Effect of membrane solubilization on the inhibition of rat and hamster liver microsomal type I 11ß-hydroxysteroid dehydrogenase by bile acids.

The aim of this study was to investigate the differences between rats and hamsters, Two of the most widely used experimental animals, with respect to the effects of microsomal membrane solubilization on the inhibition of liver 11ß-hydroxysteroid dehydrogenase (11ß-HSDI) enzyme by bile acids. Liver microsome fractions were prepared, and the 11ß-HSDI enzymatic activity was measured using cortisone as a substrate. The substrate and various concentrations of bile acids were added to the assay mixtures. After incubation, the products were extracted and analyzed using high-performance liquid chromatography. To investigate the effect of detergent on the inhibitory effects of bile acids, we conducted inhibition tests using Triton X-100-solubilized animal liver microsomes. When solubilized microsomes were used, all bile acids inhibited 11ß-HSDI from rats and hamsters to various degrees. 7α-Hydroxycholanoic acids (cholic acid and chenodeoxycholic acid) in particular had strong inhibitory activities. In hamsters, 7ß-hydroxycholanoic acid (ursodeoxycholic acid) was the strongest inhibitor among the bile acids tested, although its effect was not very strong. When nonsolubilized microsomes were used, deoxycholic acid did not inhibit but rather enhanced the enzymatic activity in both animals. Microsomal content of cholesterol and phospholipids are significantly different between rats and hamsters. Species differences in bile acid inhibition of nonsolubilized microsomes might be reflected not only by structural difference of bile acids, which affect membrane solubilization and enzyme activity directly, but also species difference in microsomal membrane lipid content.

Distribution of carbon-14 and associated radiation dose in rat fetal brain and liver after maternal injection of [14C]thymidine.

Pregnant Sprague-Dawley rats were injected intravenously with [14C]thymidine on day 13.5 of gestation, and the concentrations and radiation doses of 14C in the fetal brain and liver were determined by liquid scintillation counting and autoradiography with imaging plates. The concentrations of 14C in the whole fetal brains determined by liquid scintillation counting were 1.01% of the injected dose per gram wet weight at 6 h after injection and decreased to 0.39% g-1 at 48 h after injection. A significant accumulation of 14C was observed in the fetal liver: 3.8 and 0.51% of the injected dose per gram wet weight at 6 and 48 h after injection, respectively. Autoradiography showed that, especially at earlier periods after injection, there was remarkable concentration of 14C in the ventricular zone of the brain and the central region of the liver. With increasing time after injection, the distribution of 14C became relatively uniform. The concentrations of 14C in the ventricular zone of the fetal brain, determined by autoradiography, were much higher than those in the whole brain as determined by liquid scintillation counting. Cumulative radiation doses for 6-48 h after injection were 1.27 mGy for the whole fetus and 1.45 mGy for the whole brain. In contrast, the cumulative radiation dose for the ventricular zone of the brain which was determined by autoradiography was approximately 2.2 times that for the whole brain.

Effect of gamma-irradiation on the uptake and digestion of 125I-labeled bovine serum albumin by rat visceral yolk sac cultured in vitro.

The effect of gamma-irradiation on a major nutritional function of the yolk sac (the uptake and digestion of macromolecular materials) was studied in rat visceral yolk sacs cultured in vitro being used 125I-labeled bovine serum albumin (RISA) as the tracer protein. The uptake of RISA (per g wet weight) by rat yolk sacs irradiated with doses of 5-80 Gy was essentially the same as that in the unirradiated control yolk sacs. There were no significant differences in yolk sac uptake of RISA with respect to the radiation doses or to culture period up to 18 hours after irradiation. External gamma-irradiation with 10-80 Gy does also had no effect on the extracellular release of 125I from yolk sacs which had been taken it up as RISA. The ratios of the activity in ultrafiltrates of the medium to the total activity in the medium were slightly higher at doses of 40 and 80 Gy.

Transfer of 239Pu to mouse fetoplacental tissues.

