Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Changes in the organization and biosynthesis of cell surface acidic sugars during the phytohemagglutinin-induced blast formation of human T-lymphocytes

Use of cell electrophoresis combined with specific enzymes and varying ionic strength revealed a topological change of acidic sugars in lymphocyte membrane treated with a T-cell mitogen, phytohemagglutinin (PHA). The suggested alterations were an early translocation of hyaluronic acid to the cell periphery within 15 min of PHA addition and, 4 h later, the appearance of chondroitin sulphate in T-lymphocytes, but not in B-lymphocytes. As the contribution of chondroitin sulfate to the electrophoretic mobility increased with time up to 24 h, that of sialic acid decreased conversely. Several agents which block blast formation (2 mM ethylene glycol bis-β-aminoethylethyl-N,N,N′,N′-tetraacetic acid, 2 × 10⁻⁷ M ouabain, 0.1 μg/ml colchicine and 1 μg/ml cytochalasin B) also blocked the translocation of hyaluronic acid at the same concentrations. Chemical analysis of [¹⁴C]glycosaminoglycans by means of gel filtration followed by paper chromatography revealed a four-fold enhancement of the biosynthesis of chondroitin sulfate C after PHA stimulation. The presence of chondroitin sulfate in the cell periphery was also detected electrophoretically in T-cell type leukemia cells (MOLT-4B). These results suggest that the reorganization of glycosaminoglycans may be one of the membrane changes associated with blast formation of lymphocytes.     

Protein synthesis in the embryo ofPinus thunbergii seed III.

Dissociability of the monomer ribosomes prepared from dry and imbibed pine (Pinus thunbergii) seed embryos was analyzed in sucrose density gradient containing a high salt buffer. Abnormal dissociation into the subunits was observed with the ribosome preparation from dry seed embryos when compared with that from imbibed seed embryos, i.e. each subunit peak was broader and localized at a lower site in sucrose density gradient. This indicates some change(s) in ribosomes during imbibition of seeds. These ribosomal changes also progressedin vitro. That is, after incubation of ribosome preparation from dry seed embryos in a high salt buffer for 5 min at 30 C or in a low salt buffer for 15 hr at 0 C, complete dissociation into the normal subunits was observed. No difference was found between polyacrylamide gel electrophoresis patterns of ribosomal RNA from dry and imbibed seed embryos. These results suggest some alteration in the protein components of ribosome during imbibition of pine seeds. This paper is dedicated to Prof. Shyogo Sawamura, Utsunomiya University on his retirement in March, 1979.     

ADP-ribosylation of nuclear protein A24

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) $\text{Proc}$. $\text{Natl}$. $\text{Acad}$. $\text{Sci}$. $\text{USA}$$\text{74}$, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.     

Redox potential of plastoquinone A in spinach chloroplasts

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is ?30 mV per pH between pH 4.0 and 5.7, and ?60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Stimulation of auxin-induced elongation of cucumber hypocotyl sections by dihydroconiferyl alcohol. Dihydroconiferyl alcohol inhibits indole-3-acetic acid degradation in vivo and in vitro

The mechanism by which dihydroconiferyl alcohol (DCA) stimulatesindole-3-acetic acid (IAA)-induced elongation of cucumber hypocotylsections was studied. Although DCA did not affect the uptakeof IAA-5-³H by hypocotyl sections, the endogenous level of IAA-5-³Hin DCA-treated sections was much higher than in DCA untreatedones. IAA-5-³H in the incubation medium was degraded in thepresence of hypocotyl sections, and this degradation of IAAwas inhibited by DCA. An in vitro experiment with horseradishperoxidase revealed that DCA inhibited the IAA degrading activityof the oxidase, as did caffeic acid and ferulic acid. Theseresults suggested that DCA enhances IAA-induced cucumber hypocotylelongation by acting as an antioxidant of IAA. (Received June 4, 1975; )     

The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.     

A Versatile Chromatographic Procedure for Purifying PS II Reaction Center Complex from Digitonin Extracts of Spinach Thylakoids

A versatile, two-step chromatographic method using DEAE-Toyopearl(Toyo Soda, Japan) is described for purifying photosystem IIreaction center complex from digitonin extracts of spinach thylakoidmembranes. The method is very simple and brings about an approximatefour-fold increase in the specific activity, on a chlorophyllbasis, of 2,4-dichlorophenol-indophenol photoreduction with1,5-diphenylcarbazide (to about 2,000 µ electron equivalentsper mg chlorophyll per h), with an approximate 40 percent recoveryin chlorophyll. The SDS-polyacrylamide gel electrophoresis performedin the presence of 4 M urea in the analyzing gel shows fourpolypeptide bands of the photosystem II reaction center of about47, 43, 30 and 9 kilodaltons. The absorption and fluorescence properties, as well as the pigmentand chemical compositions and the above mentioned polypeptideprofile of the purified complex are essentially identical withthose of the preparations isolated by the previously describedmethod (Satoh 1982). The digitonin solubilization of thylakoid membranes destroysthe water splitting machinery, so that the purified complexshows no oxygen evolving activity, even although 0.6–0.7atoms of manganese per 50 chlorophyll molecules still remain. (Received March 19, 1985; Accepted July 19, 1985)