Forward Genetics Screens Using Macrophages to Identify <em>Toxoplasma gondii </em>Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

New York Medical College

To study a role of intracellular Ca2+ on protection effects in animals against intoxication, that is, autoprotection. When carbon tetrachloride (CCl4) of 0.2 ml/kg was i.p. administered daily to male Donryu rats, the serum alanine aminotransferase (ALT) activity rose to a maximal level one day later, and then decreased to the control level within three days. Thus, the animals were not received any intoxication effect of CCl4 due to second, third and fourth administrations. Thus, animals have a sensitive and non-sensitive periods against intoxication of CCl4. In non-sense period, the animals were protective against the poison. Such protection is an autoprotection. To study a role of intracellular Ca2+ on CCl4-induced autoprotection, we measured the intracellular Ca2+ in hepatocytes isolated from rats after daily CCl4 i.p. administration. Then, the peaks of the intracellular Ca2+ centered on three days and twelve hours after the first CCl4 administration from the first to fourth administration. These results show that the peak of the intracellular Ca2+ is perfectly corresponding with the trough of the serum ALT; that is, the protective period in animals against intoxication. Therefore, this result means that intracellular Ca2+ can mediate biodefense signal caused by CCl4 intoxication.

[Intracellular Ca2+ mediating biodefense signal on CCl4-induced autoprotection].

To elucidate the effects of Sho-saiko-to extract and its components, baicalin, baicalein, glycyrrhizin and glycyrrhetic acid, against the effects of longer periods of acute hepatic injury induced by CCl4, we measured serum ALT activity in male Wistar rats for five days after ip administration of CCl4 (0.2 ml/kg), and examined the daily changes of the pharmacokinetic behavior of salicylamide (SAM) for five days. Serum ALT activity rose to a maximum level within a day after administration of CCl4 and then decreased to the control level after three. Sho-saiko-to extract and its components could suppress this acute change in serum ALT activity to less than 50% of CCl4 alone. However, the pharmacokinetics of SAM showed that liver function recovers in a biphasic manner, so that plasma clearance (CL) decreased significantly at days 1 and 3 after administration of CCl4 (P<0.05). We concluded that the CL change at day 1 corresponds to the acute action of CCl4 intoxication, and that the change at day 3 is effect of physiologically reduced liver function due to the liver regeneration for tissue repair after the CCl4 hepatic injury. Sho-saiko-to extract and its components were shown to suppress acute hepatic injury induced by CCl4, and to bring about an early recovery in liver function.

Effects of Sho-saiko-to extract and its components, Baicalin, baicalein, glycyrrhizin and glycyrrhetic acid, on pharmacokinetic behavior of salicylamide in carbon tetrachloride intoxicated rats.

We have investigated the bio-defensive function of low-dose LPS pretreatment on CCl4-induced fulminant hepatic failure (FHF) in rats. In male Donryu rats treated with CCl4 at 1.0 ml/kg i.p., the serum alanine aminotransferase (ALT) increased up to a peak at 24 h. The increase in intracellular Ca2+ concentration was biphasic, i.e., a first peak at 1 h and a second at 3 days. However, when rats were treated daily with CCl4 at 1.0 ml/kg, serum ALT successively increased over several h after the second administration, but the rats died of FHF. The first Ca2+ peak occurred 1 h after CCl4 administration, but the concentration did not increase continually, until it reached the second peak. However, rats did not die when they were treated with lipopolysaccharide at 0.5 mg/kg 24 h prior to the initial dose of CCl4 at 1.0 ml/kg. The serum ALT value did not increase after administration of CCl4 at 1.0 ml/kg, but the second Ca2+ peak was sustained at > 1, 000 nM. We concluded that the second Ca2+ peak acts to protect the liver from injury.

A bio-defensive function induced by low-dose lipopolysaccharide pre-treatment against CCl4-induced fulminant hepatic failure in rats.