Cross-placental transfer of 239Pu from the mother to fetus was studied in C3H mice. The activity of 239Pu in the conceptus was measured 24 hrs after the intravenous injection of 239Pu citrate on days 10.5 to 16.5 of gestation. More 239Pu was transferred to the conceptus when the plutonium was administered in the later stages of gestation; 0.12% of the injected dose per conceptus on day 10.5 and 1.3% on day 16.5. In all the gestational stages examined, the yolk sac and decidua contained more than 89% of the total activity distributed in the conceptus. The concentration of 239Pu in the yolk sac was about two orders of magnitude greater than that in the fetus. The 239Pu concentration in the maternal liver decreased with the gestational stage. In the early gestational stages the concentration in the maternal liver was greater than that in the yolk sac; but, this relationship was reversed in the later stages.

Evaluation of the effect of gamma-irradiation on fetal erythropoiesis in rats using blood cell volume as the index.

Rat fetuses at day 14 of gestation were irradiated externally with gamma rays at doses of 0.5-8 Gy, and the effect of radiation on the transfer of the erythropoietic site with migration of stem cells from the blood islands of the yolk sac into the liver was investigated. The LD50 was about 5 Gy for 16-day-old fetuses, 2 days after irradiation. Such fetal hematological parameters as the number of blood cells in the liver and the formation rate of micronuclei in erythrocytes, also were affected by irradiation. Two types of blood cells were present in the fetal circulating blood; small blood cells originating in the fetal liver and large blood cells originating in the blood islands of the yolk sac. The number of small blood cells in the circulating blood decreased with the increase in the radiation dose; but, the number of large blood cells remained relatively constant. This suggests that external doses of irradiation of more than 1 Gy impaired the normal transfer of the hematopoietic site (stem cell migration from the blood islands of the yolk sac into the liver.

Human C-reactive protein enhances thrombus formation after neointimal balloon injury in transgenic rabbits.

BACKGROUND: High plasma levels of C-reactive protein (CRP) constitute a powerful predictive marker of cardiovascular events. Several lines of evidence suggest that CRP has prothrombogenic effects. However, whether CRP directly participates in the pathogenesis of thrombosis in vivo has not been fully clarified. OBJECTIVE: To test whether human CRP (hCRP) affects arterial thrombus formation after balloon injury of smooth muscle cell (SMC)-rich or macrophage-rich neointima. METHODS: We compared the susceptibility of transgenic (Tg) rabbits expressing hCRP (46.21 ± 13.85 mg L(-1), n = 22) and non-Tg rabbits to arterial thrombus formation after balloon injury of SMC-rich or macrophage-rich neointima. RESULTS: Thrombus size on SMC-rich or macrophage-rich neointima was significantly increased, and was accompanied by an increase in fibrin content in hCRP-Tg rabbits, as compared with non-Tg rabbits. Thrombus size did not significantly differ between SMC-rich and macrophage-rich neointima in hCRP-Tg rabbits. Tissue factor (TF) mRNA expression and activity in these neointimal lesions were significantly increased in hCRP-Tg rabbits as compared with non-Tg rabbits. The degree of CRP deposition correlated with the elevated TF expression and thrombus size on injured neointima. In addition, hCRP isolated from hCRP-Tg rabbit plasma induced TF mRNA expression and activity in rabbit cultured vascular SMCs. CONCLUSIONS: These results suggest that elevated plasma hCRP levels promote thrombus formation on injured SMC-rich neointima by enhancing TF expression, but have no additive effects in macrophage-rich neointima.

Prevalence of antibody to hepatitis E virus among wild sika deer, Cervus nippon, in Japan.

We examined 976 sika deer serum samples, 159 liver tissue samples and 88 stool samples collected from 16 prefectures in Japan, and performed ELISA and RT-PCR assays to detect antibodies to HEV and HEV RNA, respectively. Although 25 (2.6%) of 976 samples were positive for anti-HEV IgG, the antibody titers were very low. The OD values ranged between 0.018 and 0.486, forming a single distribution rather than a bimodal distribution, suggesting that the antibody detected in this study was not induced by HEV infection, or that deer have low sensitivity to HEV. HEV RNA was not detected in these samples, also suggesting that deer may not play a role as an HEV reservoir.

Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose.

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.