Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Fungal cytochrome P450s catalyzing hydroxylation of substituted toluenes to form their hydroxymethyl derivatives

The degradation of a series of nitroaromatic compounds by the lignin-degrading fungus Phanerochaete chrysosporium was examined. From 4-nitrotoluene (4-NT), several metabolic intermediates were identified. Initially, 4-NT was converted to 4-nitrobenzyl alcohol (4-NBA), followed by the oxidation reactions to form 4-nitrobenzaldehyde and 4-nitrobenzoic acid, albeit slowly. Exogenously added 4-nitrobenzaldehyde and 4-nitrobenzoic acid were predominantly reduced to 4-NBA. The fungal formation of 4-NBA was inhibited by piperonyl butoxide, a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450 in the hydroxylation of the methyl group. Similarly, 2-, and 3-nitrotoluenes and 4-chlorotoluene were converted to the corresponding arylalcohols by P. chrysosporium. On the other hand, toluene and 4-methoxytoluene were not converted. Thus, P. chrysosporium possesses an alkyl hydroxylation activity against aromatic compounds substituted with a strong electron-withdrawing group.     

Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice

Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18-22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.     

The most appropriate position and number for absolute anchorages for orthodontic tooth movements

Absolute anchorages proved to be very effective for orthodontic tooth movements. We used a 3D digitizer to record each tooth on pre-treatment diagnostic and post-treatment predictive setup models and then 3D coordinate system conversion was performed to make the coordinate values comparable. An arithmetic calculation of vector and moment based on the orthodontic forces and the tooth displacement under preliminary premises undertaken to decide the most favorable position and number for absolute anchorages. Position--For two-dimensional and three-dimensional calculations, the most appropriate positions for absolute anchorages should theoretically be on the line of resultant force (2D) and the plane (3D) where the total moment effect tends to be zero. Number--As for the number of the absolute anchorages needed, it depends on the number of target teeth. Different combinations of target teeth provide different sets of results.     

Method for evaluating metabolic functions of drugs in bioartificial liver

Lidocaine and galactose loading tests were performed on a bioartificial liver (BAL), an extracorporeal medical device incorporating living hepatocytes in a cartridge without a transport barrier across the membranes. The concentration changes were analyzed using pharmacokinetic equations to evaluate the efficacy and limitation of the proposed method. Lidocaine and galactose were found to be suitable drugs for a quantitative evaluation of the BAL functions, as they did not interact with the plasma proteins or blood vessels, making their concentrations easy to determine. The drug concentration changes after drug loading were easily analyzed using pharmacokinetic equations, and the BAL functions quantitatively expressed by pharmacokinetic parameters, such as the clearance (CL) and galactose elimination capacity (GEC). In addition, these two drugs have already been used in clinical tests to evaluate human liver functions over long periods, and lidocaineCL values andGEC values reported for a normal human liver. Thus, a comparison of theCL andGEC values for theBAL and a natural liver revealed what proportion of normal liver functions could be replaced by the BAL.     

Different mechanisms for membrane and nuclear damages in apoptosis induced by an immunosuppressant,FTY720

A novel immunosuppressant, FTY720, that was purified from cultures of Isaria sinclairii has been shown to cause apoptosis of lymphocytes, but its biochemical and molecular mechanisms are largely unknown. In this study, we investigated the signal transduction of FTY720-induced apoptosis in comparison with the Fas-induced apoptosis. Although FTY720 induced nuclear and membrane damages in a dose-dependent manner, nuclear damage, but not membrane damage, was suppressed by the caspase-3 inhibitor, DEVD-FMK. It blocked both the nuclear and membrane damages that were induced by the anti-Fas antibody. Experiments using enucleated cytoplasts also demonstrated that membrane damage was induced by FTY720. However, the ones that were induced by the anti-Fas antibody were not blocked by DEVD-FMK. Exogenously-added sphingolipids partially suppressed the FTY720-induced membrane damage. These results suggest that FTY720 induces membrane damage through the caspase-3-independent pathway that is modulated by sphingolipids.     

Localization of epidermal-type fatty acid binding protein in the thymic epithelial cells of mice

The immunoreactivity for epidermal-type fatty acid binding protein of epidermis type (E-FABP) was selectively localized in the epithelial cells of both cortex and medulla of mouse thymus. The cortical epithelial cytoreticulum was clearly visible with the intense immunoreactivity and the immunoreactive cytoreticulum extended intricately throughout the thymic cortex to enclose thymocytes. In the thymic medulla, the immunoreactivity was variable in intensity among the epithelial cells and there was a tendency that epithelial cells containing more numerous tonofilament bundles were less immunoreactive. Considering the possibility that FABPs function as intracellular carriers for unsaturated long chain fatty acids, the present finding suggests that E-FABP in the thymic epithelial cells, especially the cortical ones because of their extensive location, are intimately involved in the metabolic processes of fatty acids including production of bioactive substances, such as prostaglandin and leukotriene, which are known to exert some regulation of thymic immune responses.     

Preferential and asymmetrical accumulation of a Rac small GTPase mRNA in differentiating xylem cells of Zinnia elegans

Rac-type small GTPases are known to function in some cellular processes in plants. To further understand the involvement of Rac type GTPases in plant development, we isolated from cultured Zinnia cells a gene (ZeRAC2) encoding a new Rac-type small GTPase. ZeRAC2 mRNA accumulates preferentially in xylogenic culture and transiently at the time when visible tracheary elements appear. Experiments with ZeRAC2 recombinant proteins demonstrated that ZeRAC2 binds to and hydrolyzes GTP. A GFP-ZeRAC2 fusion protein was localized to the plasma membrane. Together with the fact that ZeRAC2 possesses a putative geranylgeranylation site at the C-terminus, this suggests that ZeRAC2 acts on the plasma membrane. In situ hybridization indicated that ZeRAC2 mRNA accumulates preferentially in xylem parenchyma and tracheary element precursor cells, and surprisingly the accumulation is restricted to the site facing developing tracheary elements.     

Characterization of an anti-decorin monoclonal antibody,and its utility

6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule, has Although it been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosaminoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5 microg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.     

Novel missense mutations in red/green opsin genes in congenital color-vision deficiencies

Lipocortin I-S100 (calcyclin) heterotetramer exhibited ATPase activity in the presence of dsDNA but not ssDNA. To demonstrate its helicase activity, an 80-mer polynucleotide complementary to the replication origin of M13mp18 was synthesized, and the oligonucleotide, (dC)(20), was ligated to either its 5'- or 3'- end for binding to lipocortin. Lipocortin I heterotetramer displaced chains of the partially Y-shaped duplexes with a dC-tail at either the 5'- or 3'- end. The chain displacement required ATP and Mg(2+). Nonhydrolyzable ATP analogues were not effective. Lipocortin I heterotetramer also catalyzed annealing of the polynucleotides to M13mp18. Ca(2+) and phospholipids but not ATP and Mg(2+) were essential for this reaction. Since the chain displacing and annealing reactions were inhibited by monospecific anti-lipocortin I or anti-S100 antibodies, the present observations suggest that the lipocortin I heterotetramer regulates unwinding and annealing of DNA by Mg(2+) (plus ATP) and Ca(2+) (and phospholipids), respectively.