2,3,7,8-tetrachlorobenzo-p-dioxin inhibits proliferation of SK-N-SH human neuronal cells through decreased production of reactive oxygen species

Oxidative stress has been known to be involved in the mechanism of toxic effects of various agents on many cellular systems. In this study we investigated the role of reactive oxygen species (ROS) in 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced neuronal cell toxicity using SK-N-SH human neuroblastoma cells. TCDD inhibited proliferation of the cells in a dose-dependent manner, which was revealed by MTT staining, counting of cells stained with trypan blue and [ 3 H]thymidine uptake assay. TCDD also suppressed the basal generation of ROS in a time- and concentration-dependent manner assessed by 2',7'-dichlorofluorescein fluorescence. In addition, TCDD induced a dose-dependent inhibition of lipid peroxidation, a biomarker of oxidative stress, whereas it significantly increased the level of glutathione (GSH), an intracellular free radical scavenger in the cells. Moreover, TCDD altered the activities of major antioxidant enzymes; increase in superoxide dismutase (SOD) and catalase, but decrease in glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Red). Pretreatment with l -buthionine- S , R -sulfoximine (BSO, 50 μM), an inhibitor of GSH synthesis, significantly prevented the TCDD-induced reduction in lipid peroxidation and cell proliferation. Interestingly, exogenous application of an oxidant, H 2 O 2 (50 μM) markedly restored the inhibited cell proliferation induced by TCDD. Taken together, these results suggest that alteration of cellular redox balance may mediate the TCDD-induced inhibition of proliferation in human neuronal cells.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

Differential potentiative effects of GABA receptor agonists in the production of antinociception induced by morphine and beta-endorphin administered intrathecally in the mouse

The effect of muscimol or baclofen injected intrathecally (i.t.) on the inhibition of the tail-flick response induced by morphine and beta-endorphin administered i.t. was studied in ICR mice. The i.t. injection of muscimol (100 ng) or baclofen (10 ng) alone did not affect the basal inhibition of the tail-flick response. Morphine (0.2 microg) and beta-endorphin (0.1 microg) caused only slight inhibition of the tail-flick response. Baclofen, but not muscimol, injected i.t. enhanced the inhibition of the tail-flick response induced by i.t. administered morphine. Both muscimol and baclofen injected i.t. significantly enhanced i.t. injected beta-endorphin-induced inhibition of the tail-flick response. Our results suggest that the GABA(B), but not GABA(A), receptors located in the spinal cord appear to be involved in enhancing the inhibition of the tail-flick response induced by morphine administered spinally. In addition, both GABA(A) and GABA(B) receptors are involved in enhancing the inhibition of the tail-flick response induced by beta-endorphin administered i.t.     

Characterization of the mitochondrial association of hepatitis B virus X protein, HBx

Chronic infection with hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC). HBx, a protein encoded by HBV is believed to contribute to the development of HCC. HBx was recently shown to associate with mitochondria. In this study, we mapped region(s) of HBx necessary for mitochondrial targeting and showed that a putative transmembrane region (aa 54-70) is required for mitochondrial association. In addition, amino acids in the putative alpha helical regions (aa 75-88 and aa 109-131) seem to aid in the mitochondrial targeting of this protein. We further show that the majority of HBx localizes to the outer mitochondrial membrane based on its sensitivity to trypsin and resistance to alkaline treatment. These studies suggest that the association of HBx with the outer mitochondrial membrane is its intrinsic property. These characterizations define transmembrane and alpha-helical regions of this viral protein as domains of mitochondrial targeting. These studies are further useful in the investigations concerning the physiological significance of the HBx's association with mitochondria and its impact on liver disease pathogenesis.     

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate

Here, we present a simple method for controlling the density of Au nanoparticles (Au NPs) on a modified silicon substrate, by destabilizing the colloidal Au NPs with 3-mercaptopropyltrimethoxylsilane (3-MPTMS) for microelectromechanical-system-based applications to reduce tribological issues. A silicon surface was pretreated with a 3-MPTMS solution, immediately after which thiolated Au NPs were added to it, resulting in their uniform deposition on the silicon substrate. Without any material property change of the colloidal Au NPs, we observed the formation of large clusters Au NPs on the modified silicon surface. Analysis by scanning electron microscopy with energy dispersive X-ray spectroscopy indicated that the addition of 3-MPTMS resulted in an alternation of the chemical characteristics of the solution. Atomic force microscopy imaging supported the notion that silicon surface modification is the most important factor on tribological properties of materials along with ligand-modified Au NPs. The density of Au NPs on a silicon surface was significantly dependent on several factors, including the concentration of colloidal Au NPs, deposition time, and concentration of 3-MPTMS solution, while temperature range which was used throughout experiment was determined to have no significant effect. A relatively high density of Au NPs forms on the silicon surface as the concentrations of Au NPs and 3-MPTMS are increased. In addition, the maximum deposition of Au NPs on silicon wafer was observed at 3 h, while the effects of temperature variation were minimal.     

