Biomedical subjects
Publications and source records attributed to T Kikuchi.
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Effects of uni- or bi-lateral resection of the sciatic nerve followed by calcium deficiency on the bone structure of the adult male rat were studied and the mechanism of the development of osteoporosis was discussed. After the evaluation of the thickness of the cortex and trabecula of the lower leg bone by X-ray morphometry, the bone was excised, calcium and phosphate contents of the bone were analyzed and histopathological and ultrastructural examinations were made on the calcaneus and the lower leg bone. Reduction of the bone trabecular area and of the thickness of the cortex, a loss of dry weight and of calcium and phosphate contents and decrease in the number of osteoblasts and osteocytes were induced to the bone with sciatic denervation. The ultrastructural changes of the osteogenic cells were also observed. The osteoblasts turned to be smoothly surfaced showing only a few cytoplasmic projections were connected loosely each other. These changes were enhanced by calcium deficiency. We concluded from these results that the disturbance of nerve connection and the calcium deficiency contributed to the development of experimental osteoporosis of rats.
The effect of oxygenated sterols on platelet aggregation induced by thrombin and ADP has been studied. All oxygenated sterols tested with a hydroxyl group on the side chain enhanced thrombin-induced aggregation at 25 microM. In the case of ADP-induced aggregation, however, only 22S-hydroxycholesterol[(22S)-5-cholestene-3 beta,22-diol] enhanced the aggregation, and 22R-hydroxycholesterol[(22R)-5-cholestene-3 beta,22-diol], 24S-hydroxycholesterol[(24S)-5-cholestene-3 beta,24-diol], and 25-hydroxycholesterol[5-cholestene-3 beta,25-diol] inhibited it. These effects were observed within 10 min after the addition of oxygenated sterols to platelet suspensions in plasma.
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The estimated growth rates for five lines of human fibroblasts (Human Genetic Mutant Cell Repository designations 0011, 2936B , 0038A , 2912A , and 3529), during the first few population doublings after establishment of the lines, were within the expected range, i.e., equivalent to doubling times from 16 to 28 hr, with a mean of 20 hr. The lines were derived from donors aged 8 fetal weeks, 20 days, 9 years, 26 years, and 66 years, respectively. The growth rates of the five lines declined as an exponential function of the population doubling level in all cases. The rate of decline of the growth rate varied for different lines and appeared to be related to the life span of the lines, which in turn was related to donor age. After 30 population doublings, the population doubling times had increased 1.3, 1.3, 1.7, 3.4, and 4.7 times for the five cell lines of the corresponding replicative life spans of 65, 57, 56, 31, and 28 population doublings. Sensitivity of the fibroblast lines to the cell growth-inhibitory effect of beta-interferon was independent of the population doubling level of the lines, i.e., interferon depressed the population growth rate of low population doubling level cells as much as that of middle or late population doubling level cells for any given line. However, the fibroblast lines showed differences in their sensitivity to the cell growth-inhibitory effect of interferon, probably as an expression of genotypic differences among the lines.
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Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.
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A method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.
The structural relationship between calpain I (low Ca2+-requiring) and calpain II (high Ca2+-requiring) molecules and their respective larger (80K) and smaller (30K) subunit proteins of several non-muscular tissues and cells was studied by testing immunological cross-reactivities. In addition to qualitative analyses by a conventional double immunodiffusion method, quantitative data were obtained, for the first time, by enzyme-linked immunosorbent assays using affinity-purified anticalpain I and anti-calpain II immunoglobulins. The enzyme sources included rat kidney, porcine kidney and erythrocytes, and human erythrocytes. It was concluded that the 30K subunits are immunologically almost indistinguishable between calpains, either I or II, not only from the same but also from different sources, while the 80K subunits of different origins are immunologically related to variable extents but always with discrimination between calpain I and calpain II. Similarity of the 30K subunit proteins and dissimilarity of the 80K counterparts were further substantiated by their chromatographic and electrophoretic behaviors.
An intracerebroventricular injection of d-coclaurine (50 micrograms), a benzyltetrahydroisoquinoline alkaloid extracted from Magnolia salicifolia, produced a slight increase in 3,4-dihydroxyphenylacetic acid level and a significant increase in homovanillic acid level in the mouse striatum. Another alkaloid d-reticuline (200 micrograms) increased only homovanillic acid level. An intracerebroventricular pretreatment with d-coclaurine (50 micrograms) did not antagonize suppressive effect of apomorphine on l-dopa formation produced by gamma-butyrolactone (750 mg/kg i.p.) plus aromatic amino acid decarboxylase inhibitor, NSD-1015 (100 mg/kg i.p.). These results suggest that d-coclaurine blocks postsynaptic but not presynaptic dopamine receptors in the mouse striatum.
Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.
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Serum rubella IgG and IgM antibodies were determined in normal healthy women and in rubella patients using rubella Enzyme Linked Immunosorbent Assay (ELISA) kits. Fifty four per cent (67/125) of healthy women had IgG rubella antibodies. Positive correlation was found between rubella ELISA IgG antibody and rubella hemagglutination inhibition (HI) test. Elevation of rubella IgM antibody was demonstrated during acute phase of the rubella infection. It was concluded that the ELISA of rubella antibody can be useful in surveying the immune status of a population before performing vaccination and in diagnosing rubella during acute phase.
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A very rare case of paraganglioma of the bladder is reported. The patient was a 51-year-old man. He was admitted to our hospital on June 18th, 1981, with the complaint of gross hematuria. He had no attack of hypertension and we could not find in him any parasympathetic symptom except hematuria. Upon urological examination, he was suspected to have stage B cancer of bladder. He received partial cystectomy with uneventful course during the operation on June 30th, 1981 and was discharged on the 15th postoperative day. The postoperative histological diagnosis was paraganglioma of the urinary bladder. He was confirmed to have normal levels of catecholamine and its metabolites in the urine after 4 months postoperatively. At present, he is asymptomatic.
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