Toshiyuki Matsunaga

Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation.

Intestinal alkaline phosphatase (AP), as a host defense factor, was first investigated in vivo using rats orally exposed to lipopolysaccharide (LPS). After the oral administration of LPS to rats, serum LPS content was increased within 2 hr and then decreased to 6 hr. In contrast, when L-phenylalanine (L-Phe), an inhibitor of intestinal-type AP isozymes, was simultaneously administered with LPS, serum LPS content significantly increased from 1 hr and the area under the concentration-time curve of serum LPS was augmented approximately 2-fold, suggesting that APs in the gastrointestinal tract reduced serum LPS content. In addition, LPS toxicity diminished by a treatment in vitro with intestinal APs, were recovered by the treatment in the co-presence of L-Phe. In the experiment using human aortic endothelial cells (HAECs), we observed that the cell viability decreased in a dose-dependent manner of LPS-exposure, and the LPS dose, exhibiting 50% viability of the cells, was 0.05 microg/ml. When the cells were exposed to LPS pretreated with 50 nIU/ml of intestinal AP at pH 10.0 and 8.0, the 50% viability was at 2.0 microg/ml of LPS. These results strongly suggest that the APs reduced the toxicity of LPS, as a host defense factor against LPS.

Changes in receptor activator of nuclear factor-kappaB, and its ligand, osteoprotegerin, bone-type alkaline phosphatase, and tartrate-resistant acid phosphatase in ovariectomized rats.

We investigated time‐course changes in the expression of receptor activator of nuclear factor‐kappaB (RANK), its ligand (RANKL), osteoprotegerin (OPG), bone‐type alkaline phosphatase (BAP), and tartrate‐resistant acid phosphatase (TRAP) in ovariectomized (OVX) rats. Samples of sera and coccyges were used for analysis of the enzyme activities and expression levels of proteins and mRNAs, and an immunohistochemical analysis was also performed. Serum BAP activity increased to 158.6% of the pre‐operation value at 1 week after OVX, and then decreased to 38.7% at 8 weeks after OVX. On the other hand, the serum TRAP activity increased to 130.9% of the pre‐operation level at 1 week after OVX, and was maintained at a high level, compared with the pre‐operation level. The patterns of BAP and TRAP activity in the coccyges specimens were similar to those seen in the sera. The expression profiles of TRAP, RANK, and RANKL proteins in the coccyx specimens were similar to the pattern of serum TRAP activity, while the profiles of the BAP and OPG proteins were similar to the pattern of serum BAP activity in OVX rats. The changes in the mRNA expression levels of the osteogenic proteins were similar to those for protein expression. These biochemical changes in OVX rats were confirmed by immunohistochemical studies. Our results suggest that not only osteoclastogenesis accelerated but also osteoblastogenesis transiently increased during the early phase of osteoporosis.

Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP‐I and rIAP‐II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP‐I and rIAP‐II displayed a typical α/β topology, but the crown domain of rIAP‐I contained an additional β‐sheet, while the embracing arm region of rIAP‐II lacked the α‐helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP‐I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP‐II. Using metal‐depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP‐I, whereas in duodenum the percentage of rIAP‐I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP‐I, but not rIAP‐II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal‐binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal‐binding site 3 and water molecules. These findings suggest that the side‐chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP‐I.

Expression of α-amylase isozymes in rat tissues

Gene expressions of α-amylase isozymes in rat tissues were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR), followed by EcoRI digestion. This procedure is based on evidence that an RT-PCR product from mouse pancreas RNA is sensitive to EcoRI, but not the product from the salivary gland or liver RNAs. The method was applied to the analysis of α-amylase expression in rat liver after partial hepatectomy, in which a potent expression of pancreas type isozyme was observed. However, no expression of the pancreatic isozyme in the regenerating liver was found. We also analyzed the expression of α-amylase gene in several additional rat tissues. In intestine, stomach, testis and skeletal muscle, the corresponding PCR products were amplified, but few were detected in heart or spleen. Intestine and stomach expressed a pancreatic isozyme of α-amylase. Analyses of the α-amylase activity and protein indicated the presence of the enzyme in those tissues. Immunohistochemical analysis also indicated that the amylase proteins were specifically present in epithelial cells of rat intestinal mucosa. This is a convenient method for identification of α-amylase isozyme mRNA in rodent tissues.

Expression of α-amylase gene in rat liver: Liver-specific amylase has a high affinity to glycogen

The reactivity of rat liver α‐amylases with maltotriose (G3), maltopentose (G5) and glycogen has been investigated. Liver amylases were found to be glycosylated and to have a molecular mass of 50 kDa by Western blotting using an anti‐human salivary amylase antibody. The glycosylated liver amylases were found to be capable of G3‐ and G5‐hydrolysis and of glucose formation, as demonstrated by thin‐layer chromatography. When the amylase preparation was exposed to different concentrations of glycogen and run on a cellulose acetate membrane, the mobilities of rat liver amylases significantly decreased with tailing directly from the point of origin. In contrast, rat salivary amylases were not so much. These results indicate that rat liver amylases have a strong affinity to glycogen. We confirmed the expression of liver‐specific amylases in rat liver by reverse transcriptional‐polymerase chain reaction (RT‐PCR); PCR products showed one band of an expected size of 474 bp using primers tested in the present study. A partial nucleotide sequence was then determined. When compared with the gene of mouse liver amylase, the substitution of 26 bases out of 434 bases was elucidated. The present data demonstrate the presence of liver‐specific amylases in rats.

