Shigeaki Sato

Use of nitrite and hypochlorite treatments in determination of the contributions of IQ-type and non-IQ-type heterocyclic amines to the mutagenicities in crude pyrolyzed materials

The mutagenic heterocyclic amines Glu-P-2, MeA alpha C and Phe-P-1, which possess a 2-aminopyridine structure in their molecule (non-IQ-type mutagens), were found to be inactivated by nitrite treatment under acidic conditions, as observed previously with Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C. In contrast, MeIQx, 4,8- and 7,8-DiMeIQx, which were originally isolated from fried beef or heated model mixtures of creatinine, amino acids and glucose, and which have a 2-aminoimidazole moiety in their molecules (IQ-type mutagens), were very resistant to nitrite treatment like IQ and MeIQ. Both types of mutagenic heterocyclic amines were completely inactivated by treatment with hypochlorite. This differential inactivation of mutagenic heterocyclic amines by nitrite and hypochlorite was used in determination of the contributions of IQ-type and non-IQ-type mutagens to the total mutagenicities of various pyrolyzed materials. The percentage contributions of IQ-type mutagens to the mutagenicities of broiled sardine, fried beef, broiled horse mackerel, cigarette smoke condensate and albumin tar were 88, 75, 48, 6 and 4, respectively.

The presence of aldolase C in rat hepatoma

Abstract Aldolase A and hybrid molecules between aldolases A and C in a strain of Yoshida ascites hepatoma, AH143A of rat were found by electrophoresis on a cellulose acetate membrane. Results of column chromatography and inhibition by anti-aldolase A confirmed the presence of A 3 C hybrid molecule in this hepatoma. The significance of the aldolase C peptide in the hepatoma is discussed in relation to dis-differentiation.

Short chain intermediate in DNA replication of Ehrlich ascites tumor cells

Abstract The mode of DNA replication was investigated in vivo with Ehrlich ascites tumor cells. Alkaline and neutral sucrose density gradient centrifugations showed that nascent DNA chains, pulse-labelled with [3H]thymidine, were shorter than the bulk of the DNA. These short chains of DNA were gradually converted to longer ones and finally to material similar to the bulk of the DNA. Hydroxyapatite column chromatography showed that the nascent DNA chains were single-stranded but as the pulse time was lengthened, they changed to double-stranded DNA. These findings are similar to those obtained on bacterial systems and on some mammalian cells cultured in vitro.

RNA bound to nascent DNA in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells were labeled with 3H-uridine in,vitro for a short period and the nascent DNA fraction was isolated using nitrocellulose column chromatography. After heat-denaturation, the nascent DNA fraction was analyzed by isopycnic centrifugation in cesium sulfate. Its 3H-radioactivity separated into two peaks, one with a buoyant density of 1.47 which was slightly heavier than single-stranded DNA, and the other with the same density as free RNA (ϱ=1.66). The radioactivity of lighter fraction was converted to an acid-soluble form by treatments with RNase and alkali. Moreover, this radioactive material showed the same characteristics as nascent DNA on centrifugation in a neutral sucrose density gradient and hydroxylapatite column chromatography. These findings suggest that newly synthesized short RNA chain is covalently bound to nascent DNA and serves as a primer for DNA replication in these cells.

Hexokinase isozyme patterns of human uterine tumors

Hexokinase isozyme patterns of human uterine carcinomas and myomas were studied using cellulose acetate membrane electrophoresis. Uterine carcinomas of the cervix and corpus contained type II hexokinase as well as type I, unlike normal uterine cervical epithelium and endometrium which contained only type I. The hexokinase activities of carcinoma tissues were also higher than those of control tissues. Uterine myomas contained only type I hexokinase, like normal myometrium, and had similar specific activity to the latter. The abnormality in the pattern of hexokinase isozymes in human tumors and the application of this in clinical diagnosis of human cancers are discussed.

