Tetsuichi Shibata

Age-Dependent Changes of Collagen and Elastin Content in Human Aorta and Pulmonary Artery

Collagen and elastin in the arterial wall are thought to show some age-dependent changes, and to relate with development of the arteriosclerotic lesion. Both aortas and the pulmonary trunks were collected from 137 autopsy cases. Biochemical determination of collagen and elastin content was carried out. Collagen and elastin of the aorta and the pulmonary artery showed no significant age-dependent changes. Elastin content of the pulmonary artery increases with age in the adult. This paper seems to be the first report of simultaneous estimation of collagen and elastin content of both the aorta and the pulmonary artery.

KIH-802, an acetohydroxamic acid derivative of 2-nitroimidazole, as a new potent hypoxic cell radiosensitizer: radiosensitizing activity, acute toxicity, and pharmacokinetics

SummaryThe radiosensitizing activity, acute toxicity, and pharmacokinetics of a new hypoxic cell radiosensitizer, potassium 2-nitroimidazole-1-acetohydroxamate (KIH-802), were compared with those of misonidazole (MISO) and etanidazole (SR-2508). The radiosensitizing activity of KIH-802 was slightly higher than that of MISO and SR-2508 in vitro and was similar to or slightly higher than that of MISO or SR-2508 in vivo. The acute toxicity of KIH-802 was slightly higher than that of MISO. The concentrations of KIH-802 in the brains and peripheral nerves of mice were as low as those of SR-2508 and lower than those of MISO.

Assay for type III collagenolytic activity in lung cancer tissue

We developed a method for measuring the activity of type III collagenolytic enzyme in lung cancer tissue, using as substrate, type III collagen purified from human placenta. In this method [3H]propionate is used for labeling type III collagen, with bacterial collagenase used for making the standard curve. It, therefore, becomes possible to compare type III collagenolytic activity with those of other collagen subtypes (types I and IV). As this method is a fibril assay it is not susceptible to trypsin or other proteases. The average type III collagenolytic enzyme activity was higher in squamous cell carcinoma than in adenocarcinoma, while that of lung cancer tissue exceeded that of normal lung tissue. The activity of type III collagenase increased with the progression from one disease stage to the next.

Introduction of a New Concept: "d-Orbital Participation in Asyammetric Recognition" (DOPAR) for Use with CD Absorption Data of Dithiolane Derivatives

An empirical correlation between the signs of Circular Dichroism (CD) absorption (at 237 and 252 nm) and the absolute configuration of two asymmetric carbons in a compound containing a dithiolane ring was elucidated by use of two types of molecular orbital calculation (MOC) (CNDO/2 and ab initio with STO-3G set). These used the atom coordinates from X-ray crystallography of α-tetrahydro-l-α-santonin ethylene dithioacetal. The contributions of both the 3d-orbital of sulfur atom and the 2p-orbitals of asymmetric carbons in LUMO (excitated state) in the results of MOC were shown to be relatively large. These results suggested that d-orbital participation in asymmetric recognition (DOPAR) would help explain the CD absorption data of the compound. The calculated/observed ratios of two transition energy differences in the CD absorption were 2.7 in CNDO/2 and 3.5 in the ab initio, respectively.

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.

An approach to the profiling analysis of urinary prostaglandins by simple extraction methods

No useful profiling analyses of prostaglandins (PGs) in biological matrix have been reported. We present here a quantitative profiling analysis of urinary PGs determined by GC or GC-MS using a new simple extraction method. In both cases ODS silica column (Sep-Paks, Waters) was used for extraction of PGs from human urine. This column was pretreated with 20 mlethanol followed by 20 mlwater before use. The 20 mlsample was acidified with formic acid to pH 3.0 and passed through a ODS silica column using a glass syringe. The column was eluted successively with 20 mlethanol/water (15/85), 20 mlpetroleum ether and 10 mlmethyl formate. The recovery rate estimated using3H-PGs in methyl formate fraction was as follows. 6-keto-PGF1α: 68%, PGE2: 60%, PGF2α: 65%. The samples were derevatized to 6-keto-PGF1α-MOX-TMS-Bz, PGE2-MOX-TMS-Bz and PGF2α-TMS-Bz after extractive alkylation using THAH and benzyl bromide. GC peaks of the PG derivatives appeared at the same retention time as external standards. The quantitative determination of endogenous PGs were thus achieved by GC. d7-6-keto-PGF1α, d7-PGE2and d8-PGF2αwere added as internal standards to the human urine, which was treated with the same procedure as above. PGs in the methyl formate fraction were derivatized to 6-keto-PGF1αMOX-Me-TMS, PGE2-Me-TMS and PGF2α-Me-TMS. The sample was analysed by GC-MS (JEOL D-300) using selected ion monitoring.In conclusion, we have developed a new simple extraction method for PGs using a convenient ODS silica column chromatography in combination with the extractive alkylation. It seems to be very useful for the quantitative profiling analysis of urinary PGs.