COMP-Ang1 ameliorates leukocyte adhesion and reinforces endothelial tight junctions during endotoxemia

Endotoxemia is characterized by multiple dysfunctions of the micro-vascular endothelium. Of these, vascular leakage is an initial event that aggravates vascular dysfunction can lead to systemic vascular collapse and organ failure. Thus, prevention of vascular leakage may ameliorate endotoxin-induced dysfunctions of blood vessels. Here we examine the effect of an angiopoietin-1 variant, COMP-Ang1, on endotoxin-induced vascular leakage in mice. COMP-Ang1 significantly reduced endotoxin-induced vascular leakage in the lung, heart, and kidney, but not in liver or intestine. Interestingly, COMP-Ang1 attenuated endotoxin-induced lung damage, presumably due to reduced infiltration of macrophages. Moreover, COMP-Ang1 restored the level of PECAM-1 expression, which is significantly reduced by endotoxin challenge. This study suggests that COMP-Ang1 reduces endotoxin-induced vascular leakage by restoration of cellular junctions and subsequent attenuation of leukocyte infiltration.     

Nitrated fatty acids prevent TNFα-stimulated inflammatory and atherogenic responses in endothelial cells

Nitration products (nitroalkenes) of linoleic acid (LNO₂) and oleic acid (OA-NO₂) can act as endogenous PPARγ ligands with electrophilic properties to exert anti-inflammatory effects on atherosclerotic plaques in the vasculature. Here, we show that OA-NO₂ and LNO₂ prevent tumor necrosis factor α (TNFα)-stimulated inflammatory and atherogenic responses in human umbilical vein endothelial cells (HUVECs). Both OA-NO₂ and LNO₂ prevented TNFα-stimulated release of the cytokines, IL-6, IL-8, IL-12/p40, IFNγ, MCP-1, and IP-10, and inhibited NF-κB activation. OA-NO₂ and LNO₂ also blocked TNFα-induced expression of the adhesion molecules, ICAM-1, VCAM-1, and E-selectin, and suppressed monocyte adhesion to HUVECs. In each case, OA-NO₂ was more potent and efficacious than was LNO₂, possibly due to increased stability in aqueous media. Collectively, these results substantiate a new functional role for nitrated fatty acids, demonstrating that OA-NO₂ and LNO₂ exert an anti-inflammatory function against the inflammatory cascade initiated by the representative pro-inflammatory cytokine, TNFα.     

Mitochondrial DNA Sequence Variation of the Swallowtail Butterfly, Papilio xuthus, and the Cabbage Butterfly, Pieris rapae

We analyzed a portion of mitochondrial COI gene sequences (658 bp) to investigate the genetic diversity and geographic variation of the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae), and the cabbage butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Papilio xuthus showed a moderate level of sequence divergence (0.91% at maximum) in 15 haplotypes, whereas Pi. rapae showed a moderate to high level of sequence divergence (1.67% at maximum) in 30 haplotypes, compared with other relevant studies. Analyses of population genetic structure showed that most populations are not genetically differentiated in both species. The distribution pattern of both species appears to be consistent with category IV of the phylogeographic pattern sensu Avise: a phylogenetic continuity, an absence of regional isolation of mtDNA clones, and extensive distribution of close clones. The observed pattern of genetic diversity and geographic variation of the two butterfly species seem to reflect the abundant habitats, abundant host plants, and flying abilities in connection with the lack of historical biogeographic barriers.     

Measurement of body segment parameters using dual energy X-ray absorptiometry and three-dimensional geometry: An application in gait analysis

Body segment parameters (BSP) are essential input for the computations in kinetics of motion applied in the field of biomechanics. These data are usually obtained from population-specific predictive equations which present limitations in being representative of the population under study. More recently, medical imaging techniques have been adopted but are limited to two-dimensional (2-D) measurements or required extensive tomographic images for three-dimensional (3-D) reconstruction. We proposed an in vivo method to measure 3-D BSP using X-ray imaging and 3-D exterior geometry. Criterion values of the BSP were determined using magnetic resonance imaging (MRI) which has previously been validated. Errors for all BSP values were less than 2% when values derived from our method were compared to the criterion values. We found no significant difference between our method and four selected BSP models in both stance and swing phase. Significant phase effects were observed for our method and other BSP models between stance and swing phase. Significant differences (p<0.05) between root mean square error (RMSE) ranged from 0.0177 to 0.0234 and 0.0234 to 0.097 Nm kg^?1 for the knee and hip joints, respectively. However, these BSP variations brought about effects on moment output that were less than 0.09 Nm kg^?1. Our findings suggest joint kinetic computations during normal gait are relatively insensitive to BSP variations. However, the influence of BSP cannot be undermined in movements that generate higher acceleration at the limbs. Considering the accuracy of our method, it could be used as a novel in vivo method to obtain direct 3-D BSP measurements.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.