Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.
Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Δ9)-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Δ11 fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Δ11 was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (α-elongation). In wild-type cells, the C16:1Δ11 elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Δ mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Δ mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular...
Sokatch, J. R. (University of Oklahoma School of Medicine, Oklahoma City). Alanine and aspartate formation during growth on valine-C14 by Pseudomonas aeruginosa. J. Bacteriol. 92:72–75. 1966.—Pseudomonas aeruginosa grown with dl-valine-4,4′-C14 synthesized alanine labeled mainly in carbons 1 and 3, indicating that the isopropyl carbons of valine were the precursors of pyruvate for alanine formation by a pathway which did not involve randomization of isotope. Alanine from cells grown on valine-1-C14 contained isotope only in the carboxyl carbon, suggesting another route to pyruvate from valine by carbon dioxide fixation. Oxidation of valine to propionyl-coenzyme A (CoA), as it occurs in animal tissues, followed by the oxidation of propionyl-CoA to acrylyl-CoA, lactyl-CoA, and pyruvate, would account for the isotope data. Cells grown on valine oxidized valine, isobutyrate, and propionate immediately, whereas cells grown on acetate did not oxidize valine or isobutyrate and required an induction period before propionate was oxidized. P. aeruginosa grown with propionate-1-C14 or propionate-2-C14 formed alanine-1-C14 and alanine-2-C14, respectively, which agrees with the contention that at least part of the propionate is oxidized via the acrylate pathway. Aspartate formed from valine-1-C14 was labeled only in the carboxyl carbons...
Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C14. The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C14 and methyl palmitate-1-C14. This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.
Finnerty, W. R. (University of Iowa, Iowa City), and R. E. Kallio. Origin of palmitic acid carbon in palmitates formed from hexadecane-1-C14 and tetradecane-1-C14 by Micrococcus cerificans. J. Bacteriol. 87:1261–1265. 1964.—Degradation of the palmitic acid moiety of cetyl palmitate and myristyl palmitate formed from hexadecane-1-C14 and tetradecane-1-C14 by Micrococcus cerificans was carried out. The patterns of C14 labeling in palmitic acid from cetyl palmitate showed that hexadecane is oxidized at the C1 position, and cetyl alcohol and palmitic acid thus formed are directly esterified. Palmitic acid arising from tetradecane and esterified to tetradecanol appeared to have been synthesized by the addition of two carbon atoms to an existing 14-carbon atom skeleton. Considerable mixing of C14 occurred in the C1 and C2 positions of palmitic acid thus synthesized.
Stjernholm, Rune L. (Western Reserve University, Cleveland, Ohio) and Frank Flanders. Metabolism of d-ribose-1-C14 and C14-labeled d-gluconate in an enzyme system of the genus Propionibacterium. J. Bacteriol. 84:563–568. 1962.—Ribose-1-C14 and potassium gluconate labeled in different positions were incubated with cell-free extracts of Propionibacterium shermanii. The resulting propionate, acetate, and succinate were isolated and the C14 distribution determined by degradation. It is proposed that the extensive randomization observed is caused by the conversion of the labeled substrates to fructose-6-phosphate via the transketolase-transaldolase sequence followed by the Embden-Meyerhof pathway, and that the triosephosphates produced by these metabolic routes are metabolized via pyruvate to succinate and propionate.
Baldwin, R. L. (Michigan State University, East Lansing), W. A. Wood, and R. S. Emery. Conversion of glucose-C14 to propionate by the rumen microbiota. J. Bacteriol 85:1346–1349. 1963.—Rumen microbiota enriched on three different diets calculated to present different levels of available carbohydrate were incubated with glucose-1-C14, glucose-2-C14, and glucose-6-C14 to determine the contribution of the randomizing (succinate) and nonrandomizing (acrylate) routes to propionate. The propionate was labeled as though 70 to 100% was formed via the randomizing route and 0 to 30% via the nonrandomizing route. The contribution of the acrylate pathway increased with higher carbohydrate availability of the diet. These results are discussed with respect to earlier data using lactate-2-C14 and lactate-3-C14, and a unifying concept for both sets of data is presented.
Baldwin, R. L. (Michigan State University, East Lansing), W. A. Wood, and R. S. Emery. Conversion of lactate-C14 to propionate by the rumen microflora. J. Bacteriol. 83:907–913. 1962.—Rumen microflora enriched on five different diets calculated to present increasing carbohydrate or lactate availability were used to determine the contribution of the randomizing (succinate) and nonrandomizing (acrylate) routes to propionate with lactate-2-C14 and -3-C14 as substrates. Propionate was labeled as though 70 to 90% was formed via the nonrandomizing route. This percentage was highest on diets containing high levels of carbohydrate or lactate or both. Evidence for the presence of succinic dehydrogenase, acetokinase, phosphotransacetylase, and coenzyme A transphorase was obtained with cell-free extracts. Propionate-2-C14 and lactate-2-C14 were converted by extracts to the activated derivatives of acrylate, lactate, propionate, and acetate.