Detection of oxidized high-density lipoprotein.

This paper reviews working procedures for the separation and detection of oxidized high-density lipoproteins (ox-HDL) and their constituents. It begins with an introductory overview of structural alterations of the HDL particle and its constituents generated during oxidation. The main body of the review delineates various procedures for the isolation and detection of ox-HDL as well as the purification and separation of phosphatidylcholine metabolites and denatured apolipoproteins in the particle. The useful methods published more recently are picked up and the utility of the separation techniques is described. The last section covers a clinical evaluation of changes in these factors in ox-HDL as well as future directions of ox-HDL research.

Characterization of the epitopes specific for the monoclonal antibody 9F5-3a and quantification of oxidized HDL in human plasma

Background: We previously isolated a monoclonal antibody, 9F5-3a, that is specific for HDL oxidized by CuSO4. Methods: We examined the characteristics of the 9F5-3a epitope by Western blot and measured the concentration of oxidized HDL in human plasma by enzyme-linked immunosorbent assay using this antibody. Results: The monoclonal antibody specifically reacted with oxidized HDL in a mixture of HDL, LDL and modified lipoproteins. Oxidation of the HDL particles accelerated cross-linkage of apolipoproteins caused by lipid peroxidation, and the cross-linked apolipoprotein AI selectively reacted with the 9F5-3a antibody. Mean (standard deviation) plasma concentrations of oxidized HDL were 127 (50) μg/L in 23 healthy controls, 191 (65) μg/L in 30 patients with non-insulin-dependent diabetes mellitus (P < 0.01 versus healthy controls) and 200 (87) μg/L in 25 patients with coronary artery disease (P < 0.01 versus healthy controls). The concentrations of oxidized HDL did not correlate with the concentrations of thiobarbituric acid-reactive substances. Conclusions: The results indicate that determination of oxidized HDL concentration may be useful for identifying patients with atherosclerotic disease.

Caspase 3 activation in nasal capillary in patients with epistaxis

OBJECTIVE We investigated whether an apoptosis of nasal microvessels contributes to probable mechanism of the onset of epistaxis. STUDY DESIGN AND SETTING Nasal septal mucosa of Littles areas taken from patients without (n = 19) and with (n = 26) epistaxis were examined. Active caspase-3 in the mucosa was detected according to the methods of immunohistochemistry and Western blotting. On Western blot analysis of the homogenates of the mucosa, we also sought probable signaling factors after caspase-3 activation. RESULTS Marked activation of caspase-3 was detected in the capillaries and its neighboring muscle cells of Littles area from patients with epistaxis, and the activation was due to enhanced expression of procaspase-3 protein and progressive cleavage of the precursor. As a result of Western blotting of signaling factors, enhanced expressions of caspase-9 and Bax protein in the homogenates of Littles area in epistaxis group were found compared with those in control group. Increased levels of cytochrome c released into a cytosol were also detected in the capillaries in epistaxis group. CONCLUSION In the present study, caspase-3 activation was found in the capillaries of Littles area from patients with epistaxis, suggesting that an apoptosis of capillaries may contribute to a mechanism of the onset of epistaxis. Moreover, alterations of some apoptotic factors such as caspase-9, Bax, and cytochrome c in the tissues demonstrated participation of mitochondrial disturbance in one of the apoptotic mechanisms. SIGNIFICANCE Further explorations of the pathobiologic mechanism of capillary apoptosis can lead not only to an identification of risk factors in the onset of epistaxis but also to the development of medical therapy of epistaxis.

Electrophoretically unique amylases in rat livers: Phylogenic and ontogenic study on the mammalian liver

Liver amylase activity in rodents was assayed with Blue Starch as substrate, and found to be higher than in humans or pigs. Based on the result of concanavalin A affinity chromatography, we found that the sugar moieties of amylase molecules increased in parallel with amylase activity in the tested mammals. However, the amounts of amylase proteins determined by Western bloting with anti‐human salivary‐type antibody as the probe, were similar to the levels in mammalian livers. Moreover, a similar expression of amylase mRNA was also detected in the mammalian livers by a reverse transcriptional‐polymerase chain reaction using primers specific for the human salivary and/or pancreatic amylase complementary DNA (cDNA) sequences. The amylase was detected at the catalytic activity, protein molecule and mRNA levels in rat liver at all ages from fetus to adult. Salivary‐type liver amylase activity increased up to one week after birth, and was maintained at the adult level thereafter. However, based on the results of the electrophoretic mobility test, livers with accelerated amylase activity, e.g., at 2–4 weeks after birth or during liver regeneration after partial hepatectomy, were also found to express an amylase electrophoretical identical to pancreatic‐type amylase in addition to salivary‐type activity. These results suggest that the liver may express an etopic amylase in a certain condition.

Constituents of the Human Body

This chapter describes sugars, glycoproteins, essential amino acids, lipids and enzymes, and their roles as the constituent parts of the human body. Differences between monosaccharides and polysaccharides are described. Glycoproteins consist of sugars attached to proteins; the synthetic pathway of the sugar chain of asparagine-linked sugar moieties is described. The typical roles of 20 amino acids and the taste of amino acids are covered. Concerning fatty acids, the structure of cholesterol and sphingolipids and localization on the cell biomembrane are described. Enzymatic reactions, reaction specificity, the classification of enzymes and isozymes, and the reaction velocity of enzymes are presented. The role played by the cell biomembranes is described.