It is already known that a tubular structure will appear on the surface of a factured dentin when etched with a weak acid [1]. The present preliminary report deals with the introduction of a scanning electron microscope for the elucidation of the appearance of these tubular structures.

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The present report, which is a sequel to Part 4 previously published that dealt with an antidotal action of nicotinism by terpin hydrate compounds [1], further attempts to clarify the toxic action of nicotine on the cardiac function and the relation of terpin hydrate compounds as an anti-nicotine agent. At the same time, as a preliminary experiment to a future study, effect of the cigarette smoke in terms of the electrocardiogram was investigated, use of the electrocardiogram for this purpose being fairly rare.

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HoDGE[1] reported the determination of lipids in human gingiva. FISHER et al.[2] investigated into the cholesterol contents in bovine and human gingiva. STERN et al.[3] observed the incorporation of sodium acetate-1-¹⁴C into cholesterol, palmitic acid and oleic acid in human gingiva. OTAKE et al.[4] also reported the incorporation of sodium acetate-1-¹⁴C into the lipid fractions in guinea pig gingiva. Recently, KANDA[5] reported the incorporation of radioactivity from sodium acetate-1-¹⁴C into the lipid fractions in gingiva of guinea pig fed on ascorbic acid deficient diet, and incorporated radioactivity in the free fatty acid fraction of scorbutic animals was increased about twice than that of normal. However, no work has been done to study the patterns of fatty acid biosynthesized in scorbutic guinea pig gingiva.
The present report describes the patterns of free fatty acids biosynthesized from sodium acetate-1-¹⁴C in scorbutic guinea pig gingiva.
Sodium acetate-1-¹⁴C (specific activity : 45.4 mCi/mM) was obtained from New England Corp. Other materials were also available commercially. Compressed diet RC5 for the experimental animals was obtained from Oriental Yeast Industrial Company, Ltd. Male guinea pig, weighing from 300 to 350 g were used. The animals were fed on a scorbutogenic diet for 23 days. The diet was prepared by heat treatment of the compressed diet RC5 at 100°C for 2 hours. The animals were killed by decapitation, and gingiva was quickly excised and placed in ice-cold 0.25 M sucrose solution. Sixty mg of gingiva was incubated at 37°C for 3 hours in 2.0 ml of the Krebs-Ringer phosphate buffer (pH 7.4) containing 100 μCi of sodium acetate-1-¹⁴C. The tissue was removed from the reaction mixture was washed with 3 x 100 ml of distilled water. Then, the tissue was homogenized with 10 ml of chloroform-methanol (2:1), and the lipids were extracted for 20 hours at room temperature. The organic phase was separated by centrifugation, and the precipitate was washed 20 ml of the same organic solvent. The combined organic solvent was dried under a nitrogen
The evidence suggested that in gingiva of the scorbutic animals, biosynthesis of unsaturated fatty acid of C₁₈ is inhibited but that of saturated fatty acid of C₁₈is stimulated.

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It has been reported that cell-free extracts of adult rat brain incubated with mevalonic acid-2-¹⁴C synthesize radioactive nonsaponifiable materials consisting largely of squalene, and the synthesis of digitonin-precipitable sterols from mevalonic acid is depressed by nicotinamide[1]. The evidence suggested that the cyclization of squalene was inhibited by nicotinamide. BOWEN et al.[2] reported that the cyclization of squalene in yeast was also inhibited by nicotinic acid. Serum cholesterol levels were reduced by nicotinic acid but not nicotinamide[3]. However, the mechanisms of this inhibition are not known.
This paper reports the stimulation of formation of squalene from mevalonic acid in cell-free extracts in adult rat brain by nicotinamide.
DL-Mevalonic acid-2-¹⁴C lactone (specific activity : 5.58 mCi/mM) was obtained from Daiichi Pure Chemicals Co., Ltd., and before use, lactone was converted to the acid by alkali. Squalene-¹⁴C (specific activity : 21.2 mCi/mM) used was prepared by rat liver enzyme according to the method of GOODMAN[4]. Male rats of Donryu strain weighing about 250 g were used. The rats were sacrificed by decapitation, and their brains were quickly excised. The brain homogenate and the components of incubation mixture were prepared according to the method of KELLEY et al. [1] except 0.1 μCi of mevalonic acid-¹⁴C or 0.05 μCi of squalene-¹⁴C as substrate was used. Incubation was carried out for 2 hours at 37°, and the reaction was stopped by the addition of 3.0 ml of water- ethanol (1:1) containing 15% KOH (w/v) and 20 μg each of carrier squalene and cholesterol, after which the mixture was heated for 1 hour at 90°. The mixture was added 6 ml of distilled water and extracted three times each time with 20 ml of petroleum ether. The combined petroleum ether was washed three times, each time with 20 ml of distilled water and evaporated to dryness. The dried nonsaponifiable extract obtained was dissolved in a small volume of benzene, and was applied on a silica gel plate for thin-layer chromatography. n-Hexene-ethyl acetate (9 :1) was used as an ascending solvent. After development, the plate was sprayed with 0.1 % 2, 7'-dichlorofluorescein ethanol solution to detect the carriers. The spots on the plate were determined by ultraviolet absorption. Then, the radioactive areas on the plate were determined using a thin-layer radiochromatogram scanner, and the radioactive products were scraped off and eluted from the gel by three washes with ethyl acetate.
The ethyl acetate was transferred into a counting vial and dried under a stream of nitrogen. Radioactivity of each sample was assayed with a liquid scintillation counter.
The results are summarized in Tables 1 and 2.
The activity of the cell-free extracts of rat brain for the incorporation of mevalonic acid-2-¹⁴C into squalene-¹⁴C was markedly increased about 7 times by nictinamide. However, nicotinamide had no significant stimulatory effects on the formation of cholesterol-¹⁴C from squalene-¹⁴C.

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