Kanie, Matuso (Kagoshima University, Kagoshima, Japan), Shigeo Fujimoto, and J. W. Foster. Chemical degradation of dipicolinic acid-C14 and its application to biosynthesis by Penicillium citreo-viride. J. Bacteriol. 91:570–577. 1966.—A chemical degradation of dipicolinic acid-C14 has been worked out, enabling determination of the specific radioactivity of the carboxyl-carbons (carbons-7 and -8), and of the following carbons of the pyridine ring: carbons-2 and -6 combined, carbons-3 and -5 combined, and carbon-4. The degradation was applied to dipicolinic acid synthesized by washed, submerged mycelium of the mold from glucose and C14O2, and from glucose-1-C14, -2-C14, and -6-C14. The distribution of radioactivity within the labeled dipicolinic acids is consistent with operation of respiratory cycles and with the incorporation of one molecule of CO2 in the pyridine acid. A C3 compound is a primary building block. The C7 chain is believed to result from a C3 plus C4 condensation, pyruvic acid and aspartic acid β-semialdehyde being proposed as likely precursors. Other aspects of the biosynthesis of C7 open-chain compounds and of dipicolinic acid are discussed.
Hauschild, Andreas H. W. (University of Toronto, Toronto, Ontario, Canada). Incorporation of C14 from amino acids and peptides into protein by Clostridium perfringens type D. J. Bacteriol. 90:1569–1574. 1965.—Uptake of C14 from C14-labeled amino acids and peptides by Clostridium perfringens was measured in culture media containing acid or papain hydrolysates of C14-labeled Chlorella protein. Between 2 and 4 hr of growth, the rate of C14 uptake from peptides was higher than from free amino acids. Peptides extracted from cells with hot ethyl alcohol contained six to nine times more C14 after 4 hr of growth with C14-labeled peptides than with C14-labeled amino acids. Incorporation of C14-labeled glycine, serine, threonine, alanine, and proline into both cellular and exocellular protein was two to five times higher when these were supplied as components of dialyzable peptides rather than as free amino acids.
Merdinger, Emanuel (Roosevelt University, Chicago, Ill.), and Rosalind H. Frye. Distribution of C14 from glucose-1-C14 in the lipid fractions of Debaryomyces hansenii. J. Bacteriol. 91:1831–1833. 1966.—Debaryomyces hansenii cells were grown in a medium containing yeast extract, malt extract, glucose, sodium chloride, and nutrient salts, to which glucose-1-C14 was added. The lipids extracted from the cells were fractionated by use of a single column packed with silicic acid. Of the total C14 added to the culture medium, the neutral lipid fractions contained 21.06% while the phospholipid portions contained only 0.89%. The highest amount of C14 among the neutral lipids was found in the fraction containing the hydrocarbons (11.64%). Among the phospholipids, the highest amount (0.66%) was found in phosphatidyl serine and phosphatidyl ethanolamine.
Resonance Raman spectroscopy is used to examine the possibility that C14-C15 single bond isomerizations of the retinal prosthetic group are involved in the photochemical reactions of bacteriorhodopsin. Normal mode calculations show that the vibration that contains predominantly C14-C15 stretch character is approximately equal to 70 cm-1 lower in frequency in the 14-s-cis conformer than in the s-trans case. This geometric effect is insensitive to out-of-plane twists and should be observed in the sterically hindered 13-cis, 14-s-cis retinal protonated Schiff base, which has been proposed as the chromophore in the K and L intermediates of bacteriorhodopsin. Resonance Raman spectra were obtained of K625 by using the low temperature (77 K) spinning-cell technique. Isotopic substitutions with 13C and 2H show that significant C14-C15 stretch character is observed in normal modes at approximately equal to 1185-1195 cm-1. The relatively high frequency of the C14-C15 stretch argues that K625 contains a 13-cis, 14-s-trans chromophore. Similarly, isotopic derivatives show that L550 has a localized C14-C15 stretch at 1172 cm-1, consistent with a 14-s-trans chromophore. These results argue that the primary step in bacteriorhodopsin is a C13=C14 trans----cis photoisomerization that does not involve C14-C15 s-cis structures.
