Yukihisa Fujita

Evaluation of role of mast cells in the development of liver fibrosis using mast cell-deficient rats and mice

BACKGROUND/AIMS Several studies have suggested that mast cells participate in the development of liver fibrosis in rodent models. In this study mast cell-deficient mutant Ws/Ws rats and W/Wv mice were used to examine whether mast cells are involved in the development of liver fibrosis. METHODS Liver fibrosis was induced in rats by bile duct resection (BDR), and by intraperitoneal injections of carbon tetrachloride (CCl4) or porcine serum, and in mice by intragastric administrations of CCl4, and BDR. The degree of fibrosis was evaluated by measuring the hydroxyproline content (microg/mg tissue) of the liver as an index of the collagen content. The density of mast cells (number/cm2 liver section) was determined by counting mast cells in liver sections stained with alcian blue. RESULTS In the liver of control non-mutant (+/+) rats, mast cells were found principally in portal areas, and their average density was 200-300/cm2 liver section. BDR, and treatments with CCl4 and porcine serum increased the density of mast cells in the liver of +/+ rats several-fold, and induced liver fibrosis, increasing the liver hydroxyproline content markedly. BDR, and treatments with CCl4 and porcine serum also induced liver fibrosis in Ws/Ws rats, increasing the liver hydroxyproline content to a similar or higher level than that in +/+ rats. However, the average densities of mast cells in the liver of Ws/Ws rats after BDR and treatment with CCl4 and porcine serum were at most 10.2/cm2 liver section. The density of mast cells in the liver of control +/+ mice was extremely low (average, less than 2), and neither BDR nor treatment with CCl4 caused any significant increase in their density, whereas these treatments induced liver fibrosis and markedly increased the liver hydroxyproline content. Furthermore, treatment with CCl4 induced fibrosis in the liver of W/Wv mice similarly to that in +/+ mice, but the density of mast cells in the liver of W/Wv mice was very low (average, less than 1), and was not increased by treatment with CCl4. CONCLUSIONS The present results indicate that mast cells play no role in the development of liver fibrosis in rats and mice.

Effect of hydration on the thermal denaturation of lysozyme as measured by differential scanning calorimetry.

The thermal denaturation of lysozymc in aqueous alcohol solution has been investigated by differential scanning calorimetry. The alcohols employed were methanol, ethanol, isomeric propyl alcohols and butyl alcohols as monohydric alcohols, and ethylene glycol and glycerol as polyhydric alcohols. In monohydric alcohols, the temperature of denaturation, Td, of lysozyme decreased linearly with increasing alcohol concentration, which became pronounced with an increasing in the hydrophobic character. The enthalpy of denaturation, ΔHd, of lysozyme showed a complex dependence on the solvent composition; the ΔHd first increased with increasing alcohol concentration and then started decreasing at different concentrations for each alcohol. The branching of the alkyl chain decreased the destabilizing effect of the alcohol on the native conformation of the protein. In polyhydric alcohols, both Td and ΔHd increased with increasing alcohol concentration. The polyhydric alcohols stabilized the native conformation of the ...

A simple procedure for the extraction of the native chromophore of visual pigments : The formaldehyde method

The chromophore of visual pigments was efficiently extracted in its original isomeric conformation by dichloromethane/hexane in the presence of excess formaldehyde. This new method of extraction (the formaldehyde method) in combination with HPLC is useful for the determination of the chromophore composition of visual pigments. The chromophore isolated by this method is biologically active and can be used for regeneration experiments.

Self-augmentation Effect of Male-specific Products on Sexually Differentiated Progesterone Metabolism in Adult Male Rat Liver Microsomes

It is well known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific or -predominant manner by adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16α-hydroxyl and 6β-hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5α-reduced products by female-predominant 5α-reductase in the female. These sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control. In the present study, we show that unlabeled 16α-hydroxyprogesterone and 6β-hydroxyprogesterone inhibited the 5α-reductive [3H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione not only stimulated the 5α-reductive metabolism producing themselves but also inhibited the male-specific oxidative metabolism. This finding compels us to propose a novel hypothesis that adult male rat liver microsomes may possess a self-augmentation system regulated by the male-specific products on sexually differentiated steroid metabolism, besides regulation by gene expressions of the related enzymes.

Thermal stability of alkylated and hydroxyalkylated lysozymes

Abstract Five derivatives of lysozyme which were modified in four of the original six lysine residues were prepared by reductive alkylation with three aliphatic aldehydes of different chain lengths and two hydroxyaldehydes of different hydroxyl-group contents. The thermal stability of these derivatives was investigated by DSC. The alkylated derivatives were less stable than unmodified lysozyme, depending on the size of the introduced alkyl groups. However, there was no significant difference in the thermal stability among unmodified and two hydroxylated lysozymes. The change in the thermal stability upon lysine modification of lysozyme is remarkably dependent on the qualitative factors of the entering substituents, and on other factors such as steric hindrance.

Studies on 16α-Hydroxylation of Steroid Molecules and Regioselective Binding Mode in Homology-Modeled Cytochrome P450-2C11

We investigated the 16α-hydroxylation of steroid molecules and regioselective binding mode in homology-modeled cytochrome P450-2C11 to correlate the biological study with the computational molecular modeling. It revealed that there was a positive relationship between the observed inhibitory potencies and the binding free energies. Docking of steroid molecules into this homology-modeled CYP2C11 indicated that 16α-hydroxylation is favored with steroidal molecules possessing the following components, (1) a bent A-B ring configuration (5β-reduced), (2) C-3 α-hydroxyl group, (3) C-17β-acetyl group, and (4) methyl group at both the C-18 and C-19. These respective steroid components requirements were defined as the inhibitory contribution factor. Overall studies of the male rat CYP2C11 metabolism revealed that the above-mentioned steroid components requirements were essential to induce an effective inhibition of [3H]progesterone 16α-hydroxylation. As far as docking of homology-modeled CYP2C11 against investigated steroids is concerned, they are docked at the active site superimposed with flurbiprofen. It was also found that the distance between heme iron and C16α-H was between 4 to 6 Å and that the related angle was in the range of 180 ± 45°.