Characterization of β-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl--D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 g ml–1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.     

Cytotoxic screening of medicinal and edible plants in Okinawa,Japan, and identification of the main toxic constituent of Rhodea japonica (Omoto)

The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 microg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC(50) value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC(50), 60 nM).     

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species.     

Effect of ischemic preconditioning on cerebral blood flow after subsequent lethal ischemia in gerbils

Ischemic tolerance, the phenomenon where a sublethal ischemic preconditioning protects the brain against a subsequent lethal ischemia, has been widely studied. Studies have been done on cerebral blood flow levels prior to the lethal ischemia, but the hemodynamic pattern after global ischemia with ischemic preconditioning has not been reported. Sequential changes in regional cerebral blood flow (rCBF) in gerbil hippocampus after 5 min global ischemia with or without 2 min ischemic preconditioning were studied to determine if ischemic preconditioning affects rCBF. Four different treatments were given: (1) sham-operated, (2) 2 min ischemia, (3) non-preconditioned, and (4) preconditioned. Groups (1) and (2) (both groups n = 5) were given a 24-h recovery period and the rCBF was measured for baseline values. 24 h after sham-operation (3) and 2 min ischemia (4), gerbils were subjected to 5 min ischemia followed by 1 h, 6 h, 1-day or 7-day reperfusion periods (all groups n = 5). Although no regional difference was observed in the recovery pattern of rCBF, the values of rCBF were significantly higher in the preconditioned group throughout whole brain regions including hippocampus. These results indicate that ischemic preconditioning facilitated the recovery of rCBF after 5 min global ischemia. It needs further study to determine whether the protecting effects of preconditioning relate to the early recovery of rCBF or not. However, our results could be interpreted that the early recovery of rCBF may lead to benefits for cell survival in the CA1 neuron, probably facilitating other protecting mechanisms.     

Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.     

Phytoplankton productivity in a pond of brownish-colored water in a Japanese lowland marsh, Naka-ikemi

To understand the characteristics of the ecosystem in Japanese lowland marsh, we investigated chlorophyll-a (Chl. a), photosynthesis and respiration of a phytoplankton community in a brownish-colored pond in Naka-ikemi marsh, Tsuruga, Fukui Prefecture. Chl. a concentrations and volumetric gross primary production rates ranged between 1.3–57.0 μg Chl. a l⁻¹ and 148–1619 μg C l⁻¹ day⁻¹ during the study period. Higher values of Chl. a and primary production rates were clearly observed from June to September, when the dominant algae were the phytoflagellates, Peridinium (Dinophyceae) and Cryptomonas (Cryptophyceae), with swimming ability. The trophic status of the pond water of Naka-ikemi marsh was defined as being in eutrophic condition based on the biomass and productivity of phytoplankton. However, depths of Z _1% showing the productive layer in this study site were relatively narrower than those observed in the hyper-eutrophic Lake Suwa with frequent cyanobacterial water bloom. Factor-attenuating underwater light intensity in Naka-ikemi marsh was presumed to be colored dissolved organic matter. Thus, not only phytoplankton primary production, but also allochthonous organic matter supplied from the catchment area seems to be the dominant factor in the whole energy budget of the pond. In conclusion, we regarded the pond ecosystem in Naka-ikemi marsh to be in a eutrophic–dystrophic condition.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.