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.     

Genetic studies on site-specific endodeoxyribonucleases in Bacillus subtilis: multiple modification and restriction systems in transformants of Bacillus subtilis 168

Summary We transformed B. subtilis 168 with DNA from B. subtilis IAM1231, IAM1192 and ATCC6633. When we examined the restriction activities of the transformants in vivo and in vitro using phage 105C we found the following: (1) Cells of either IAM1231 or IAM1192 have two modification and restriction systems (Bsu1231(1)-system and Bsu1231(II)-system in IAM1231, and Bsu1192(I)-system and Bsu1192(II)-systems in IAM1192), and cells of ATCC6633 have only one system (Bsu6633-system). (2) The restriction enzymes of all of these five systems are site-specific endonucleases. (3) The nucleotide sequence specificities of the enzymes involved in Bsu1231(I)-system, Bsu1192(I)-system and Bsu6633-system are the same; and those of Bsu1231(II)-system and Bsu1192(II)-system are the same. The sequence specificities of these two groups are different from each other and also different from those of the Bsu168-system of B. subtilis 168, the BsuR-system of B. subtilis R and the Bsu1247(I)-and Bsu1247(II)-systems which are systems of B. subtilis IAM1247. (4) Transformants possessing four different modification and restriction systems (Bsu1231(I)-, Bsu1247(I)-, BsuR- and Bsu168-systems) were constructed. (5) Transformation of two derivatives of 168 that were m _R ⁺ r _R ⁺ by DNA from IAM1231 produced 16 transformants that had the Bsu1231(II) restriction system, but had lost the BsuR system. Transformation of a derivative of 168 that was m _1247(II) ⁺ r _1247(II) ⁺ by DNA from m _1231(II) ⁺ r _1231(II) ⁺ -or m _R ⁺ r _R ⁺ -derivative of 168 produced about 100 each of transformants that had the Bsu1231(II)-restriction system or the BsuR-restriction system. But all these transformants lost the Bsu1247(II)-system.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Co-oxidations of 1,3-diphenylisobenzofuran by the Haber-Weiss reaction. Is singlet oxygen concerned in this oxidation?

The reaction of $\text{O}{}_2{}^{\mathrm{??}}$ with H₂O₂ in the presence of 1,3-diphenylisobenzofuran was studied. o-Dibenzoylbenzene was obtained in 82 % yield, which decreased to 52 % when dimethoxyethane was presence. Additions of β-carotene or 1,4-diazabicyclo-[2,2,2]-octane also inhibited the production of o-dibenzoylbenezene. These results show that singlet oxygen may be a considerable species generated by the Haber-Weiss reaction.     

Purification and characterization of DNA-relaxing enzyme from Haemophilus gallinarium.

A DNA-relaxing enzyme capable of concerted nicking and closing of DNA backbone bonds has been purified from Haemophilus gallinarum by two chromatographic steps and gel filtration. The enzyme efficiently catalyzes the removal of superhelical turns from a negatively twisted DNA and requires Mg2+ for this activity. Slight removal of superhelical turns from a positively twisted DNA generated by binding of ethidium bromide is found, but only at high enzyme concentrations. The DNA-relaxing activity is inhibited markedly with heat-denatured DNA, whereas native DNA and RNA have almost no affect on this activity.     

Anomeric preferences ofd-glucose uptake and utilization by cerebral cortex slices of rats

On aerobic incubation of rat cerebral cortex slices with anomers ofd-glucose and with 2-deoxy-d-glucose (2DG) for 5 min, the disappearance of -d-glucose from the incubation mixture was greater than that of -d-glucose and both anomers had a greater rate of disappearance than that of 2DG. In addition, there were significantly greater consumption of oxygen and production of lactate with the -anomer than with the -anomer. In similar experiments with³H-labeledd-glucose anomers and [1-³H]-3-O-methyl-d-glucose (3MG), the accumulation of [1-³H]--d-glucose (up to 5 min) by rat cerebral cortex slices was greater than that of [1-³H]--d-glucose. Although initially lower than that of the anomers, the accumulation of [1-³H]-3MG increased at a greater rate and, by 5 min of incubation, was greater than that of both glucose anomers. This preferential accumulation was seen to disappear when the slices were preincubated with 2DG (hexokinase inhibitor) or when the temperature of incubation was reduced to 20°C. Under those conditions the data with the glucose anomers were similar to those obtained with 3MG. Our data then suggested that the greater accumulation of -d-glucose than of -d-glucose by the slices was probably not due to differences in transport through brain cell membranes but rather to the preferential metabolism of the -d-glucose.     

Continuous survey of the distribution of RNA coliphages in Japan

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5-yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities. Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II:III = 3:1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5-yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.     

Distribution of ribonucleic acid coliphages in south and east Asia.

We investigated the distribution of ribonucleic acid (RNA) coliphages in the Philippines, Singapore, Indonesia, India, and Thailand by collecting sewage samples from domestic drainage in November 1976. Of the 221 samples collected from domestic drainage, 50 contained RNA phages (52 strains). By serological analysis, 46 of the 52 strains were found to belong to group III. It can thus be said that the most prevalent RNA phages in Southeast Asia (at least, in the Philippines, Singapore, and Indonesia) were group III phages. Investigations of sewage samples collected from domestic drainage in Japan indicate that the most prevalent RNA phages in mainland Japan (north of Kyushu) are group II phages, whereas group III phages are predominant in the southern part of Japan (south of Amamiohshima Island). We therefore propose a borderline between Kyushu and Amamiohshima Island for the geographical distribution of RNA coliphages in the domestic drainage of South and East Asia. Moreover, one strain (ID2) was inactivated to some extent with the antisera of four groups of RNA phages. This is thought to be significant from the evolutionary viewpoint.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.