Tsugikazu Komoda

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.

Bezafibrate has an antioxidant effect: Peroxisome proliferator-activated receptor α is associated with Cu2+, Zn2+-superoxide dismutase in the liver

Administration of bezafibrate in rats significantly reduced the levels of plasma thiobarbituric acid-reactive substances (TBARS) in comparison with those obtained in rats fed a soy or lard chow. Moreover, an elevation of in vitro conjugated diene production and linoleic acid levels in the high-density lipoproteins and low-density lipoproteins induced by a soy or lard chow, was reduced by bezafibrate administration. In addition, the liver Cu2+, Zn2+-superoxide dismutase (SOD) gene expression showed a significant positive correlation with the liver peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA level (R=0.769, p<0.0001). This unique characteristic of bezafibrate, which possesses both a hypolipidemic effect and antioxidant activity, may be beneficial in preventing vascular complications in hyperlipidemia.

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes.

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.

Sugar-chain heterogeneity of human alkaline phosphatases: differences between normal and tumour-associated isozymes

The sugar-chain heterogeneity of alkaline phosphatases (ALPs) from various human organs was investigated by using the serial lectin affinity technique. This technique revealed a possible structure of the sugar chain(s) of ALP isozymes and clarified a difference in affinity on the lectin column not only among three genetically different isozymes (liver/bone/kidney, intestinal and placental types) but also among liver, bone, and kidney ALPs. Lectin-binding profiles of ALPs in these human organs closely resembled those in the corresponding organs of the rat, as reported previously, suggesting that heterogeneities in sugar chains of ALPs have a specificity for the respective organs rather than being species-specific. Lectin-binding profiles of tumour-produced placental and liver ALPs were significantly different from those of ALPs in the respective normal organs. However, the two altered ALPs exhibited similar lectin-binding affinities. Isoelectric focusing analysis showed essentially no difference in protein charge between the normal and tumor-produced ALPs. Moreover, tumour-produced ALPs had the same N-terminal amino acid sequence and peptide mapping as normal ALPs. From these results, it is possible to suggest that organ-specific sugar chains in ALP isozymes are changed into those peculiar to tumours in association with malignant transformation.

Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells

We examined the localization of oxidized high-density lipoprotein (HDL) in atheromatous plaques and the oxidized HDL binding sites on endothelial cells. Histochemical analysis using CuSO4-oxidized HDL-specific 9F5-3a antibody indicated the presence of oxidized HDL in the intima of atheromatous plaques in human abdominal aortae. The cell surface binding of 125I-oxidized HDL to cultured human aortic endothelial cells (HAEC) was saturable, with an apparent dissociation constant (K d) of 1.43 μmol/L. Competition for 125I-oxidized HDL binding was strong for oxidized HDL, moderate for native HDL and low for acetylated low-density lipoprotein (LDL) or oxidized LDL. Using oxidized HDL as a ligand for blotting, a major 130-kDa band was detected in HAEC. These results suggest that oxidized HDL and its putative binding protein are present in atheromatous plaques and endothelial cells, respectively.

Serum lipid metabolism abnormalities and change in lipoprotein contents in patients with advanced-stage renal disease.

BACKGROUND Arteriosclerosis is the major cause of death in patients with chronic renal failure. There is much interest in the lipid metabolism of patients treated with hemodialysis. METHODS We analyzed low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in chronic renal failure (CRF) patients according to patients on hemodialysis (HD), patients with diabetic nephropathy before initiation of dialysis (DN), and patients with chronic glomerulonephritis in the conservative stage (CGN); and compared the lipid metabolic abnormalities in patients on hemodialysis and those not yet on hemodialysis. We also analyzed the qualitative abnormalities of LDL and HDL and their relationship with the pathological stages. RESULTS Electrophoretic patterns identified small LDL particles and small HDL particles in the three groups, and the degree of denaturation was more enhanced in CRF patients in the conservative stage than in HD patients. For LDL susceptibility to oxidation LDL (oxLDL) by addition of Cu(2+), the lag time was approximately 57 min in healthy controls and CGN patients, but was prolonged to approximately 75 min in HD and DN patients. For HDL susceptibility to oxidation HDL (oxHDL), HD, DN and CGN patients showed lag times shorter than those found in healthy control subjects. These results showed that LDL and HDL in the serum of CRF patients were in a state of enhanced susceptibility to oxidative modification. In Western blot analysis using anti-human-denatured LDL and anti-human-oxidized HDL monoclonal antibodies, bands of low molecular oxLDL at 150-197 kDa were detected in all CRF patients, with marked tailing in CGN patients. Similarly, bands of small oxHDL particles at 110 and 120 kDa were found in HD, DN and CGN patients. CONCLUSIONS Oxidative modification of both LDL and HDL occurs in patients with advanced CRF resulting in small lipoproteins. Increased production of oxLDL and oxHDL is the main cause of lipid metabolic abnormality in CRF patients.

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.

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.

Different lectin affinities in rat alkaline phosphatase isozymes: Multiple forms of the isozyme isolated by heterogeneities of sugar moieties

Differences among rat alkaline phosphatases from various organs were established by using the serial lectin affinity technique. Elution profiles of isozymes with various lectin columns were significantly different from each other, and it was possible to distinguish between isozymes by this technique. It has been shown by many workers that a high-mannose-type and/or hybrid-type sugar chain is contained in the fraction bound strongly to concanavalin A-Sepharose. The duodenal alkaline phosphatase had a low content of this fraction, although the content of this fraction obtained from duodenal explants was increased markedly when explants were cultured with swainsonine, which is an inhibitor of alpha-mannosidase II, and this leads to the accumulation of high-mannose-type and hybrid-type sugar chains in the pathway of sugar chain processing. From the present results, it is suggested that differences in the elution profiles of isozymes may be due to the structural differences of sugar chains in alkaline phosphatases.

A possible mechanism of induction and translocation into blood stream of rat alkaline phosphatase activity by bile duct ligation.

We investigated the effects of bile duct ligation on alkaline phosphatase (ALP) activities in liver, calvarium, duodenum, and ileum in rats and its possible mechanism of action. ALP isozyme activities in the ligated rats were significantly elevated in the liver and duodenum, while those in the ileum and calvarium were markedly decreased. The ALP isozyme activity elevated by the ligation was obviously suppressed by prior administration of indomethacin, an inhibitor of prostaglandin synthesis. Moreover, phorbol ester also elevated the ALP activity as well as the phosphatase level in the ligated rat. However, other drugs, such as an inhibitor of protein kinase C and calmodulin, showed different effects: calmodulin stimulated an 11.0-, 1.3-, or 1.5-fold increase in ALP activity in the ileum, duodenum, or calvarium, respectively; whereas the hepatic enzyme activity was not affected. The induction by calmodulin was markedly different from that by the ligation. Moreover, imipramine, an inhibitor of protein kinase C, had little effect. These results suggest that prostaglandin is a possible ALP inducer in ligated rats, probably working by elevating the cAMP level. On the other hand, the ligation induced simultaneously de novo synthesis of the membranous and soluble ALP isozymes; and the release rate of the soluble enzyme was greater than that of the membranous isozymes, indicating that the soluble enzyme might be a main source of the induced serum ALP. Lectin affinity chromatography indicated that the soluble enzyme or induced serum enzyme may contain more fucose than that of the membranous one, suggesting that the sugar moiety in the ALP molecule may relate to the clearance of ALP from or its release into the circulation.