Ultrastructural and immunohistochemical analysis of cholangiocarcinoma in immunized Syrian golden hamsters infected with Opisthorchis viverrini and administered with dimethylnitrosamine.

Utilizing the experimental model in Syrian golden hamsters, we explored the role of immunization in carcinogenesis. The animals, which were infected with liver flukes (Opisthorchis viverrini), and administered a subcarcinogenic dose of dimethylnitrosamine, developed cancer. Pre-immunizing with a crude somatic antigen did not reduce cancer development, but accelerated carcinogenesis. Histopathological analysis of the cancer tissues was done once at week 30 and again at week 39 using H and E staining, immunostaining for the p53 tumor suppressor phosphoprotein, and electron microscopy. Thirty weeks after immunization, the immunized hamsters developed tubular adenocarcinoma at a higher rate (71.43%) than the non-immunized group (20.00%). This rate (20.00%) increased to 63.64% by week 39. The small foci cancer in the non-immunized group decreased in frequency from 80.00% (at week 30) to 36.36% (by week 39), suggesting the small foci cancer progressed to tubular adenocarcinoma during the 9-week interval. Most of the observed tubular adenocarcinoma was well differentiated. Nearly all hamsters that tested positive for cancer also tested positive for p53 immunostaining in the epithelia of the small bile ducts. The positive reaction for p53-immunostaining was localized in the rough endoplasmic reticulum, Golgi apparatus and perinuclear membranes. The electron micrographs of these positive p53-immunostained cells showed characteristics of early cancer. The detection of p53 in early cancer development makes it a candidate as a tumor marker.     

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.     

Dopamine beta-hydroxylase like immunoreactive cells in the frog taste disc

We immunohistochemically examined the existence of dopamine beta-hydroxylase (DBH), a noradrenalin (NA)-synthesizing enzyme from dopamine, in the taste disc of frog, Rana catesbeiana. DBH-like immunoreactive cells were located in the intermediate layer in the taste disc; the cells showed an apical process reaching the surface of the disc and one or several basal processes. Cells with a thick apical process and those with a thin apical process were both immunoreactive: these cells corresponded to type II and III receptor cells of the frog taste disc. Immunoreactive granules were observed in the cytoplasm of those cells. In the frog taste disc, only type III cells are reported to have afferent synapses with the nerve via basal processes but those basal processes have not been reported in type II cells. In the present study, we found that type II-like cells possessed a long basal process extending toward the basal lamina. Mucous (type Ia) cells, wing (type Ib) cells, and glia-like sustentacular (type Ic) cells were all immunohistochemically unreactive. The present observations support the argument that NA (or adrenalin) may work as a chemical transmitter in the frog taste organ.     

Rhamnolipid-dependent spreading growth of Pseudomonas aeruginosa on a high-agar medium: marked enhancement under CO2-rich anaerobic conditions

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions.     

Discovery of novel and orally active NR2B-selective N-methyl-D-aspartate (NMDA) antagonists, pyridinol derivatives with reduced HERG binding affinity

Novel NR2B antagonists with an amide tether were found by an approach to avoid pharmacophoric similarity to dofetilide. Structure-activity relationship investigation led to N-[cis-4-hydroxy-4-(5-hydroxypyridin-2-yl)cyclohexyl]-3-henylpropanamide as an orally active NR2B-subtype selective N-methyl-D-aspartate (NMDA) receptor antagonist with very weak HERG (human ether-a-go-go related gene) binding (IC(50)> 30 microM). This compound exhibited potent in vivo anti-allodynic activity in the mouse partial sciatic nerve ligation (PSL) model (minimum effective dose=10 mg/kg, po).     

The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast

Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.