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.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

PPARγ ligands induce growth inhibition and apoptosis through p63 and p73 in human ovarian cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists, including thiazolidinediones (TZDs), can induce anti-proliferation, differentiation, and apoptosis in various cancer cell types. This study investigated the mechanism of the anticancer effect of TZDs on human ovarian cancer. Six human ovarian cancer cell lines (NIH:OVCAR3, SKOV3, SNU-251, SNU-8, SNU-840, and 2774) were treated with the TZD, which induced dose-dependent inhibition of cell growth. Additionally, these cell lines exhibited various expression levels of PPARγ protein as revealed by Western blotting. Flow cytometry showed that the cell cycle was arrested at the G1 phase, as demonstrated by the appearance of a sub-G1 peak. This observation was corroborated by the finding of increased levels of Bax, p21, PARP, and cleaved caspase 3 in TGZ-treated cells. Interestingly, when we determined the effect of p53-induced growth inhibition in these three human ovarian cancer cells, we found that they either lacked p53 or contained a mutant form of p53. Furthermore, TGZ induced the expression of endogenous or exogenous p63 and p73 proteins and p63- or p73-directed short hairpin (si) RNAs inhibited the ability of TGZ to regulate expression of p21 in these cells. Thus, our results suggest that PPARγ ligands can induce growth suppression of ovarian cancer cells and mediate p63 and p73 expression, leading to enhanced growth inhibition and apoptosis. The tumor suppressive effects of PPARγ ligands may have applications for the treatment of ovarian cancer.     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

Brownian dynamics simulations of ion transport through the VDAC

It is important to gain a physical understanding of ion transport through the voltage-dependent anion channel (VDAC) because this channel provides primary permeation pathways for metabolites and electrolytes between the cytosol and mitochondria. We performed grand canonical Monte Carlo/Brownian dynamics (GCMC/BD) simulations to explore the ion transport properties of human VDAC isoform 1 (hVDAC1; PDB:2K4T) embedded in an implicit membrane. When the MD-derived, space-dependent diffusion constant was used in the GCMC/BD simulations, the current-voltage characteristics and ion number profiles inside the pore showed excellent agreement with those calculated from all-atom molecular-dynamics (MD) simulations, thereby validating the GCMC/BD approach. Of the 20 NMR models of hVDAC1 currently available, the third one (NMR03) best reproduces both experimental single-channel conductance and ion selectivity (i.e., the reversal potential). In addition, detailed analyses of the ion trajectories, one-dimensional multi-ion potential of mean force, and protein charge distribution reveal that electrostatic interactions play an important role in the channel structure and ion transport relationship. Finally, the GCMC/BD simulations of various mutants based on NMR03 show good agreement with experimental ion selectivity. The difference in ion selectivity between the wild-type and the mutants is the result of altered potential of mean force profiles that are dominated by the electrostatic interactions.     

Overexpression of tumor necrosis factor receptor-associated protein 1 (TRAP1), leads to mitochondrial aberrations in mouse fibroblast NIH/3T3 cells

Cancer cells undergo uncontrolled proliferation, and aberrant mitochondrial alterations. Tumor necrosis factor receptorassociated protein 1 (TRAP1) is a mitochondrial heat shock protein. TRAP1 mRNA is highly expressed in some cancer cell lines and tumor tissues. However, the effects of its overexpression on mitochondria are unclear. In this study, we assessed mitochondrial changes accompanying TRAP1 overexpression, in a mouse cell line, NIH/3T3. We found that overexpression of TRAP1 leads to a series of mitochondrial aberrations, including increase in basal ROS levels, and decrease in mitochondrial biogenesis, together with a decrease in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA levels. We also observed increased extracellular signal-regulated kinase (ERK) phosphorylation, and enhanced proliferation of TRAP1 overexpressing cells. This study suggests that overexpression of TRAP1 might be a critical link between mitochondrial disturbances and carcinogenesis. [BMB Reports 2014; 47(5): 280-285]     

Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.

Two genes, xynA and xynB, encoding xylanases from Paenibacillus sp. KCTC 8848P were cloned and expressed in Escherichia coli, and their nucleotide sequences were determined. The xylanases of E. coli transformants were released into the extracellular culture fluid in the absence of xylan. The structural gene of xynA 636 bp, encoded a protein of 212 amino acids, while the xynB gene consisted of 951 bp open reading frame for a protein of 317 amino acids. The amino acid sequence of the xynAgene showed 83% similarity to the xylanase of Aeromonas caviae, and belonged to the family 11 glycosyl hydrolases. The deduced amino acid sequence of the xynB gene, however, showed 51% similarity to the xylanase of Rhodothermus marinus, and belonged to the family 10 glycosyl hydrolases.     

Induction of scavenger receptor class B type I is critical for simvastatin enhancement of high-density lipoprotein-induced anti-inflammatory actions in endothelial cells

Changes in plasma lipoprotein profiles, especially low levels of high-density lipoprotein (HDL), are a common biomarker for several inflammatory and immune diseases, including atherosclerosis and rheumatoid arthritis. We examined the effect of simvastatin on HDL-induced anti-inflammatory actions. HDL and sphingosine 1-phosphate (S1P), a bioactive lipid component of the lipoprotein, inhibited TNF alpha-induced expression of VCAM-1, which was associated with NO synthase (NOS) activation, in human umbilical venous endothelial cells. The HDL- but not S1P-induced anti-inflammatory actions were enhanced by a prior treatment of the cells with simvastatin in a manner sensitive to mevalonic acid. Simvastatin stimulated the expression of scavenger receptor class B type I (SR-BI) and endothelial NOS. As for S1P receptors, however, the statin inhibited the expression of S1P(3) receptor mRNA but caused no detectable change in S1P(1) receptor expression. The reconstituted HDL, a stimulator of SR-BI, mimicked HDL actions in a simvastatin-sensitive manner. The HDL- and reconstituted HDL-induced actions were blocked by small interfering RNA specific to SR-BI regardless of simvastatin treatment. The statin-induced expression of SR-BI was attenuated by constitutively active RhoA and small interfering RNA specific to peroxisome proliferator-activated receptor-alpha. Administration of simvastatin in vivo stimulated endothelial SR-BI expression, which was accompanied by the inhibition of the ex vivo monocyte adhesion in aortas from TNF alpha-injected mice. In conclusion, simvastatin induces endothelial SR-BI expression through a RhoA- and peroxisome proliferator-activated receptor-alpha-dependent mechanism, thereby enhancing the HDL-induced activation of NOS and the inhibition of adhesion molecule expression.     

MMP13 is a critical target gene during the progression of osteoarthritis

Introduction

Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.

Methods

To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13^fx/fx (Mmp13^Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.

Results

The OA progression was decelerated in Mmp13^Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13^Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13^Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13^Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13^Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.

Conclusions

Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.