Successive transfer in synthetic medium of spores and mycelial fragments from original toxin-producing cultures of Helminthosporium sacchari results in attenuated cultures which do not produce the host-specific toxin helminthosporoside. When attenuated cultures are grown on material obtained from the water wash of sugarcane leaves susceptible to this fungus, the production of heminthosporoside resumes.Cpmpounds that activate toxin production in the fungus are present on the leaf surface and presumably arise via plant metabolism. One activator was identified as a novel free amine, serinol (2-amino-1,3-propanediol). It activates toxin production in attenuated cultures at 1 gM. Several experiments described in this report argue against the selection theory for the attenuation of cultures. The biological significance and some possible mechanisms for the activation of toxin biosynthesis are discussed.Variation within a pathogen population may produce highly efficient biotypes that are extremely successful parasites. Conversely, biotypes may arise in which the ability to persist parasitically is greatly reduced or eliminated. This loss of parasitic capabilities has been labeled attenuation (1).The genus Helminthosporium includes pathogens whose conidial stages are responsible for serious diseases of rice, corn, grasses, and cereals (2). Several members of the genus, including H. tyctoriae, H. carbonum, H. maydis, and H. sacchari, produce host-specific toxins. These compounds, which are fungal metabolites toxic only to the susceptible host, produce virtually all of the disease symptoms and are critical for pathogenicity of the fungi (3). Since pathogenicity in these fungi is dependent upon their host-specific toxins, the loss of toxin production and attenuation are synonymous. Cessation of toxin production has been observed in cultures of H. maydis (4), and H. carbonum and H. vwctoriae (5).Helminthosporium sacchari (Van Breda de Haan) Butler is the causal agent of the eyespot disease of sugarcane. This disease, found in most areas where sugarcane is grown (6), is characterized by the formation of eye-shaped lesions, followed by the development of reddish-brown "runners" which extend from the lesion toward the leaf tip. Runner formation is caused by the host-specific toxin helminthosporoside (7) (2-hydroxycyclopropyl-a-D-galactopyranoside), a fungal metabolite directly involved in the pathogenesis of the eye-spot disease (8).Fortuitously, we observed that when toxin-producing isolates of H. sacchari were grown on a synthetic medium and transferred a number of times through the same medium, they lost their toxin-producing ability: they became attenuated. When an attenuated isolate was transferred to a medium containing an infusion of sugarcane leaves (susceptible clone 51 NG 97), full toxin production was restored. These observations suggested that a compound present in sugarcane is required for toxin biosynthesis.This report describes the isolation, purification, and chemical characterization of one of several "...

A somatic cell mutant (CR1) of the Chinese hamster ovary cell line (CHO-KI) that is defective in the regulation of cholesterol biosynthesis can be grown under conditions in which plasma membranes from these cells display various cholesterol contents and acyl chain order parameters. The (Na++K+)stimulated adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from these cells was shown to vary in activity by a factor of 10 as the order parameter was varie and the activity exhibited an exponential dependence on this parameter. Under these conditions the number of Na+,K+-ATPase molecules was shown to remain constant by affinity labeling with [.y-32PJATP in the absence of Na+. Control experiments showed that alteration in cholesterol content without change in order arameter did not result in altered enzyme activity. It is concuded that, under our conditions, the rate of catalysis by the Na+,K+-ATPase is determined by the order parameter. These studies suggest a physical mechanism by which variation of membrane lipid composition or other factors that determine membrane lipid acyl chain order parameter can result in variation in membrane enzyme activity. Recently, Sinensky has described (1, 2) a somatic cell mutant (CR1) of the Chinese hamster ovary cell (CHO-K1) that is defective in the regulation of cholesterol biosynthesis, plasma membrane cholesterol levels, and plasma membrane acyl chain order parameter. This defect in the ability to regulate order parameter should permit ready analysis of the effects of acyl chain ordering on plasma membrane functions in whole cells or membrane preparations. In order to assess the potential of this system we have chosen to examine the dependence of the activity of (Na++K+)-stimulated adenosinetriphosphatase (Na+,K+-ATPase; ATP pbosphohydrolase, EC 3.6.1.3) on acyl chain mobility.This enzyme was chosen because studies by Kimelberg and Papahadjopoulos (3) have indicated that such a dependence on lipid acyl chain ordering was likely for this enzyme and because affinity labeling techniques exist (4) that permit the determination of the number of enzyme molecules present in a membrane preparation. In this report we show that the CR1 Na+,K+-ATPase is indeed affected by membrane fluidity in a fashion comparable to the reconstituted system studied by Kimelberg and Papahadjopoulos (3). We further show that the catalytic rate constant for this system is determined by the plasma membrane acyl chain order parameter. These studies demonstrate the utility of the CR1 system in studying the effects of membrane fluidity on cell surface functions. The advantages of the CR1 system over other approaches to varying membrane fluidity are discussed.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertzsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.EXPERIMENTAL PROCEDURE Medium and Cells. Cells were grown on medium F12 (5) supplemented with 8% fetal calf serum (F12FC8) or 8% fetal c...

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7-and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag ؉ -HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C 27 sterols observed in the SLOS blood samples were cholesterol (12-98%), 7-dehydrocholesterol (0.4-44%), 8-dehydrocholesterol (0.5-22%), and cholesta-5,7,9(11)-trien-3 ␤ -ol (0.02-5%), whereas the normal blood samples contained Ͻ 0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05-0.6%) and cholestanol (0.1-0.4%) and ϳ 0.003-0.1% each of the ⌬ 8 , ⌬ 8(14) , ⌬ 5,8(14) , ⌬ 5,24 , ⌬ 6,8 , ⌬ 6,8(14) , and ⌬ 7,24 sterols. The results are consistent with the hypothesis that the ⌬ 8(14) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the ⌬ 5,7,9(11) sterol. 19-Norcholesta-5,7,9-trien-3 ␤ -ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.-Ruan, B.

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