Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents.

1996 ◽  
Vol 40 (2) ◽  
pp. 443-447 ◽  
Author(s):  
B Favre ◽  
N S Ryder
Keyword(s):  
Biochemical Characterization ◽  
Trichophyton Rubrum ◽  
Side Chain ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
High Potency ◽  
Whole Cells ◽  
Absolute Requirement ◽  
Tertiary Amino

Squalene epoxidase (SE) is the primary target of the allylamine antimycotic agents terbinafine and naftifine and also of the thiocarbamates. Although all of these drugs are employed primarily in dermatological therapy, SE from dermatophyte fungi has not been previously investigated. We report here the biochemical characterization of SE activity from Trichophyton rubrum and the effects of terbinafine and other inhibitors. Microsomal SE activity from T. rubrum was not dependent on soluble cytoplasmic factors but had an absolute requirement for NADPH or NADH and was stimulated by flavin adenine dinucleotide. Kinetic analyses revealed that under optimal conditions the Km for squalene was 13 microM and its Vmax was 0.71 nmol/h/mg of protein. Terbinafine was the most potent inhibitor tested, with a 50% inhibitory concentration (IC50) of 15.8 nM. This inhibition was noncompetitive with regard to the substrate squalene. A structure-activity relationship study with some analogs of terbinafine indicated that the tertiary amino structure of terbinafine was crucial for its high potency, as well as the tert-alkyl side chain. Naftifine had a lower potency (IC50, 114.6 nM) than terbinafine. Inhibition was also demonstrated by the thiocarbamates tolciclate (IC50, 28.0 nM) and tolnaftate (IC50, 51.5 nM). Interestingly, the morpholine amorolfine also displayed a weak but significant effect (IC50, 30 microM). T. rubrum SE was only slightly more sensitive (approximately twofold) to terbinafine inhibition than was the Candida albicans enzyme. Therefore, this difference cannot fully explain the much higher susceptibility (> or = 100-fold) of dermatophytes than of yeasts to this drug. The sensitivity to terbinafine of ergosterol biosynthesis in whole cells of T. rubrum (IC50, 1.5 nM) is 10-fold higher than that of SE activity, suggesting that the drug accumulates in the fungus.

scholarly journals The introduction of the C-22–C-23 ethylenic linkage in ergosterol biosynthesis

1968 ◽  
Vol 106 (3) ◽  
pp. 623-626 ◽  
Author(s):  
M Akhtar ◽  
M. A. Parvez ◽  
P. F. Hunt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Good Yield ◽  
The Other ◽  
Side Chain ◽  
Ergosterol Biosynthesis ◽  
Hydrogen Atoms ◽  
Whole Cells ◽  
C 23 ◽  
Methylene Derivative

Methods for the chemical synthesis of [23−3H2]lanosterol, [23,25−3H3]24-methyldihydrolanosterol and [24,28−3H2]24-methyldihydrolanosterol are described. It is shown that, in the biosynthesis of ergosterol from [26,27−14C2,23−3H2]lanosterol by the whole cells of Saccharomyces cerevisiae, one of the original C-23 hydrogen atoms is lost and the other is retained at C-23 of ergosterol. It is also shown that 24-methyldihydrolanosterol is converted into ergosterol in good yield and without prior conversion into a 24-methylene derivative. On the basis of these results possible pathways for the formation of the ergosterol side chain from a 24-methylene side chain are discussed.

scholarly journals Biological, Biochemical, and Molecular Characterization of a New Clinical Trichophyton rubrum Isolate Resistant to Terbinafine

2006 ◽  
Vol 50 (6) ◽  
pp. 2234-2236 ◽  
Author(s):  
Colin S. Osborne ◽  
Ingrid Leitner ◽  
Bettina Hofbauer ◽  
Ceri A. Fielding ◽  
Bertrand Favre ◽  
...  
Keyword(s):  
Amino Acid ◽  
Missense Mutation ◽  
Molecular Characterization ◽  
Amino Acid Substitution ◽  
Trichophyton Rubrum ◽  
Mechanisms Of Action ◽  
Clinical Strain ◽  
Squalene Epoxidase

ABSTRACT We have characterized a new clinical strain of Trichophyton rubrum highly resistant to terbinafine but exhibiting normal susceptibility to drugs with other mechanisms of action. Resistance to terbinafine in this strain is caused by a missense mutation in the squalene epoxidase gene leading to the amino acid substitution F397L.

scholarly journals Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

2003 ◽  
Vol 69 (9) ◽  
pp. 5423-5432 ◽  
Author(s):  
Armelle Cochu ◽  
Christian Vadeboncoeur ◽  
Sylvain Moineau ◽  
Michel Frenette
Keyword(s):  
Biochemical Characterization ◽  
Sugar Transport ◽  
Streptococcus Thermophilus ◽  
Phosphate Group ◽  
Phosphotransferase System ◽  
Whole Cells ◽  
Genes Encoding ◽  
Regulatory Functions

ABSTRACT In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.

