Samson R. Dutky

Melanin biosynthesis in Pyricularia oryzae: Site of tricyclazole inhibition and pathogenicity of melanin-deficient mutants

Abstract Tricyclazole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) inhibits melanin synthesis in Pyricularia oryzae at concentrations less than 0.01 μg/ml. The primary site of inhibition in the biosynthetic pathway occurs between scytalone and vermelone. Accumulation of several metabolites derived from melanin precursors along branch pathways is associated with inhibition of melanin biosynthesis. At low tricyclazole concentrations (0.01–1 μg/ml), predominant accumulation of 2-hydroxyjuglone and 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone (3,4,8-DTN) occurs as a result of the primary block between 1,3,8-trihydroxynaphthalene and vermelone. As the concentration of tricyclazole is increased from 1 to 10 μg/ml, flaviolin accumulation is markedly enhanced, whereas that of 3,4,8-DTN and 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone is depressed, indicating possible secondary sites of inhibition in the main and branch pathways. Five melanin-deficient mutants of P. oryzae that phenotypically resemble the tricyclazole-treated wild-type strain were nonpathogenic or rarely infected two rice varieties. Three of the mutants studied were genetically defective in the melanin biosynthetic pathway at the site blocked by tricyclazole in the wild type. The wild-type strain converted both scytalone and vermelone to melanin; whereas the three mutants and the tricyclazole-treated wild type converted only vermelone to melanin. The data suggest a relationship between melanin biosynthesis and pathogenicity in P. oryzae.

NMR spectra of C-24 isomeric sterols

Abstract Cholesterol and four pairs of C-24 isomeric sterols, campesterol-22,23-dihydrobrassicasterol, α-spinasterol-chondrillasterol, stigmasterol-poriferasterol, and sitosterol-22,23-dihydroporiferasterol were studied by NMR spectroscopy and their spectra are presented. The NMR spectra of three of the pairs of isomeric sterols recorded at 100 MHz could be differentiated from each other, although at 60 MHz only the spectra of campesterol (24α-methylcholesterol) and 22,23-dihydrobrassicasterol (24β-methylcholesterol) showed differences. Sitosterol and 22,23-dihydroporiferasterol, the pair of sterols that showed no differences in their NMR spectra are readily differentiated by the physical properties of their acetates. The practical application of NMR spectroscopy to several problems concerning the C-24 isomeric sterols is demonstrated.

26-Hydroxyecdysone: New Insect Molting Hormone from the Egg of the Tobacco Hornworm

Five kilograms of tobacco hornworm eggs (48 to 64 hours old) afforded 26.5 milligrams of a new crystalline insect molting hormone identified as 26-hydroxyecdysone. The three known insect ecdysones—α-ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone—were also present but in much smaller quantities. The new hormone is the predominant molting hormone in the hornworm during this stage of embryonic development. These results takent in context with the current knowledge of the chemistry and biochemistry of the molting hormones during postembryonic development in the hornworm indicate quantitative and qualitative differences in the biosynthetic-mnetabolic pathways as well as in the ecdysones in different developmental stages of this insect.

Triparanol inhibition of sterol biosynthesis in Chlorella emersonii

Abstract Nine sterols, in addition to the five previously found, have been identified by means of GLC and mass spectral analysis in triparanol-treated cells of Chlorella emersonii. They are: 24-methylene-pollinastanol, 24-methylenecycloartanol, 24-dihydroobtusifoliol, cycloeucalenol, obtusifoliol, 14α-methyl-(24S)- 5α-stigmast-8-en-3β-ol, 4α,14α-dimethyl-(24S)-5α-stigmast-8-en-3β-ol, 5α-ergosta-7,22-dien-3β-ol and (24S)- 5α-stigmasta-7,25-dien-3β-ol. The presence of most of these sterols in both untreated and triparanol-treated cultures has led to a proposed scheme of sterol biosynthesis in Chlorella emersonii.

Ecdysone metabolism: Ecdysone dehydrogenase-isomerase

An enzyme system that converts α-ecdysone to its hormonally less active 3α-epimer was detected only in the midgut of the tobacco hornworm,Manduca sexta (L.). This system appears to be specific for the ecdysones and may represent a metabolic control point for regulating molting hormone activity.

3-epi-20-hydroxyecdysone from meconium of the tobacco hornworm

Abstract A new ecdysteroid, 3-epi-20-hydroxyecdysone, with biological activity 1 10 to 1 15 that of α-ecdysone has been isolated and identified from the meconium of the tobacco hornworm. The possible role or function of this steroid as an inactivation product and/or regulator of molting hormone titer is discussed.

Sterol composition and phytosterol utilization and metabolism in the milkweed bug

Analysis of the sterols of the milkweed bug,Oncopeltus fasciatus (Dallas) and dietary sunflowerseeds revealed that there is little, if any, conversion of dietary C28 or C29 phytosterols to cholesterol in this phytophagous insect. The dietary sterols are apparently utilized with little alteration both during development to the adult stage and egg production, and cholesterol comprises <1% of the sterols in either adult males and females or in the eggs. The significance of these findings are discussed in light of the recent discovery that the C28-ecdysone, makisterone A, is the predominant molting hormone in the enbryonated egg of the milkweed bug.

14α-methyl-5α-ergost-8-en-3β-ol and 14α-methyl-5α-ergosta-8,24(28)-dien-3β-ol from triparanol-treated Chlorella emersonii☆

Abstract From triparanol-treated cultures of Chlorella emersonii , two new sterols, 14α-methyl-5α-ergost 8-en-3β-ol and 14α-methyl-5α-ergosta-8,24(28)-dien-3β-ol were isolated and identified. These two sterols represent 7 and 3 per cent, respectively, of the total sterols from treated cultures, though only the former was discernible in gas chromatograms of non-treated cultures. The triparanol-treated cultures also contained the naturally occurring sterols, ▵ 7 -ergostenol, ▵ 7 -chondrillastenol and chondrillasterol, but at concentrations less than that present in control cultures. The synthesis of 14α-methyl-5α-ergost-8-en-3β-ol is described.

Ecdysone 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.)

An ecdysone 20-hydroxylase enzyme system that converts α-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue.

The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity.

Six naturally occurring C27 ecdysteroids were isolated and identified from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. In order of decreasing quantities the hormones were: 20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, 20hydroxyecdysone, 3-epi-20,26-dihydroxyecdysone, 3-epi-ecdysone, and ecdysone. 20-Hydroxyecdysone, in an earlier study, was the major molting hormone present at peak titer during pupal-adult development. The major ecdysteroid present during embryonic development in this insect, 26-hydroxyecdysone, was not detected. The copresence of all six of these ecdysteroids from a single developmental stage of an insect provides information on the metabolic interrelationships that exist among these steroids and on their possible function(s) in insects. The 3alpha-ecdysteroids were far less active than the 3 beta-epimers in the house fly assay. The significance of epimerization is discussed.