Enhanced expression of glutamate decarboxylase 65 improves symptoms of rat parkinsonian models

In this study, we report the amelioration of parkinsonian symptoms in rat Parkinson's disease (PD) models, as a result of the expression of glutamate decarboxylase (GAD) 65 with a modified cytomegalovirus (CMV) promoter. The transfer of the gene for gamma-amino butryic acid (GAD), the rate-limiting enzyme in gama-amino butrylic acid (GABA) production, has been investigated as a means to increase inhibitory synaptic activity. Electrophysiological evidence suggests that the transfer of the GAD65 gene to the subthalamic nucleus (STN) can change the excitatory output of this nucleus to inhibitory output. Our in vitro results also demonstrated higher GAD65 expression in cells transfected with the JDK promoter, as compared to cells transfected with the CMV promoter. Also, a rat PD model in which recombinant adeno-associated virus-2 (rAAV2)-JDK-GAD65 was delivered into the STN exhibited significant behavioral improvements, as compared to the saline-injected group. Interestingly, we observed that these behavioral improvements were more obvious in rat PD models in which rAAV2-JDK-GAD65 was injected into the STN than in rat PD models in which rAAV2-CMV-GAD65 was injected into the STN. Moreover, according to electrophysiological data, the rAAV2-JDK-GAD65-injected group exhibited more constant improvements in firing rates than did the rAAV2-CMV-GAD65-injected group. These data indicate that the JDK promoter, when coupled with GAD65 expression, is more effective with regard to parkinsonian symptoms than is the CMV promoter.     

CASPASE-8 gene is inactivated by somatic mutations in gastric carcinomas

Several lines of evidence indicate that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-8 activation plays a central role in the initiation phase of apoptosis. The aim of this study was to explore the possibility that genetic alteration of CASPASE-8 gene is involved in the development of human cancers, including gastric cancers. We have analyzed the entire coding region of human CASPASE-8 gene for the detection of somatic mutations in 162 gastric carcinomas (40 early and 122 advanced cancers), 185 non-small cell lung cancers, 93 breast carcinomas, and 88 acute leukemias by PCR-single-strand conformation polymorphism. Of the cancers analyzed, 13 cancers harbored CASPASE-8 somatic mutations. Interestingly, all of the mutations were detected in the advanced gastric cancers (10.7% of the 122 samples). We expressed the tumor-derived caspase-8 mutants in 293T, 293, and HT1080 cells and found that most of the mutants (9 of the 10 mutations tested) markedly decreased the cell death activity of caspase-8. In addition, in the cells with the inactivating caspase-8 mutants, cleavage of poly(ADP-ribose)polymerase was markedly reduced compared with that of wild-type caspase-8. The occurrence of CASPASE-8 mutation and the inactivation of cell death activity by the mutants suggest that CASPASE-8 gene mutation may affect the pathogenesis of gastric cancers, especially at the late stage of gastric carcinogenesis.     

Regulation of tumor angiogenesis by fastatin, the fourth FAS1 domain of betaig-h3, via alphavbeta3 integrin

We previously reported that the FAS1 domains of betaig-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with alphavbeta3 integrin and regulate angiogenesis. In the present study, we show that the fourth FAS1 domain, designated fastatin, inhibits endothelial adhesion and migration, not only to betaig-h3, but also fibronectin and vitronectin, in a RGD-dependent manner. Fastatin and other FAS1 domains suppress endothelial cell tube formation and in vivo neovascularization in a Matrigel plug assay. The antiangiogenic activity of fastatin is associated with antitumor activity in mouse tumor models. Fastatin additionally induces apoptosis in several cells expressing alphavbeta3 integrin, including endothelial cells. Binding of fastatin to alphavbeta3 integrin inhibits phosphorylation of focal adhesion kinase, Raf, extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin. Fastatin is thus the first endogenous angiogenesis regulator identified that inhibits both endothelial cell migration and growth by binding to alphavbeta3 integrin. Our data suggest that FAS1 domains from all possible forms of the four human FAS1 family proteins are potential endogenous regulators for pathologic angiogenesis. Moreover, FAS1 domains such as fastatin may be developed into drugs for blocking tumor angiogenesis.     

