Search PubMed⌕ Search

Biomedical subjects

L J Goad

Publications and source records attributed to L J Goad.

At least 37 records · Page 2Linked to original sources
PubMed1988

Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes.

Promastigotes of 36 World Health Organization reference (and other) strains of 6 species and 10 subspecies of Leishmania were cultured in the presence of 3 antimycotic azole drugs (ketoconazole, itraconazole, fluconazole) and their population growth determined. A representative of each subspecies was also analyzed for its sterol composition. For all strains the order of azole drug activity with respect to both growth and sterol biosynthesis inhibition was itraconazole greater than or equal to ketoconazole greater than fluconazole. The inhibitory actions of the three azole drugs were greater on L. donovani and L. braziliensis subspecies and on L. mexicana amazonensis than on L. aethiopica, L. major, L. tropica and L. mexicana mexicana. The nature of the changes in sterol composition caused by the drugs was the same for all strains. The normal, major endogenous sterols of the promastigotes (5-dehydroepisterol and ergosterol) were reduced in amount to 1-2% of the total free sterols and were replaced by endogenous 14 alpha-methyl sterols and exogenous cholesterol. The changes occurred rapidly, were drug concentration dependent and coincided with growth inhibition. Six strains of those Leishmania species less sensitive to the azole drugs could be subcultured indefinitely at reduced growth rates in the presence of a ketoconazole concentration causing the same extraordinary alterations in sterol composition. This suggested that the bulk membrane functions of sterols in leishmanias can be served by 14 alpha-methyl sterols and cholesterol, albeit imperfectly, while traces of 14 alpha-desmethyl sterols are needed for uncharacterized metabolic functions.

Animals↗
PubMed1987

Paclobutrazol inhibition of sterol biosynthesis in a cell suspension culture and evidence of an essential role for 24-ethylsterol in plant cell division.

Growth of a celery (Apium gravidens) cell suspension culture was inhibited by the synthetic plant growth regulator paclobutrazol. Paclobutrazol caused a reduction in the incorporation of [2-14C]acetate into the 4-demethyl sterols (campesterol, sitosterol, stigmasterol) but radioactivity accumulated in the 4 alpha-methylsterols. The accumulating 4 alpha-methylsterols were identified as obtusifoliol and cycloeucalenol indicating that paclobutrazol was inhibiting sterol biosynthesis by blocking 14 alpha-demethylation. The inhibition of celery cell growth by paclobutrazol could be partially overcome by addition of cholesterol to the culture medium. However, addition of stigmasterol restored growth to the control value suggesting an essential role for a 24-ethylsterol to support plant cell division.

Acetates↗
PubMed1987

Strategy for the analysis of steryl esters from plant and animal tissues.

Methods are described for the analysis of intact steryl esters present in complex mixtures isolated from plant or animal tissues. A preliminary examination by analytical thin-layer chromatography (TLC) and capillary column gas chromatography-mass spectrometry (GC-MS) under electron impact (EI) ionisation reveals the complexity of the mixture and the nature of the steryl moieties. Preparative TLC is then utilised to separate the steryl esters into two broad groups, containing fatty acyl moieties of shorter (C2-C8) or longer chain length (C10-C22). The shorter-chain fatty acyl steryl esters are separated by adsorption high-performance liquid chromatography (HPLC) on a LiChrosorb Silica-60 column. The steryl esters with longer-chain fatty acyl moieties are analysed by reversed-phase HPLC on either an Ultrasphere ODS, 5-micron, or a S3 Spherisorb ODS, 3-micron, column. Steryl esters with unsaturated fatty acyl moieties are eluted with the shorter-chain fatty acyl steryl esters. The presence of the unsaturated fatty acyl esters can be monitored by analytical argentation TLC, which will also reveal the degree of unsaturation. The steryl esters are fractionated into the saturated, mono-, di-, tri- and polyene acyl types by preparative medium-pressure liquid chromatography on a column of 10% AgNO3-silica gel. Each of these steryl ester types can then be resubmitted to reversed-phase HPLC or analysed by GC-MS on a short fused-silica capillary column with a bonded phase of the OV-1 type. GC-MS on a magnetic-sector instrument under negative-ion chemical ionisation conditions with ammonia as the reagent gas produces fragment ions for both the steryl and fatty acyl moieties, thus permitting identification of the individual intact steryl esters. These various methods are illustrated by analyses of the steryl ester mixtures obtained from human plasma, barley seedlings, palm oil and rape seed oil.

Animals↗
PubMed1987

Capillary gas chromatography/mass spectrometry of cholesteryl esters with negative ammonia chemical ionization.

