MyD88 in donor bone marrow cells is critical for protection from acute intestinal graft-vs.-host disease.

To understand the role of myeloid differentiation factor 88 (MyD88) expressed by donor bone marrow (BM) in the pathophysiology of graft-vs.-host disease (GVHD), we investigated the effects of transplantation of MyD88-deficient T cell-depleted BM (MyD88KO TCD-BM) on the severity of GVHD. Transplantation with MyD88KO TCD-BM aggravated GVHD; serious gut damage was evident, with high infiltration of T cells into the intestines of recipients and markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs). MDSCs from MyD88KO mice were defective in inducing donor T-cell apoptosis and inhibiting T-cell proliferation. Supplementation of transplanted mice with MDSCs from wild-type mice, but not MyD88KO mice, attenuated GVHD severity with reduced intestinal T-cell infiltration in MyD88KO TCD-BM recipients. Pretreatment of BM donors with lipopolysaccharide to increase MDSC levels and MyD88 transcription in the TCD-BM transplant alleviated GVHD severity and intestinal T-cell infiltration. The T cell/MDSC ratios were correlated with intestinal GVHD severity in both animal models and human patients. This study indicates that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal intestinal GVHD.

Functional relevance of human adh polymorphism.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were C. J. Peter Eriksson and Tatsushige Fukunaga. The presentations were (1) 4-Methylpyrazole as a tool in the investigation of the role of ADH in the actions of alcohol in humans, by Taisto Sarkola and C. J. Peter Eriksson; (2) ADH2 polymorphism and flushing in Asian populations, by Wei J. Chen, C. C. Chen, J. M. Ju, and Andrew T. A. Cheng; (3) Role of ADH3 genotypes in the acute effects of alcohol in a Finnish population, by Hidetaka Yamamoto, Kathrin Kohlenberg-Müller, and C. J. Peter Eriksson; (4) Clinical characteristics and disease course of alcoholics with different ADH2 genotypes, by Mitsuru Kimura, Masanobu Murayama, Sachio Matsushita, Haruo Kashima, and Susumu Higuchi; (5) ADH2 polymorphism, alcohol drinking, and birth defects, by Lucinda Carr, D. Viljoen, L. Brooke, T. Stewart, T. Foroud, J. Su, and Ting-Kai Li; and (6) ADH genotypes and alcohol use in Europeans, by John B. Whitfield.

In vitro galactosylation of rhodopsin and opsin: kinetics, properties and characterization.

At best, only trace amounts of galactose have been detected as constituents of rhodopsin as analysed by several laboratories. Nevertheless, the enzymatic galactosylation of rhodopsin proceeds readily in vitro, a process which can be catalysed by galactosyltransferases from several sources. Little information is available, however, concerning the properties of the in vitro reaction. We have examined characteristics of the latter process with the hope of shedding light on the capacity of the retina to carry out this reaction. Kinetic properties of the galactosyltransferases of bovine and embryonic chick retinas, bovine milk and rat liver-Golgi were examined using rhodopsin, opsin, N-acetylglucosamine and ovalbumin as exogenous acceptors. All of these studies demonstrated the very limited activity of the galactosyltransferases of the retina as compared to the milk and rat liver systems. The subcellular distribution of the galactosyltransferases of bovine retina was examined. The influence of compounds that might modulate the reaction was also examined. alpha-Lactalbumin, a modifier of the galactosyltransferase in milk, acted as a competitive inhibitor of the galactosylation of opsin. Analogs of vitamin A, shown to inhibit galactosyltransferases in other systems, did not have this effect on the galactosylation of opsin. Similarly, mixing experiments could not demonstrate the presence of endogenous material that inhibited the reaction in the retina. The conformation of the visual pigment was shown to influence the reaction. After bleaching by visible light, opsin was preferred over rhodopsin as an acceptor of galactose by the galactosyltransferases of bovine and embryonic chick retinas and by rat liver. This distinction was only minimally demonstrated by the milk enzyme. The galactosylation of ovalbumin was unaffected by conditions of light or dark by any of the enzymes. While the mode ratio of galactose to rhodopsin after catalysis by the milk enzyme was about 1.6, this ratio was only about 0.01 after reaction with the enzyme from bovine retina. The linkage of galactose in enzymatically galactosylated rhodopsin and opsin was beta(1-4).

Reductively methylated, tritiated rhodopsin of high specific activity; a convenient sensitive tracer for use in the radioimmunoassay of rhodopsin.

Bovine rhodopsin was subjected to reductive methylation in the dark using formaldehyde and high specific activity sodium borotritide. After purification by gel filtration and affinity chromatography on Concanavalin A-Sepharose, the product retained its immunoreactive properties. [3H]-Reductively methylated rhodopsin (specific activity, 32 Ci/mmole) was suitable for use in radioimmunoassays for rhodopsin, having many advantages over radioiodinated rhodopsin for this purpose. The site of the reductive methylation was shown to be the non-active site lysines with the production of tritiated N-epsilon-dimethyllysine and tritiated N-epsilon-methyllysine in a molar ratio of about 1.3:1, respectively. In terms of stability, ease of preparation, and specificity, tritiated, reductively methylated rhodopsin presents itself as a preferable ligand to radioiodinated rhodopsin in many applications, such as the radioimmunoassay.

Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver, kidney, and lung.

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochrome b5 contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline. N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 and b5. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue specificity of induction of hamster monooxygenase activity by ethanol.

The effects of ethanol on liver, kidney and intestine monooxygenases were studied using hamsters chronically fed with isocaloric control and ethanol-containing liquid diets. The inductive effects of ethanol on liver and kidney aniline hydroxylase activities began to approach plateau level after the animals were fed ethanol for two weeks. Intestinal aniline hydroxylation was refractory to ethanol induction. In control and ethanol-fed hamsters, CO-difference spectra of hepatic and extrahepatic microsomes differed in absorption maxima. Chronic alcohol consumption caused significant increases of cytochrome P-450 and cytochrome b5 contents of liver and kidney microsomes. The increases of the heme proteins were associated with the induction of aniline hydroxylase, N-nitrosodimethylamine demethylase and 7-ethoxycoumarin 0-deethylase activities. In contrast to the liver and kidney, intestinal microsomal cytochromes P-450 and b5 contents in ethanol-treated animals were lower than the controls. Ethanol pretreatment was without effect on intestinal monooxygenase activities toward the metabolism of aniline, N-nitrosodimethylamine, 7-ethoxycoumarin and benzo(a)pyrene. Gel electrophoresis of tissue microsomes from control and ethanol-treated hamsters revealed that ethanol treatment enhanced the intensity of the protein band(s) in the cytochrome P-450 molecular weight region in the liver and kidney, but not in the intestine. These results demonstrate that in hamsters the response of monooxygenase to ethanol may vary from tissue to tissue and it is difficult to make a generalization regarding the inducing property of ethanol. The differential effect on cytochrome P-450 may be an important factor in determining the interaction between ethanol and xenobiotic metabolism in animal tissues.