Biochemical Characterization of Propylglyoxal Bis(guanylhydrazone). Facile Synthesis of Monoalkylglyoxal Bis(guanylhydrazones)

1985 ◽  
Vol 40 (11-12) ◽  
pp. 839-842 ◽  
Author(s):  
Hannu Elo ◽  
Raija Laine ◽  
Leena Alhonen-Hongisto ◽  
Juhani Jänne ◽  
Ilpo Mutikainen ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cellular Uptake ◽  
Biochemical Characterization ◽  
Prior Exposure ◽  
Side Chain ◽  
Facile Synthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Decarboxylase Inhibition ◽  
Striking Contrast

Abstract Propylglyoxal Bis(guanylhydrazone). Ethylglyoxal Bis(guanylhydrazone), Adenosylmethionine Decarboxylase Inhibition. Tumor Cells, Cellular Uptake Propylglyoxal bis(guanylhydrazone) sulfate, a novel analog of the well-known antileukemic drug methylglyoxal bis(guanylhydrazone), has been prepared from 2,2-dibromopentanal, and the compound has been characterized biochemically. Although it is a powerful inhibitor of S-adenosylmethionine decarboxylase, its Ki, value (0.2 μᴍ) is considerably higher than that of ethylglyoxal bis(guanylhydrazone) (0.06 μᴍ). The compound is only poorly taken up by tumor cells, and its accumulation is not stimulated by a prior exposure of the tumor cells to di-fluoromethylornithine, a compound that causes polyamine depletion. Thus, the uptake charac­ teristics of the compound are similar to those of ethylglyoxal bis(guanylhydrazone), but in striking contrast to those of methylglyoxal and glyoxal bis(guanylhydrazones). Since the configuration of the double bonds in glyoxal, methylglyoxal and propylglyoxal bis(guanylhydrazones) has been shown to be identical, the different uptake characteristics are probably only due to differences in side chain size and/or hydrophobicity.

scholarly journals Mutation in the Squalene Epoxidase Gene ofTrichophyton interdigitaleandTrichophyton rubrumAssociated with Allylamine Resistance

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Shivaprakash M. Rudramurthy ◽  
Shamanth A. Shankarnarayan ◽  
Sunil Dogra ◽  
Dipika Shaw ◽  
Khurram Mushtaq ◽  
...  
Keyword(s):  
Antifungal Agents ◽  
Susceptibility Testing ◽  
Trichophyton Rubrum ◽  
Antifungal Susceptibility ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Content Type ◽  
Recent Attention

ABSTRACTDermatophytosis, the commonest superficial fungal infection, has gained recent attention due to its change of epidemiology and treatment failures. Despite the availability of several agents effective against dermatophytes, the incidences of chronic infection, reinfection, and treatment failures are on the rise.Trichophyton rubrumandTrichophyton interdigitaleare the two species most frequently identified among clinical isolates in India. Consecutive patients (n= 195) with suspected dermatophytosis during the second half of 2014 were included in this study. Patients were categorized into relapse and new cases according to standard definitions. Antifungal susceptibility testing of the isolatedTrichophytonspecies (n= 127) was carried out with 12 antifungal agents: fluconazole, voriconazole, itraconazole, ketoconazole, sertaconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine, griseofulvin, and luliconazole. The squalene epoxidase gene was evaluated for mutation (if any) in 15T. interdigitaleand 5T. rubrumisolates exhibiting high MICs for terbinafine. A T1189C mutation was observed in fourT. interdigitaleand twoT. rubrumisolates. This transition leads to the change of phenylalanine to leucine in the 397th position of the squalene epoxidase enzyme. In homology modeling the mutant residue was smaller than the wild type and positioned in the dominant site of squalene epoxidase during drug interaction, which may lead to a failure to block the ergosterol biosynthesis pathway by the antifungal drug.

Biochemical characterization of 3-ketoacyl-acyl carrier protein synthase II from leek epidermis

2000 ◽  
Vol 28 (6) ◽  
pp. 610-613 ◽  
Author(s):  
F. Domergue ◽  
D. Post-Beittenmiller
Keyword(s):  
Fatty Acid Synthase ◽  
Biochemical Characterization ◽  
Acyl Carrier Protein ◽  
Allium Porrum ◽  
Carrier Protein ◽  
Substrate Specificities ◽  
Absolute Requirement ◽  
Acyl Chains ◽  
Different Tissues

In order to define its possible involvement in production of stearic acid for wax biosynthesis, the presence of 3-ketoacyl acyl synthase II (KAS II) activity was investigated in different tissues of leek (Allium porrum L.) leaves. KAS II activity was identified in sheath and lamina epidermis, as well as in underlying parenchyma. In all three tissues, activity was inhibited by 50% on addition of l00μM cerulenin, and showed an absolute requirement for acyl-ACP substrates. More interestingly, the different tissues did not display similar KAS II substrate specificities. Parenchyma and lamina epidermis tissues presented typical KAS II activities, since C18 0-ACP was the exclusive product. In contrast, in sheath epidermis, KAS II activity resulted in the synthesis of acyl-chains up to 22 carbons in length, suggesting the existence in this tissue of an unusual KAS II. This activity was sufficient to elongate all of the palmitoyl-ACP produced by the fatty acid synthase, suggesting that C18 0 is the substrate of the microsomal elongases involved in wax biosynthesis.

Plant Pathology Diagnosed by X-Ray Microanalysis

1987 ◽  
Vol 45 ◽  
pp. 974-975
Author(s):  
J. H. Resau ◽  
N. Howell ◽  
S. H. Chang
Keyword(s):  
Electron Microscopy ◽  
Plant Pathology ◽  
Biochemical Characterization ◽  
Nutrient Deficiency ◽  
Green Leaf ◽  
Parasitic Infestation ◽  
X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

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