Mixtures of both synthetic and naturally occurring (human plasma cholesteryl esters have been examined by capillary gas chromatography/mass spectrometry (GC/MS). A magnetic sector mass spectrometer was used and a variety of ionization modes were assessed with a view to obtaining structural information on intact cholesteryl esters. By employing ammonia as reagent gas, with negative ion scanning, spectra were produced from which the nature of steryl and fatty acyl moieties could be readily deduced. Analyses were performed at an ion source temperature of 300 degrees C in order to maintain the integrity of the gas chromatographic profile. The technique described is of general use for the GC/MS analysis of steryl esters, particularly in conjunction with magnetic sector instruments.

Ammonia↗
PubMed1986

Effects of ketoconazole on sterol biosynthesis by Trypanosoma cruzi epimastigotes.

Reproduction of Trypanosoma cruzi epimastigotes in a liver infusion-tryptose-serum medium was inhibited by the antimycotic agent ketoconazole. Effective drug concentrations also blocked the biosynthesis of the parasite's fungal-type sterols at the stage of C-14 demethylation, as demonstrated by radioisotopic, chromatographic and mass spectrometric methods. Coincidently, a 4,4,14 alpha-trimethyl sterol, 24-methylenedihydrolanosterol, accumulated and came to replace as much as three fourths of the free sterol content of the cells. Precedents from studies with fungi are invoked to suggest that the cytotoxicity of ketoconazole for T. cruzi is a consequence of the inability of 24-methylenedihydrolanosterol to perform the membrane lipid bilayer functions of the normal epimastigote sterols.

Animals↗
PubMed1986

Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells.

Murine macrophage tumor cells infected with Leishmania mexicana mexicana were exposed to the antimycotic drug ketoconazole and to [2-14C]mevalonate, then the amastigotes were isolated, collected, purified, and their free sterols were analyzed by chromatographic and mass spectrometric methods. Control amastigotes contained as products of de novo biosynthesis C28 4-desmethyl sterols (episterol, 5-dehydroepisterol), C29 4-desmethyl sterols (stigmasta-7,24 (28)-dien-3 beta-ol, stigmasta-5,7,24(28)-trien-3 beta-ol), 4-methyl sterols (4 alpha, 14 alpha-dimethylzymosterol, obtusifoliol) and a 4,4-dimethyl sterol (lanosterol). Present also were macrophage sterols (cholesterol, desmosterol) and a putative product of the C-24 alkylation of desmosterol by amastigotes (24-methylenecholesterol). Amastigotes from macrophages exposed to ketoconazole showed notable changes in the proportions, concentrations and specific activities of their free sterols; increased for 4 alpha, 14 alpha-dimethylzymosterol and decreased for the endogenous C28 and C29 4-desmethyl sterols. Such changes were observed at a ketoconazole concentration as low as 0.01 microgram ml-1. By contrast, uninfected macrophages accumulated only small amounts of lanosterol of high specific activity at a ketoconazole concentration of 10 micrograms ml-1. the ketoconazole-induced alterations in amastigote sterols parallel those previously reported in fungi and L. m. mexicana promastigotes, and suggest a biochemical mechanism for the anti-leishmanial activity of the drug in which changes in sterol composition are linked to disturbances of cell membrane structure and function, and hence to cytotoxicity.

Animals↗
PubMed1985

Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes.

Leishmania mexicana mexicana promastigotes grown with cholesterol, supplied in natural products as the free sterol and as cholesteryl esters, were exposed to [2-14C]mevalonate and to the antimycotic drug ketoconazole. Growth was inhibited and cholesterol and 14 alpha-methyl sterols accumulated in free and esterified forms (cholesterol much greater than 4 alpha,14 alpha-dimethylcholesta-8,24-dien-3 beta-ol much greater than 14 alpha-methylcholesta-8,24-dien-3 beta-ol congruent to 14 alpha-methylergosta-8,24(28)-dien-3 beta-ol much greater than 4 alpha,14 alpha-dimethylergosta-8,24(28)-dien-3 beta-ol; identified by capillary gas chromatography/mass spectrometry, and by 1H and 13C nuclear magnetic resonance spectrometry). The 14 alpha-methyl sterols were preferentially labelled with 14C. The cholesterol was unlabelled and substituted for a substantial fraction of the major product of sterol biosynthesis, ergosta-5,7, 24(28)-trien-3 beta-ol (5-dehydroepisterol), but did not replace it and did not offer remarkable protection against either growth inhibition or alteration of sterol biosynthesis. Promastigotes grown with [6-2H]cholesterol or [4-14C]cholesterol did not contain labelled forms of Leishmania sterols, or other sterols. The chromatographic and spectrometric sterol analyses and the isotopic tracer findings suggested that ketoconazole impaired the cytochrome P-450 dependent 14 alpha-demethylation of lanosterol, that cholesterol was neither biosynthesized nor metabolized, and that the physiological functions of 5-dehydroepisterol had sterol structural requirements not entirely met by cholesterol. In all these studies, L. mexicana mexicana demonstrated a sterol biochemistry remarkably similar to that of fungi. This recommends an increase in interest in antimycotic drugs as chemotherapeutic agents for leishmanial infections.

