Kazuya Nakakuki

Establishment of a kinetic model of dialysis-related amyloid fibril extension in vitro

β2-microglobulin (β2M) is a major structural component of dialysis-related amyloid fibrils (fAβ2M). In order to make clear the mechanism of fAβ2M deposition in vivo, as well as to assess the effects of several biological factors on it, it is essential to build up a kinetic experimental system to analyze fAβ2M formation in vitro. We first determined the optimum conditions for quantitative jluorometry of fAβ2M with thioflavine T (ThT). Optimum fluorescence measurements of fAβ2M were obtained at the excitation and emission wavelengths of 455 nm and 485 nm, respectively, with the reaction mixture containing 3 μM ThT and 50 mM of glycine-NaOH buffeer, pH 8.5. We then focused our study on the extension phase of fAβ2M formation in vitro. When fAβ2M were incubated with monomeric β2M, the extension of fAβ2M was observed with electron microscopy. Quantitative fiuorometry revealed that: (a) extension of fAβ2M proceeded by a pseudo-first order exponential increase as measured by the fluorescence of ThT; (b) the rate ...

High frequency of human papillomavirus infection in carcinoma of the urinary bladder

Background and Methods. The prevalence of type 6, 11, 16, 18, and 33 human papillomavirus (HPV) was investigated with the polymerase chain reaction (PCR) on formalin‐fixed, paraffin wax‐embedded material, including 48 neoplastic and 21 normal urinary bladder specimens. The PCR‐amplified DNA were analyzed by gel electrophoresis and dot blot and Southern blot hybridization. Some tissues were tested further by nonisotopic in situ hybridization.

Fluorometric Examination of Tissue Amyloid Fibrils in Murine Senile Amyloidosis: Use of the Fluorescent Indicator, Thioflavine T

Thioflavine T, a fluorescent indicator of amyloid fibrils (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989), has been tested in unfractionated tissue homogenates. With 250 nM thioflavine T, liver homogenates from a 17-month-old SAM-P/1 (senescence accelerated mouse-prone), which contained amyloid fibrils in murine senile amyloidosis (fASSAM) fluoresced brightly, whereas normal liver homogenates showed a negligible fluorescence. The following evidence confirmed that the fluorescence in the unfractionated preparations specifically represented fASSAM. The fluorescence of fASSAM deposited liver tissue was diminished to the level of the normal liver when the structure of the amyloid fibrils was disrupted by guanidine-HCl. Second, about 90% of the fluorescence of fASSAM deposited liver tissue was fractionated in the water-soluble fraction, in which amyloid fibrils are generally enriched. We then determined the concentration of fASSAM in the tissue, from the fluorescence and using the standard curve described in the above mentioned report. Lower limits of fASSAM determination were about 1 microgram/mg of tissue. A marked regional heterogeneity of fASSAM deposition was observed in the liver. We observed a linear correlation between fASSAM concentration and percentage of amyloid positive area in the liver. Age-dependent increases in fASSAM concentrations and total fASSAM contents were noted in the liver and spleen of 11 to 15-month-old SAM-P/1. There was also a positive correlation between the organ weight and fASSAM concentrations in both organs. Thus, thioflavine T is of practical use for the determination of fASSAM concentrations in the tissue.

SIX CASES OF RARE MALIGNANT TUMORS OF THE LIVER

Akitsugu OJIMA, Taketoshi SUGIYAMA, Toshio TAKEDA, Fumitada HAZAMA, Kazuya NAKAKUKI, * Yuji UESUGI, Akio MIYAZAKI, Yasuyuki SUZUKI and Masakazu FUKUSHIMA Department of Pathology, Faculty of Medicine, Kyoto University Six cases of rare malignant tumors originating in the liver, mesenchyma1 and epithelial, encountered in our department during the past five yeilrs and the incidence in Japan are briefly reported in this paper.

Kinetic Properties of Amyloid Fibril Polymerization in Vitro

We investigated the polymerization kinetics of murine senile amyloid fibrils(fASSAM) in vitro. When sonicated fASsAM was incubated with its constituent monomer protein (ASSAM), the extension of amyloid fibrils was observed in an electron microscopic analysis. Quantitative fluorometric analysis with thioflavine T revealed that 1) extension of amyloid fibrils occurred by a pseudo-first-order exponential increase in the fluorescence of thioflavine T; 2) rate of extension was maximal around pH7.5 and inhibited with the increase in KCl or NaCl concentration in the reaction mixture; 3) rate of polymerization was proportional to the product of the fASSAM number concentration and the ASSAM concentration; 4) net rate of extension was the sum of the rates of polymerization and depolymerization. These results show that extension of amyloid fibrils proceeds by consecutive association of precursor proteins onto the ends of existing fibrils.