The fermentation of uniformly labeled glucose-C14, glucose-1-C14, -2-C14, and -6-C14, xylose-1-C14, cellulose-1-C14, -2-C14, and -6-C14, and lactate-2-C14 by rumen fluids from cows fed all-hay, hay and concentrate (50:50), and all-concentrate diets was investigated. The results obtained suggested that the Embden-Meyerhof glycolytic pathway is the major pathway of hexose utilization, that the major pathway of xylose fermentation involves hexose synthesis, and that the contributions of the nonrandomizing (acrylate) pathway of propionate formation during glucose, xylose, and cellulose fermentations are 4.5, 8.0, and 10.5%, and 24.6, 25.8, and 17.2%, respectively, by rumen fluids from the cows fed all-hay and all-concentrate rations.
The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0–20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 Å height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations...
The non-hepatic tissues in a perfused "carcass" (caudal half of the rat) maintain some physiological functions for as long as 5 to 6 hours of perfusion, including good clearance of lysine-ε-C14 and glucose from the perfusate, and synthesis of both tissue and plasma proteins. The perfused "carcass" tissues incorporate only small amounts of lysine-ε-C14 into the plasma proteins to an extent not markedly affected by the presence of the gastrointestinal tract, pancreas, spleen, or kidneys. This activity is found only in the globulin fraction obtained by sodium sulfate fractionation. No significant activity was detected in the plasma fibrinogen or albumin fractions. C14-labeled plasma proteins obtained from the eviscerated surviving rat 6 hours after intravenous lysine-ε-C14 have been separated by zone electrophoresis. The gamma globulins contain most of the C14 activity, with small but measurable activity in the beta and alpha globulins, and no activity in the albumin fraction.
The distribution of C14 was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C14 in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C14 in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C14, glucose-1-C14, and glucuronolactone-U-C14, was administered. The highest values were obtained after the administration of glucose-6-C14; glucuronolactone-U-C14 gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C14 assays and autoradiograms indicate that C14 is also present in other parts of the comb. As seen in autoradiography, the concentration of C14 was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies...
Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea...
Conformational changes of the retinal chromophore about the C14-C15 bond in bacteriorhodopsin (BR) have been proposed in models for the mechanism of light-driven proton transport. To determine the C14-C15 conformation in BR's L550 intermediate, we have examined the resonance Raman spectra of BR derivatives regenerated with retinal deuterated at the 14 and 15 positions. Vibrational calculations show that the C14-2H and C15-2H rocking modes form symmetric (A) and antisymmetric (B) combinations in [14,15-2H]retinal chromophores. When there is a trans conformation about the single bond between C14 and C15 (14-s-trans), a small frequency separation or splitting is observed between the A and B modes, which are found at approximately equal to 970 cm-1. In 14-s-cis molecules, the splitting is large, and the Raman-active symmetric A mode is predicted at approximately equal to 850 cm-1. In addition, the monodeuterium rock should appear at an unusually low frequency (920-930 cm-1) in the 14-2H-labeled 14-s-cis molecules. These patterns are insensitive to computational details: similar results are predicted by a modified Urey-Bradley force field and by MNDO (modified neglect of differential overlap) calculations for twisted chromophores and for highly delocalized protonated Schiff base cations. Time-resolved resonance Raman spectra were obtained of BR's L550 intermediate regenerated with [14-2H]-...
The metabolism of homologous plasma proteins, labeled with lysine-ε-C14, after oral administration to dogs has been investigated. The speed of the various processes involved is indicated by the maximum rate of C14 O2 excretion which is attained within 1 to 4 hours, the prompt appearance of protein activity in the plasma and disappearance of non-protein activity from it, both virtually complete in 7 to 10 hours, as well as the rapid incorporation of a large percentage of the fed-C14 into tissues. There are no essential differences between the behavior of labeled plasma and that of an amino acid digest containing ε-C14 labeled lysine when these two materials are given orally. At the end of 48 hours after labeled plasma feeding, a CO2 elimination of 16 to 28 per cent of the fed C14 is noted. In contrast, after 48 hours following labeled plasma by vein, a CO2 elimination of only 2.5 per cent is recorded—almost a 10 to 1 ratio. We believe this, together with the data concerning plasma and tissue protein activity, represents a significant difference in the metabolic process. The evidence favors a complete breakdown of plasma protein to the amino acid level when given orally but not when given by vein.
A study of the rates of incorporation in vivo of labeled amino acids into tissue proteins was undertaken to find the best time at which intermediates in the process might be found. The rates observed were so fast that we decided to investigate the process in some detail. The amino acids were L-α-aminoadipic-6-C14 acid, glycine-1-C14, L-histidine-2-C14-imidazole, L-leucine-1-C14, and L-lysine-1-C14.