Animals↗
PubMed1984

Sterols of Leishmania species. Implications for biosynthesis.

The major sterol of promastigotes of stocks of Leishmania tropica, L. donovani and 3 subspecies of L. mexicana has been identified as ergosta-5,7,24(28)-trien-3 beta-ol; and of an L. major stock as ergosta-7,24(28)-dien-3 beta-ol. 24-Methylcholesta-5,7,22-trien-3 beta-ol and 24-ethylcholesta-5,7,22-trien-3 beta-ol were minor constituents, and traces of ergosta-5,7,22,24(28)-tetraen-3 beta-ol and a C27-diene were also recognized in some species. Lanosterol and 4,4-dimethylcholesta-8,24-dien-3 beta-ol were detected in all species studied, and squalene was identified in a stock of L. tropica. The sterol composition of members of the genus Leishmania and the sterol biosynthetic pathways it implies are characteristic of yeast and other fungi.

Animals↗
PubMed1980

The effect of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor ML-236b on phytosterol synthesis in Acer pseudoplatanus tissue culture.

Sycamore cell cultures were incubated with various labelled sterol precursors. ML-236B, a fungal metabolite, caused virtually total inhibition of acetate or leucine incorporation into sterols, while mevalonate incorporation was unaffected. Sterol synthesis from endogenous precursors, measured by incorporation of [Me-14C] methionine into the side chain, continued at a reduced rate for at least 6 h after addition of the inhibitor.

Carbon Radioisotopes↗
PubMed1975

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.

Animals↗
PubMed1975

The conversion of cholest-5-en-3beta-ol into cholest-7-en-3beta-ol by the echinoderms Asterias rubens and Solaster papposus.

1. The echinoderms Asterias rubens and Solaster papposus (Class Asteroidea) metabolize injected [4(-14)C]cholest-5-en-3beta-ol to produce labelled 5alpha-cholestan-3beta-ol and 5alpha-cholest-7-en-3beta-ol. 2. Conversion of 5alpha-[4(-14)C]cholestan-3beta-ol into 5alpha-cholest-7-en-3beta-ol was demonstrated in A. Rubens. 3. Incubations of A. rubens with [4(-14)C]cholest-4-en-3-one resulted in the production of labelled 5alpha-cholestan-3-one, 5alpha-cholestan-3beta-ol and 5alpha-cholest-7-en-3beta-ol. 4. [4(-14)C]Sitosterol was metabolized by A. rubens to give 5alpha-stigmastan-3beta-ol and 5alpha-stigmast-7-en-3beta-ol. 5. The significance of these results in relation to the presence of alpha7 sterols in starfish is discussed.

Animals↗
PubMed1974

Progesterone metabolism by the echinoderms Asterias rubens and Marthasterias glacialis.

The echinoderms Asterias rubens and Marthasterias glacialis metabolize injected [4-(14)C]progesterone to give labelled 3beta-hydroxy-5alpha-pregnan-20-one and 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. These radioactive products are converted by the animals into conjugated forms that are soluble in aqueous methanol, and which have mobilities on t.l.c. similar to the asterosaponins.

Animals↗
PubMed1974

Sterol biosynthesis by the sea urchin Echinus esculentus.

1. The 4-demethyl sterols of Echinus esculentus consisted of cholesterol as the major component, with lower concentrations of nine other C(26), C(27), C(28) and C(29) Delta(5) sterols. 2. [2-(14)C]Mevalonic acid was readily incorporated by the urchin into squalene, lanosterol and desmosterol but only to a small extent into cholesterol. 3. [26-(14)C]Desmosterol did not appear to be reduced to give cholesterol, but conversion of 5alpha-[2-(3)H(2)]lanost-8-en-3beta-ol into cholesterol was observed. 4. No C-24 dealkylation of [4-(14)C]sitosterol or metabolism of [4-(14)C]cholesterol could be detected.

Animals↗
PubMed1973

The sterols of the echinoderm Asterias rubens.

1. Twenty-two sterols were identified in the starfish Asterias rubens (Phylum, Echinodermata; Class, Asteroidea). 2. The major 4-demethyl sterols had a Delta(7) bond and the C(27) compound 5alpha-cholest-7-en-3beta-ol predominated over other mono- and di-unsaturated sterols belonging to the C(26), C(27), C(28) and C(29) series. 3. Small amounts of cholest-5-en-3beta-ol and 5alpha-cholestan-3beta-ol were also present. 4. The minor sterols identified all contained either one or two methyl groups at C-4 and are considered to be potential biosynthetic precursors of 5alpha-cholest-7-en-3beta-ol. 5. Three sterols possessing a 9beta,19-cyclopropane ring were also isolated and were probably derived by the starfish from a dietary source.

Animals↗