Immunochemical studies of a purified antigen from Micropolyspora faeni.

A pure antigen fraction was isolated from the crude culture filtrate of Micropolyspora faeni by gel filtration and affinity chromatography. The isolated antigen has a mol. wt of approximately 16,000 and an isoelectric point of pH 3.8. The major amino acid content of this fraction includes glycine, glutamic acid, aspartic acid and alanine. This antigen fraction reacted with the sera of all 15 farmer's lung patients and 20 asymptomatic farmers with circulating anti-M. faeni antibodies. An ELISA method was developed using the purified antigen to detect specific circulating antibodies against M. faeni in farmer's lung patients.

Preparation, properties and metabolism of 5,6-monoepoxyretinoic acid.

1. Methyl retinoate has been converted into methyl 5,6-monoepoxyretinoate by reaction with monoperphthalic acid. The epoxy acid ester on alkaline hydrolysis gave 5,6-monoepoxyretinoic acid. 2. Treatment of the 5,6-monoepoxy compounds with ethanolic hydrochloric acid gave the corresponding 5,8-epoxy (furanoid) compounds. 3. With lithium aluminium hydride, the acid and the ester groups were selectively reduced to primary alcohols. 4. Administration of methyl 5,6-monoepoxyretinoate intraperitoneally and subcutaneously had good growth response in vitamin A-deficient rats. 5. 5,6-Monoepoxyretinoic acid, when given intraperitoneally as the sodium salt, was 157% as active as all-trans-retinyl acetate. 6. Methyl 5,6-monoepoxyretinoate was hydrolysed to the epoxy acid by rat-liver homogenate. It had 35% of the biological activity of all-trans-retinyl acetate in the rat when given orally.

Metabolism and biological potency of retro-retinyl acetate in the rat.

1. retro-Retinyl acetate was shown to exert its biological activity by conversion into vitamin A. 2. When administered orally, retro-retinyl acetate was hydrolysed to retro-retinol in the intestine, isomerized to retinol and esterified before being transported to the liver for storage. 3. Administration of the compound at as high a dose as 4.0mg./day for 4 days led to the accumulation of both vitamin A and retro-vitamin A in the liver. The amount of retro-vitamin A in liver gradually decreased until it was almost completely converted into vitamin A in 18 days. 4. Intraperitoneal administration of the compound led to the accumulation of both vitamin A and retro-vitamin A in liver and other tissues. No vitamin A was detected in any tissue of rats receiving retro-retinyl acetate intraperitoneally after enterectomy. 5. The small intestine is the major site of conversion of retro-vitamin A into vitamin A. The conversion could also be demonstrated by everted intestinal sacs. 6. The biological potency of retro-retinyl acetate determined by the rat-growth assay was 20.5% that of all-trans-retinyl acetate, when given orally.

Separation and allosteric properties of two forms of UDP-glucuronate carboxy-lyase.

DEAE-cellulose chromatography of partially purified preparations of UDP-glucuronate carboxy-lyase from wheat germ results in the separation of two forms of the enzyme. Both are fully active in the absence of added DPN, have indistinguishable molecular weights (210,000), but differ in charge and kinetic properties. Both are cooperatively activated by UDP-glucuronate, however Enzyme 1 is activated at lower concentrations than Enzyme 2. At low substrate concentrations (less than or equal to 5 micron), both enzymes are activated by UDP-glucose, 2 mM concentrations of activator increasing the activity of Enzyme 1 2-fold and of Enzyme 2 2.5-fold. UDP-xylose allosterically inhibits both enzymes. At substrate concentrations equal to the apparent Km values, inhibition of Enzyme 1 is much greater than that of Enzyme 2 (83 and 28% at 0.33 mM inhibitor concentration). The data suggest that synthesis of UDP-xylose is controlled both by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. The existence of a "more active" and a "less active" species of the enzyme suggests the possibility of two interconvertible forms of the same protein and the involvement of such interconversion in further regulation of UDP-xylose biosynthesis. However it is equally possible that both represent true isoenzymes.

UDP-glucuronate carboxy-lyase in cultured chondrocytes.

UDP-glucuronate carboxy-lyase has been demonstrated in chick chondrocytes in tissue culture. It occurs in the particulate fraction, and its activity is stimulated by exogenous NAD. The enzyme is allosterically activated by UDP-glucuronate and inhibited by UDP-xylose, n Values of 2.8 indicate positive cooperativity of at least three interacting sites on the enzyme. These data suggest that UDP-xylose concentration in chondrocytes is regulated by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. Activity levels of the enzyme during growth of the cells peak towards mid-log phase and decline thereafter, closely paralleling levels of chondroitin sulfate glycosyltransferases determined previously (Schwartz, N. B. (1976) J. Biol. Chem. 251, 3346-3351). Thus, it appears that during chondrocyte development a common mechanism governs induction of glycosyltransferases and of UDP-glucuronate carboxy-lyase.