Purification and properties of α,α-trehalase from the mucosa of rat small intestine

Abstract α,α-Trehalase (EC 3.2.1.28, α,α-trehalose glucohydrolase) was solubilized from the microvillous membrane of the intestinal mucosa of rats with Triton X-100 and butanol. It was purified 6350-fold by gel filtration on Sephadex G-150 and chromatography on DE-52 and hydroxyapatite. The purified enzyme, with a specific activity of about 127 units per mg of protein, showed almost a single band of protein and activity on polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 96 000 on Sephadex G-150 and 90 000 and sodium dodecyl sulfate-polyacrylamide gel electrophoressi. Its pH optimum was 5.5–5.7 and its Km value for trehalose was 5.4 mM. Its activity was inhibited 30 and 100% by 1 mM p-chloromercuribenzoate and 0.1 mM HgCl2, respectively and 30% by 1 mM MgCl2. Moreover, its activity was inhibited completely by 10 mM tris(hydroyxmethyl)aminomethane and about 60% by 10 mM sucrose and cellobiose. The enzyme showed a high specificity for trehalose.

Mutagenicity of smoke condensates from joss sticks

Smoke condensates obtained from 8 Japanese brands of joss sticks were assayed for mutagenicity on Salmonella typhimurium TA100 and TA98 with and without metabolic activation by S9 mix. An average of 22 mg of smoke condensate was obtained per joss stick weighing about 0.3 g. Smoke condensates obtained from all the joss sticks tested showed definite mutagenicity with a linear dose response on both tester strains of bacteria with metabolic activation. Without S9 mix, the smoke condensates from some of the joss sticks also showed positive mutagenicity on TA100 at 0.15 or 0.3 mg/plate. The revertant numbers over the background counts induced by 1-mg samples of the smoke condensates from joss sticks with S9 mix were 140--310 with TA100 and 90--200 with TA98. These values were one-fifth to half that of cigarette smoke condensate with TA100 and one-fourteenth to one-fifth that with TA98. However, it was calculated that, when joss sticks are burnt continuously in a closed space under the usual conditions, the mutagenicity of their smoke per unit volume of air is similar to that of the smoke produced by the smoking of two cigarettes per hour. Extracts of unburnt joss sticks with methanol, chloroform or dimethylsulfoxide had no mutagenicity, showing that the burning process produces the mutagenic substance(s).

Purification of mouse pepsinogens by pepstatin-affinity chromatography

Pepsin and pepsinogen, especially those of hog and human, are the most extensively studied acid proteases and the primary structure of hog pepsin has been established [l] . However, although mice and rats are widely used experimental animals, little is known about their pepsin and pepsinogen. We purified two forms of mouse pepsinogen (PgI and PgII) to homogeneity, using pepstatin-Sepharose 4B affinity chromatography, gel fntration and Sephadex G-100 and DEAE-Sepharose CL6B column chromatography. This is the first report on the purification of mouse pepsinogens. Moreover, this result provides the evidence that pepsinogens per se have an ability to bind to pepstatin, a specific acid protease inhibitor [2,3].

Properties of nascent dna of ehrlich ascites tumor cells obtained by nitrocellulose column chromatography.

Abstract Ehrlich ascites tumor cells were labeled with 3 H-thymidine in vitro and their DNA was extracted by treatments with sodium dodecyl sulfate and NaCl. Bulk DNA and nascent DNA were separated by nitrocellulose column chromatography. Nascent DNA was characterized as short, single-stranded DNA by sucrose density gradient centrifugation, hydroxylapatite column chromatography and isopycnic centrifugation in cesium chloride. The properties of nascent DNA obtained by nitrocellulose column chromatography in this experiment were considerably different from those reported by other workers. The reasons for the difference are discussed and the value of this method for preparation and characterization of nascent DNA is emphasized.

Elevation of serum albumin concentration in analbuminemic rats by administration of 3′-methyl-4-dimethylaminoazobenzene

Abstract Albumin-like protein was detected in the serum of analbuminemic rats which have a mutation affecting albumin mRNA processing and genetically lack serum albumin. This protein was identified as rat serum albumin on the basis of molecular weight, immunological property and digestion patterns with proteases. The serum albumin level in analbuminemic rats was 5 μg/ml 4 weeks after birth and increased slightly during aging. By continuous administration of a hepatocarcinogen, 3′-methyl-4-dimethylaminoazobenzene, the level of serum albumin in the mutant rats increased over that of untreated rats and reached a maximum of 32 μg/ml at the 15th week of the carcinogen administration, whereas that of untreated rats was 16 μg/ml at the same time.