We investigated the tolerance of mice to carbon tetrachloride (CCl(4))-induced fulminant hepatic failure (FHF) in mice with daily intraperitoneal (i.p.) injection of high-dose CCl(4) (4 ml/kg). The tolerance was induced by preliminary i.p. administration of a low dose of CCl(4) (1 ml/kg) 24 h prior to the initial high-dose administration of CCl(4) (4 ml/kg). These mice were protected from death because of FHF induced by subsequent daily high-dose injection of CCl(4). This protection appears to result from a priming effect of the innate immune activity of CCl(4). The crude lipid extracted from the liver of low-dose CCl(4) injected mice also protected mice from death because of FHF. Examination of the innate immunological system using OK-432 (picibanil) revealed that the protection results from a priming effect of the innate immune activity with the low-dose CCl(4) or the crude lipid extract.

Innate host-defensive function of a hepatic lipid fraction produced in low-dose carbon tetrachloride-pretreated mice.

We studied the effect of sodium selenate on insulin resistance of Goto-Kakizaki (GK) rats. Rats were kept on standard laboratory chow with and without i.p. injections of sodium selenate (0.173 mg/kg body weight) for 14 days, and then subjected to the glucose clamp. The glucose clamp studies confirmed an improvement in insulin-stimulated glucose disposal in GK rats treated with sodium selenate, with respect to both insulin sensitivity and responsiveness. This amelioration of insulin resistance may be partly due to a direct effect of the sodium selenate on peripheral tissues. 2-Deoxyglucose uptake in sodium selenate-treated adipocytes was increased and the insulin findings suggest that sodium selenate increases not only insulin sensitivity but also insulin responsiveness. Sodium selenate also accelerated glucose incorporation into adipocytes of rats, suggesting that the action of sodium selenate is peripheral. Interestingly, sodium selenate at a high concentration (about 1 mmol/L) was more effective than insulin in enhancing glucose uptake. The present study suggested that sodium selenate treatment led to substantial improvement in peripheral insulin resistance.

Significant improvement of insulin resistance of GK rats by treatment with sodium selenate.

The aim of this study was to examine enhancing effect of L: -histidine into cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier. Uptake of L: -histidine into LMECs markedly increased with the addition of ZnSO(4) (0.1 mmol/L), and this enhanced uptake of L: -histidine was drastically reduced in the presence of the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH). However, the uptake of L: -histidine together with ZnSO(4) was not reduced by the addition of metabolic inhibitor, 2,4-dinitrophenol, or sodium ion replacement. Moreover, the addition of the system N-substrate, L: -glutamic acid γ-monohydroxamate did not significantly decrease the uptake of L: -histidine with 143 mmol/L Na (+) + 1 mmol/L BCH. These results indicated that system-N transporter does not play a role in the uptake of L: -histidine in the presence of ZnSO(4), suggesting that only system-L transporter is involved in the uptake of L: -histidine, although L: -histidine in the absence of ZnSO(4) was taken up by at least two pathways of Na(+)-dependent system-N and Na(+)-independent system-L processes into rat LMECs. The uptake of L: -histidine into rat LMECs in the presence of ZnSO(4) was also found to be unaffected by pH (5.0-7.4), indicating that uptake of L: -histidine into LMECs by the addition of zinc may not be involved in the H(+)-coupled transporters.

Enhancing effect of zinc on L-histidine transport in rat lung microvascular endothelial cells.

The aim of this study was to examine whether cultured rat thoracic aortic endothelial cells (TAECs) have the ability to metabolize the tertiary amine, imipramine. In rat TAECs, imipramine was biotransformed into N-demethylate and N-oxide by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively. The intrinsic clearance (V max/K m) for the N-oxide formation was approximately five times as high as that for the N-demethylate formation, indicating that oxidation by CYP was much higher than that by FMO. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to N-demethylate in rat TAECs using the respective anti-rat CYP antibodies (anti-CYP2C11 and anti-CYP3A2). The presence of CYP2C11 and CYP3A2 proteins was also confirmed in cultured rat TAECs using a polyclonal anti-CYP antibody and immunofluorescence microscopy. In contrast, the formation rate of N-oxide at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent K i value of 0.80 μmol/L, demonstrating that N-oxidation was catalyzed by FMO in rat TAECs. These results suggest that rat TAEC enzymes can convert substrates of exogenous origin such as imipramine, indicating that TAECs have an important function for metabolic products, besides hepatic cells.

Yukari Ueda

Department of Microbiology and Immunology

New York Medical College

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity

Yukari Ueda

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New York Medical College

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity

Department of Microbiology and Immunology