Detection of Campylobacter jejuni in dairy farm environmental samples using SYBR Green real-time polymerase chain reaction

The aim of this study was to evaluate a SYBR Green based real-time PCR assay using well-characterized primers to detect Campylobacter jejuni in naturally contaminated dairy farm environmental samples. Specificity of the assay was determined with 62 C. jejuni strains and 120 non-C. jejuni strains. Peak melting temperature obtained with melting curves specific for C. jejuni was 77.5 degrees C. Standard curves were constructed using mean threshold cycle (C(T)) and various concentrations of C. jejuni ranging from 10(0) to 10(8) colony forming units (CFU)/mL, which resulted in a linear relationship between C(T) and log input DNA. Correlation coefficients of standard curves based on pure culture of C. jejuni in broth and spiked cells in lagoon water were R(2) = 0.995 (slope = 3.21) and R(2) = 0.988 (slope = 3.22), respectively, and sensitivity limits were <10 and >10(3) CFU/mL, respectively. After 24-h enrichment, total C. jejuni counts of all samples spiked with 10(0) CFU/mL reached >10(5) CFU/mL, and the detection limit was improved from >10(3) CFU/mL to <10 CFU/mL of inoculum in broth. Eighty-two dairy farm environmental samples, including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding material, were analyzed. The real-time PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of these samples being culture positive for C. jejuni. All samples that were positive by standard culture methods were also positive by the real-time PCR method. Mean C( T ) values of 48-h enriched cultures for 17 PCR-positive/culture-positive samples and eight PCR-positive/culture-negative samples were 21.4 +/- 3.6, and 34.6 +/- 1.5 (p < 0.0001), respectively. C( T ) values for negative samples were >38.0. These results indicate that the SYBR Green real-time PCR assay provides a specific, reproducible, and simple method for detecting C. jejuni in dairy farm environmental samples.     

Laboratory and field validation of the GC-NPD method for the measurement of formaldehyde in the workplace

Formaldehyde is classified as a suspected or probable human carcinogen by several organizations. Since conventional sampling and analytical methods for airborne formaldehyde show relatively poor sensitivity, an improved method is needed. The aim of this study was to develop a new analytical method for measuring the airborne formaldehyde concentrations in workplaces and to evaluate the performance of the method through laboratory and field tests. The method employs a sampling tube containing silica gel coated with 2, 4-dinitrophenylhydrazine (2,4-DNPH), which produces 2,4-DNPH-formaldehyde derivative with formaldehyde. Then the 2,4-DNPH-formaldehyde derivative is analyzed using gas chromatography (GC) equipped with a nitrogen-phosphorus detector (GC-NPD). In laboratory tests, the new method, referred to as the GC-NPD method, was as sensitive as the National Institute for Occupational Safety and Health (NIOSH) analytical method, which uses high-performance liquid chromatography equipped with ultraviolet detector. The total analytical precision and 95% confidence limit of the estimated total standard error for the GC-NPD method were 0.009 and +/- 12.0%, respectively, which satisfied the OSHA sampling and analytical criteria. In field tests, the overall uncertainty of the GC-NPD method was 11.2%, which satisfied the NIOSH criteria for sampling and analytical methods. The GC-NPD method with a 2,4-DNPH coated adsorbent sampler for the determination of airborne formaldehyde concentration showed good performance with acceptable accuracy and precision.     

Antiinflammatory effects of chiisanoside and chiisanogenin obtained from the leaves of Acanthopanax chiisanensis in the carrageenan- and Freund's complete adjuvant-induced rats

To find the antiinflammtory constituents of Acanthopanax chiisanensis (Araliaceae) leaves, phytochemical isolation procedures were performed by activity-guided fractionation in carrageenan- and Freund's complete adjuvant (FCA) reagent-induced rat models, respectively. In the two assay system, the MeOH extract (100 and 250 mg/kg, p.o.) showed significant antiinflammtory effects. Since BuOH extract among the fractionated extracts exhibited the most potent effect, it was subjected to column chromatography to yield a main triterpene glycoside, chiisanoside (1). This compound was hydrolyzed in alkaline solution to find the biological activity of produced aglycone, chiisanogenin (1a). Oral treatment with compounds 1 and 1a produced significant antiinflammtory effects at 10 and 30 mg/kg dose, and 1a was more potent than 1. The antiiflammtory effects of the two compounds were supported by the reduction of carrageenan-induced lipid peroxidation and hydroxy radical in serum. Furthermore, treatment with 1 and 1a significantly reduced rheumatoid arthritis (RA) and C-reactive protein (CRP) factors in the rat induced by Freund's complete adjuvant reagent. Compounds, 1 and 1a, inhibited xanthine oxidase activity and increased superoxide dismutase (SOD), glutathione peroxidase and catalase indicating that both compounds scavenged reactive oxygen species (ROS).     

Induction of nuclear factor-kappaB activation through TAK1 and NIK by diesel exhaust particles in L2 cell lines

Diesel exhaust particles (DEPs) are known to induce allergic responses in airway epithelial cells, such as the production of various cytokines via nuclear factor-kappa B (NF-kappaB). However, the intracellular signal transduction pathways underlying this phenomenon have not been fully examined. This study showed that DEP induced NF-kappaB activity via transforming growth factor-beta activated kinase 1 (TAK1) and NF-kappaB-inducing kinase (NIK) in L2 rat lung epithelial cells. DEP induced the NF-kB dependent reporter activity approximately two- to three-fold in L2 cells. However, this effect was abolished by the expression of the dominant negative forms of TAK1 or NIK. Furthermore, it was shown that DEP induced TAK1 phosphorylation in the L2 cells. These results suggest that TAK1 and NIK are important mediators of DEP-induced NF-kappaB activation.     

