Mitsugu Tomioka

α-tocopherol affects the urinary and biliary excretion of 2,7,8-trimethyl-2(2′-carboxyethyl)-6-hydroxychroman, γ-tocopherol metabolite, in rats

In this study, we investigated a change in the excretory content of 2,7,8-trimethyl-2(2′-carboxyethyl)-6-hydroxychroman (γ-CEHC), a γ-tocopherol (γ-Toc) metabolite, in rat urine and bile by using a new high-performance liquid chromatography-elelectrochemical detection (HPLC-ECD) method. In this determination, CEHC [α- and γ-CEHC, where α-CEHC-2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychioman] in the biological specimens were treated with 3 N methanolic HCl to hydrolyze conjugates and to promote esterification. The methylated samples were extracted by n-hexane/water (1∶2). The analyses of the methyl esters of α-CEHC and γ-CEHC were performed by an HPLC-ECD using an ODS-3 column at 35°C. The mobile phase was acetonitrile/water (45∶55, vol/vol) containing 50 mM sodium perchlorate. After rat urine and bile samples, respectively, were methylated as described above, methylated biliary metabolites were identified by liquid chromatography mass spectrometry as methyl esters of γ-CEHC. Furthermore, we examined the differences in the excretion of γ-CEHC between rat urine and bile after an oral administration of γ-Toc or α- +γ-Toc by the above HPLC method. In the γ-Toc group, each vitamin E-deficient rat was given 0.5 mL of a stripped corn oil preparation containing 10 mg of γ-Toc. In the α- +γ-Toc group, the rat was given 10 mg of α-Toc and 10 mg of γ-Toc. The content of γ-CEHC in rat urine from the α- +γ-Toc group was increased more in comparison to the γ-Toc group at 18–36 h after oral administration. Moreover, the content of γ-CEHC in rat bile in the α- +γ-Toc group was increased more in comparison to the γ-Toc group at 6–18 h after oral administration. Therefore, we have suggested that γ-CEHC was shifted mainly to urinary excretion after γ-CEHC had been excreted into the bile. Furthermore, we assume that α-Toc may affect the metabolism of γ-Toc to γ-CEHC in the body.

LPS inhibits the intracellular growth of Legionella pneumophila in thioglycolate elicited murine peritoneal macrophages by iron-dependent, tryptophan-independent, oxygen-independent, and arginine-independent mechanisms.

Thioglycolate‐elicited murine macrophages from genetically susceptible A/J mice activated with lipopolysaccharide (LPS) and infected with Legionella pneumophila in vitro evince marked inhibition of intracellular growth of this bacterium. The mechanism of inhibition by LPS‐activated macrophages in terms of replication of this intracellular pathogen is unclear. LPS activation of murine macrophages induced a downshift in transferrin receptor (TfR) expression and reduction in cellular iron content, and this was correlated with augmented intracellular growth of Legionella in the cells. When LPS‐stimulated macrophages were first saturated with iron, partial reversion of L. pneumophila growth restriction was observed. However, an excess of exogenous L‐tryptophan (Trp) did not reverse this growth inhibition, nor did supplementation of the macrophage culture medium with both iron and Trp. The antilegionella activity of the macrophages induced by LPS activation was independent of reactive oxygen intermediates (ROI), since the scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on growth restriction. Likewise, notwithstanding the ability of LPS‐activated macrophages to synthesize reactive nitrogen intermediates (RNI), which was inhibited by l‐arginine analogs (NG‐monomethyl‐l‐arginine and l‐aminoguanidine), as well as by incubation in arginine‐free medium, their ability to inhibit the intracellular replication of L. pneumophila was not affected. Thus, we conclude that LPS‐activated macrophages inhibit the intracellular growth of L. pneumophila partially by iron‐dependent, Trp‐independent, and ROI‐ and RNI‐independent mechanisms. We also suggest that additional unknown mechanisms are involved, since complete reversion was not obtained. J. Leukoc. Biol. 57: 80–87; 1995.

CHEMICAL CROSS-LINKING OF PEPTIDES DERIVED FROM RECA WITH SINGLE-STRANDED OLIGONUCLEOTIDES CONTAINING 5-FORMYL-2′-DEOXYURIDINE

We report the first example of chemical cross-linking of 5-formyl-2′-deoxy-uridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.

The effect of link protein on the rheological properties of solutions of proteoglycan-hyaluronic acid aggregates

Abstract Proteoglycan monomers, prepared from bovine nasal cartilage, were reassembled with a commercial hyaluronic acid, with or without link protein. The effects of link protein on the rheological properties of proteoglycan aggregates were studied over the concentration range 0.1–2.0 g/100 ml. Aggregate formation of proteoglycan monomer with hyaluronic acid increased the dependence on shear rate and shifted the Newtonian region to lower shear rates, compared to that observed with a proteoglycan monomer solution at the same concentration. The dynamic viscoelasticity also increased. In the presence of the link protein there is no Newtonian flow region over the shear rate ranges at a concentration of 1 g/100 ml or above and the value of the loss tangent remained low over the angular frequencies examined. Link protein strengthens the intermolecular interactions increasing the relaxation time of the aggregate solution. It increases the co-operativity of the binding of the proteoglycan monomers to the hyaluronic acid chain allowing a dynamic network structure to be developed in solution. The rheology is strongly dependent on the weight ratio of the proteoglycan to hyaluronic acid.