Absorption, storage and distribution of 3-dehydrovitamin A in the rat.

1. The metabolism of 3-dehydroretinal was found to be similar to that of retinal. It alleviated all the symptoms of vitamin A deficiency, and promoted the growth of vitamin A-deficient rats. 2. When administered orally, 3-dehydroretinal was reduced in the intestine of the rat and subsequently esterified and transported to the liver, where it was stored mainly as the higher fatty acid ester. 3. Intraperitoneal administration of the compound led to the accumulation of 3-dehydrovitamin A in liver and other tissues. Subcutaneous administration of the compound showed a good growth response in the rat. 4. The ratio of 3-dehydroretinyl higher fatty acid ester to 3-dehydroretinol in liver, in the post-absorptive state, was nearly 93:7. 5. There was a linear relationship between the 3-dehydroretinol concentrations of blood and liver of rats. 6. Administration of 3-dehydroretinal at a dosage of 7.5mg./day for 3 days brought about hypervitaminosis A in the rat. 7. The maximal retention of 3-dehydrovitamin A by the kidneys was at an optimum dosage of 4.5mg./day for 3 days.

Preparation, properties and metabolism of retro-3-dehydroretinyl acetate.

1. Treatment of 3-dehydroretinyl acetate with aqueous hydrobromic acid resulted in the formation of retro-3-dehydroretinyl acetate, which, on alkaline hydrolysis, gave the corresponding alcohol. 2. retro-3-Dehydroretinyl acetate was isomerized to 3-dehydrovitamin A when fed to vitamin A-deficient rats. 3. When retro-3-dehydroretinyl acetate was administered orally, it was hydrolysed to retro-3-dehydroretinol in the rat intestine, isomerized to 3-dehydroretinol and esterified before being transported to the liver for storage. 4. When administered intraperitoneally, both 3-dehydrovitamin A and retro-3-dehydrovitamin A were accumulated in liver and other tissues, whereas after enterectomy 3-dehydrovitamin A was not detected anywhere in the body. 5. The small intestine was shown to be the major site of conversion of retro-3-dehydrovitamin A into 3-dehydrovitamin A. 6. The extent of conversion of retro-3-dehydroretinyl acetate into 3-dehydrovitamin A was much smaller than that of the conversion of retro-retinyl acetate into vitamin A. 7. The biological potency of retro-3-dehydroretinyl acetate, determined by the rat-growth assay, was 2.6% of that all-trans-retinyl acetate, when given orally.

A partially purified glycoprotein antigen from Aspergillus fumigatus.

Culture filtrate antigens of Aspergillus fumigatus were fractionated by isoelectric focusing using a pH gradient of 4-6.5. Three fractions, namely, 18, 19 and 20 were pooled and subjected to immunochemical analysis. It contained a concanavalin A binding glycoprotein. Antibody raised against this component was used to prepare an affinity column of IgG-Sepharose and was used to purify crude culture filtrate antigens. This component produced three precipitin arcs in crossed immunoelectrophoresis using anti-A. fumigatus rabbit serum. This fraction has an isoelectric point of 6.5 and showed three components in two-dimensional electrophoresis with approximate molecular weights of 20, 40 and 80 kilo daltons. This antigen reacted with patient sera in the biotin-avidin linked immunosorbent assay and showed high levels of anti-A. fumigatus IgG and IgE antibodies in allergic bronchopulmonary aspergillosis and IgG antibodies in aspergilloma. Both controls and Aspergillus skin test positive asthmatics showed only low levels of specific antibodies. Because of the purity of this antigen, its potential use as a standardized antigen in the detection of antibody is discussed.

Preparation, properties and metabolism of 5,6-monoepoxy-3-dehydroretinal.

1. 5,6-Monoepoxy-3-dehydroretinal was synthesized from 3-dehydroretinyl acetate and characterized. 2. When fed to vitamin A-deficient rats, 5,6-monoepoxy-3-dehydroretinal was converted into 5,6-monoepoxy-3-dehydrovitamin A and stored in the liver. 3. It was demonstrated that the rat possesses the necessary enzymes for the reduction and oxidation of 5,6-monoepoxy-3-dehydroretinal to the corresponding alcohol and acid respectively. 4. The biological potency of the epoxy-3-dehydroretinal by the rat-growth assay (determined by USP XIV procedure) was 1.07% of that of vitamin A.