Protection by a transdermal patch containing physostigmine and procyclidine of soman poisoning in dogs

The prophylactic efficacy of a combinational patch system containing physostigmine and procyclidine against soman intoxication was evaluated using dogs. Female beagle dogs (body weights 9-10 kg) were shaved on the abdominal side, attached with a matrix-type patch (7x7 cm) containing 1.5% of physostigmine plus 6% procyclidine for 2 days, and challenged with subcutaneous injection of serial doses (2-10 LD50) of soman. Separately, in combination with the patch attachment, atropine (2 mg/dog) plus 2-pralidoxime (600 mg/dog) or atropine plus 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 500 mg/dog) were injected intramuscularly 1 min after soman poisoning. The LD50 value of soman was determined to be 9.1 microg/kg, and high doses (> or = 1.4 LD50) of soman induced salivation, emesis, defecation and diarrhea, tremors and seizures, and recumbency of dogs, leading to 100% mortality in 24 h. The prophylactic patch, which led to mean 18.5-18.8% inhibition of blood cholinesterase activity by physostigmine and mean 7.9-8.3 ng/ml of blood concentration of procyclidine, exerted a high protection ratio (4.7 LD50), in comparison with relatively-low effects of traditional antidotes, atropine plus 2-pralidoxime (2.5 LD50) and atropine plus HI-6 (2.7 LD50). Noteworthy, a synergistic increase in the protection ratio was achieved by the combination of the patch with atropine plus HI-6 (9 LD50), but not with atropine plus 2-pralidoxime (5 LD50). In addition, the patch system markedly attenuated the cholinergic signs and seizures induced by soman, especially when combined with atropine plus HI-6, leading to elimination of brain injuries and physical incapacitation up to 6 LD50 of soman poisoning. Taken together, it is suggested that the patch system containing physostigmine and procyclidine, especially in combination with atropine and HI-6, could be a choice for the quality survival from nerve-agent poisoning.     

Preparation and characterization of solid dispersions of itraconazole by using aerosol solvent extraction system for improvement in drug solubility and bioavailability

The objective of this study was to elucidate the feasibility to improve the solubility and bioavailability of poorly water-soluble itraconazole via solid dispersions by using supercritical fluid (SCF). Solid dispersions of itraconazole with hydrophilic polymer, HPMC 2910, were prepared by the aerosol solvent extraction system (ASES) under different process conditions of temperature/pressure. The particle size of solid dispersions ranged from 100 to 500 nm. The equilibrium solubility increased with decrease (15 to 10 MPa) in pressure and increase (40 to 60 degrees C) in temperature. The solid dispersions prepared at 45 degrees C/15 MPa showed a slight increase in equilibrium solubility (approximately 27-fold increase) when compared to pure itraconazole, while those prepared at 60 degrees C/10 MPa showed approximately 610-fold increase and no endothermic peaks corresponding to pure itraconazole were observed, indicating that itraconazole might be molecularly dispersed in HPMC 2910 in the amorphous form. The amorphous state of itraconazole was confirmed by DSC/XRD data. The pharmacokinetic parameters of the ASES-processed solid dispersions, such as Tmax, Cmax, and AUC(o-24 h) were almost similar to Sporanox capsule which shows high bioavailability. Hence, it was concluded that the ASES process could be a promising technique to reduce particle size and/or prepare amorphous solid dispersion of drugs in order to improve the solubility and bioavailability of poorly water-soluble drugs.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide and cyclooxygenase-2 by chiisanoside via suppression of nuclear factor-kappaB activation in RAW 264.7 macrophage cells

In the present study, the effects of several triterpenes isolated from the leaves of Acanthopanax chiisanensis (Araliaceae), namely, chiisanoside, isochiisanoside, 22-hydroxychiisanoside and chiisanogenin (the aglycone of chiisanoside) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Of the triterpenes tested, chiisanoside was found to most potently inhibit NO and PGE2 production. In addition, chiisanoside significantly reduced the release of inflammatory cytokines like TNF-alpha and IL-1beta. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 enzyme were found to be inhibited by chiisanoside in a concentration-dependent manner. Furthermore, chiisanoside inhibited the nuclear factor-kappaB (NF-kappaB) activation induced by LPS and this was associated with a reduction in p65 protein in the nucleus and with the phosphorylations of ERK1/2 and JNK MAP kinases. Taken together, our data indicate that the anti-inflammatory properties of chiisanoside might be the result from the inhibition of iNOS, COX-2, TNF-alpha and IL-1beta expression through the down-regulation of NF-kappaB binding activity.