The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.     

Hwangryun-Hae-Dok-tang (Huanglian-Jie-Du-Tang) extract and its constituents reduce ischemia-reperfusion brain injury and neutrophil infiltration in rats

The preventive effect of Hwangryun-Hae-Dok-tang (HHDT, Huanglian-Jie-Du-Tang), a Chinese herbal medicine, and its ingredients on ischemia/reperfusion-induced brain injury was evaluated in the rat brain. HHDT consists of four herbs, namely, Coptidis rhizoma, Scutellariae radix, Phellodendri cortex, and Gardeniae fructus. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 120 min and reperfusion was continued for 22 h. HHDT (200 mg/kg), Coptidis rhizoma (100 mg/kg), Scutellariae radix (100 mg/kg), Phellodendri cortex (100 mg/kg), and Gardeniae fructus (100 mg/kg) were orally administered, promptly prior to reperfusion and 2 h after reperfusion. Baicalein, a component of Scutellariae radix, was also examined at a dosage of 50 mg/kg given 2 h apart, promptly prior to and 2 h after reperfusion. Total infarction volume in the ipsilateral hemisphere of ischemia/reperfusion rats was significantly lowered by treatment with HHDT, Scutellariae radix, and balicalein. However, the other ingredient of HHDT did not show any ameliorating effects on total infarction volume. The inhibiting effect of Scutellariae radix on total infarction volume was much higher than that of the others. In addition, HHDT, Scutellariae radix, and baicalein significantly inhibited myeloperoxidase (MPO) activity, an index of neutrophil infiltration in ischemic brain tissue at about the same rate (30%). There was marked mismatch between total infarction volume and MPO activity in the Scutellariae radix-treated rats but not in the HHDT- and baicalein-treated groups. Our findings suggest that Scutellariae radix as an ingredient of HHDT plays a crucial protective role in ischemia-induced brain injury. In addition, it is apparent that the effect of Scutellariae radix is the result, in part, of baicalein, a compound contained in Scutellariae radix.     

Differential effects of cholera toxin and pertussis toxin on the c-fos and c-jun mRNA expression in rat C6 glioma cells

Cholera toxin (CTX) increased c-fos mRNA level whereas it down-regulated the c-jun mRNA level in rat C6 glioma cells. In contrast to the action of CTX, pertussis toxin (PTX) did not affect either c-fos or c-jun mRNA level. The elevated c-fos mRNA level induced by CTX was significantly inhibited by the co-treatment with dexamethasone (DEX). However, DEX did not affect CTX-induced down-regulation of c-jun mRNA level. Cycloheximide (CHX) increased c-fos and c-jun mRNA levels. CHX caused a super-induction of CTX-induced c-fos mRNA level. Our results suggest that CTX-, but not PTX-, sensitive G-proteins may play an important role for c-fos mRNA up-regulation and c-jun mRNA down-regulation. In addition, DEX appears to have a selective inhibitory action against c-fos mRNA expression regulated by CTX. Ongoing protein synthesis inhibition is required for the superinduction of c-fos, but not c-jun, mRNA induced by CTX.     

Prevalence of Toxocara canis, Toxascaris leonina and Dirofilaria immitis in dogs in Chuncheon, Korea (2004)

The intestines and hearts of dogs were examined for Toxocara canis, Toxascaris leonina, and Dirofilaria immitis, after necropsy between June 26 and September 29, 2004 in Chuncheon, Korea. Of the 662 dogs examined, 6 were infected with T. canis (0.9%), 86 with T. leonina (13.0%). Fifty dogs were infected with D. immitis among 500 dogs examined (10.0%). Five were co-infected with T. canis and T. leonina, and three were co-infected with T. leonina and D. immitis. The cumulative positive infection rate for three species was 134/662 (20.2%). Considering previously reported seropositive rates of T. canis excretory-secretory antigen, i.e., 5% in the adult population in Korea, the possibility of toxocariasis caused by T. leonina should be reevaluated.