DNA microarray analysis of changes in gene expression induced by 1,25-dihydroxyvitamin D3 in human promyelocytic leukemia HL-60 cells

Using a DNA microarray, we analyzed about 16,600 genes for changes in expression associated with the differentiation of human promyelocytic leukemia HL-60 cells induced by 1alpha,25-dihydroxyvitamin D3 (DVD). Many of the up-regulated genes could be correlated to differentiation-associated changes toward a monocyte/macrophage lineage, and many down-regulated genes could be correlated to repressed cell growth. The present study revealed the down-regulated gene expression of importins and exportins 1, 5, 7, and exportin-tRNA. Thus, the present results confirmed our previous findings of down-regulation of exportin 1 and exportin-tRNA by DVD. Gene expression of exportin 6 is suggested to be regulated differently from that of exportins 1, 5, 7, and exportin-tRNA. The down-regulation of nuclear transport factors may be deeply associated with the differentiation of HL-60 cells induced by DVD.     

Fixation conditions affect the immunoreactivity of gustducin in rat vallate taste buds

This study aimed to examine gustducin immunoreactivities when adopting various immunostaining conditions in rat vallate taste buds. The occurrence and intensity of the immunoreactivities exhibited specific patterns in accordance with the fixation time. The immunoreactions were localized to only taste hairs, the upper part of the taste bud, after short fixation periods but then to the cell-body cytoplasm excluding the taste hairs after long fixation periods. These immunohistochemical data suggest that the staining protocols, especially the fixation time, cause discrepancies in gustducin immunoreactivities.     

Methods of preventing vinorelbine-induced phlebitis: an experimental study in rabbits

Purpose: In order to identify methods for preventing phlebitis caused by intravenous administration of vinorelbine (VNR), we established a procedure for estimating the severity of phlebitis in an animal model.Methods: Four different factors (administration rate, dilution, flushing, and infusion of fat emulsion) were evaluated for alleviation of phlebitis caused by VNR infusion. VNR was diluted with normal saline to prepare test solutions with concentrations of 0.6 mg/mL or 0.3 mg/mL for infusion into the auricular veins of rabbits. Two days after VNR infusion, the veins were subjected to histopathological examination.Results: VNR did not cause obvious loss of venous endothelial cells, the most sensitive and common feature of phlebitis, but VNR infusion led to inflammatory cell infiltration, edema, and epidermal degeneration.Tissue damage was significantly decreased by shortening the administration time and by diluting the VNR solution for infusion from 0.6 mg/mL to 0.3 mg/mL. However, there was no effect of flushing with normal saline after VNR infusion, while treatment with fat emulsion before and after VNR infusion only had a minimal effect.Conclusion: Rapid infusion and dilution are effective methods of reducing phlebitis caused by the infusion of VNR, but the efficacy of flushing with normal saline or infusion of fat emulsion was not confirmed.     

Determination of toluene in brain of freely moving mice using solid-phase microextraction technique

For the purpose of measuring the pharmacokinetics of inhaled toluene in the brain of mice, we developed a method for the direct detection of toluene by gas chromatography/mass spectrometry (GC/MS). The method uses a solid-phase microextraction (SPME) fiber inserted into the mouse' hippocampus (CA1) through a cannula fixed onto the animal. BALB/c mice were exposed to 0, 0.9, 9, 50 and 90 ppm toluene for 30 min. The toluene level detected near the hippocampus by this method after exposure to 0.9 ppm toluene in air showed no significant difference from the level found in the non-exposure control group; however, the toluene level increased significantly after exposure to concentrations of 9, 50 and 90 ppm. These increases were concentration-dependent. In addition, the pharmacokinetics of toluene in the brain of mice exposed to 50 ppm toluene showed that the toluene level decreased rapidly after the exposure, and returned to control levels after 60 min. This study describes the method which has successfully detected toluene levels in the brain of conscious, free-moving mice for the first time.     

Hepatocyte-specific Pten deficient mice

Histological findings of hepatocyte-specific Pten deficient(Pten KO) mice livers were consistent with the criteria of human nonalcoholic steatohepatitis(NASH). Therefore, Pten KO mice are a powerful animal model of NASH. In Pten KO mice livers, steatosis develops based on the increased expression of genes that promote fatty acids synthesis. The increased expression of beta, oxidation-related genes accumulates oxidative stress in Pten KO mice livers resulting in hepatitis based on lipid hyperoxidation of hepatocytes membranes. Onset and exacerbation of hepatitis are also related to endotoxins derived from intestinal bacteria. Oxidative DNA damage, hyperproliferation of hepatocytes induced by the activation of Akt and ERK, and the increasement of malignant potential based on the change of fatty acids composition in the livers contribute to the development of hepatocellular carcinoma in Pten KO mice. Since it is considered that the mechanism to induce hepatic lesions in some NASH patients is the same as that in Pten KO mice, the investigation using Pten KO mice gives us clues to clarify the pathogenesis or develop effective therapy of NASH.     

Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter

Purpose To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4). Methods Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography–tandem mass spectrometry. Results From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4. Conclusions The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.