An imidazole derivative (econazole) as an antifungal agent in cell culture systems

Summary Econazole, an imidazole derivative, was tested as an antifungal agent in different cell culture systems. In comparison with Fungizone, Econazole has the following advantageous properties: higher stability, higher solubility, better antifungal activity against contaminants of cell cultures and a wider range between minimal inhibitory to cytotoxic concentration withAspergillus fumigatus, Candida albicans andPenicillium sp., activity against gram-positive bacteria and lower price. Econazole exerts no antiviral effect and can therefore be used for virus isolation from heavily contaminated material. The antagonistic effect of serum on the antifungal effect of Econazole and Fungizone was comparable as was the inhibitory effect of both antimycotics onMycoplasma growth. In view of the above mentioned properties Econazole lactic acid can be recommended as an antifungal agent for cell culture systems at a concentration of 1 μg per ml.     

Drosophila cecropin as an antifungal agent

The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 microM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture.     

Miconazole: An effective antifungal agent for plant tissue culture

Miconazole at concentrations between 5 and 20 mgl^-1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. These fungi included 4 Oomycetes, 11 Deuteromycetes, 4 Ascomycetes and 4 common airborne contaminants of plant tissue culture. The phytotoxicity of 20 mgl^-1 miconazole was also tested against a wide range of in vitro plant cultures. Shoot cultures from 15 species showed either no response or a slight growth reduction in the presence of miconazole. Although the growth of shoot cultures in 2 other species was significantly reduced by miconazole, a positive growth rate was maintained. Callus and hairy root cultures from 5 species were more sensitive to miconazole than shoot cultures, although they also retained a positive growth rate in the presence of the antifungal agent. Reconstruction experiments demonstrated the effectiveness miconazole had for the rescue of plant material from in vitro cultures contaminated from fungi.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - EBI ergosterol biosynthetic inhibitor - IAA indole-3-acetic acid - MZ miconazole - NAA -naphthaleneacetic acid     

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.     

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3 D) culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3 D cell models. Thus, the 3 D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3 D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting. In addition, the progress of employing 3 D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3 D cell culture systems were discussed.     

The use of psyllium (isubgol) as an alternative gelling agent for microbial culture media

Psyllium (Isubgol), the mucilage husk of Plantago ovata, was successfully used as an alternative gelling agent (5% w/v as ground husk for pouring medium and 4% w/v as ground husk in combination with 0.5% w/v agar for slant) for microbial culture. Most of the undesirable features of psyllium-gelled media (slant as well as pouring) were removed by adding the u.v.-treated (20min) or oven-sterilized (120^°C for 1h) psyllium as ground husk to autoclaved medium at 50–60^°C under aseptic condition just before pouring.     

Effects of an imidazole derivative (KK-42) on development and ecdysteroid production in Locusta migratoria (Insecta, Orthoptera)

The imidazole derivative KK-42 was applied in various experimental conditions to larvae and adult females of Locusta migratoria. The effect of this compound was monitored on the development of larvae, on oocyte growth in adult females and on the development of eggs laid by these females. KK-42 had only minor effects on postembryonic development; anticipation of imaginal moult was never observed. In contrast oocyte and egg development were markedly affected by KK-42: this effect is however not related to modifications of the synthesis of ecdysteroids in the ovaries.     

Discovery of a quinoline-based phenyl sulfone derivative as an antitrypanosomal agent

A series of natural products-based phenyl sulfone derivative and their property-based analogues were investigated as potential growth inhibitors of Trypanosoma brucei. Trypanosoma brucei is a kinetoplastid protozoan parasite that causes trypanosomiasis. In this work, we found that nopol- and quinoline-based phenyl sulfone derivative were the most active and selective for T. brucei, and they were not reactive towards the active thiol of T. brucei’s cysteine protease rhodesain. A thiol reactive variant of the quinoline-based phenyl sulfone was subsequently investigated and found to be a moderate inhibitor of rhodesain. The quinoline-based compound that is not reactive towards rhodesain can serve a template for phenotypic-based lead discovery while its thiol-active congener can serve as template for structure-based investigation of new antitrypanosomal agents.     

Use of caryophyllene oxide as an antifungal agent in an in vitro experimental model of onychomycosis

Caryophyllene oxide, an oxygenated terpenoid, well known as preservative in food, drugs and cosmetics, has been tested in vitro as an antifungal against dermatophytes. Its antifungal activity has been compared to ciclopiroxolamine and sulconazole, commonly used in onychomycosis treatment and chosen because of their very different chemical structures. So, a new model has been tested, utilizing sheep hoof plates in order to simulate human nails, which are almost unobtainable for in vitro tests. Three protocols were utilized: pre-treatment. simultaneous treatment and post-treatment. Among these, the post-treatment method was the best to simulate antifungal therapy. as it permitted testing and comparing the efficiency of different antifungal drugs.This revised version was published online in October 2005 with corrections to the Cover Date.     

An evaluation of tribromoethanol (TBE) as an anaesthetic agent in the Mongolian gerbil (Meriones unguiculatus)

Mongolian gerbils injected i.p. with a 1.25% solution of tribromoethanol (TBE) quickly lost the righting reflex and showed good surgical anaesthesia, the duration of which was positively related to the dose administered. All levels (225-450 mg/kg bodyweight) of TBE injected s.c. resulted in loss of the righting reflex, though surgical anaesthesia was not attained. Following recovery from anaesthesia induced with 2.25% TBE i.p., or high doses of 1.25% TBE i.p., visceral adhesions and deaths occurred. To avoid these TBE-induced fatalities such extreme care must be taken during the preparation, storage and administration of the agent that it is not recommended as a suitable anaesthetic for general use in the gerbil.     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent

Aspergillus nidulans was used as a model organism to investigate the fungal propionate metabolism and the mechanism of growth inhibition by propionate. The fungus is able to grow slowly on propionate as sole carbon and energy source. Propionate is oxidized to pyruvate via the methylcitrate cycle. The key enzyme methylcitrate synthase was purified and the corresponding gene mcsA, which contains two introns, was cloned, sequenced and overexpressed in A. nidulans. The derived amino acid sequence of the enzyme shows more than 50% identity to those of most eukaryotic citrate synthases, but only 14% identity to the sequence of the recently detected bacterial methylcitrate synthase from Escherichia coli. A mcsA deletion strain was unable to grow on propionate. The inhibitory growth effect of propionate on glucose medium was enhanced in this strain, which led to the assumption that trapping of the available CoA as propionyl-CoA and/or the accumulating propionyl-CoA itself interferes with other biosynthetic pathways such as fatty acid and polyketide syntheses. In the wild-type strain, however, the predominant inhibitor may be methylcitrate. Propionate (100 mM) not only impaired hyphal growth of A. nidulans but also synthesis of the green polyketide-derived pigment of the conidia, whereas in the mutant pigmentation was abolished with 20 mM propionate.     

Lactate as an indicator of terminating time in insect cell culture baculovirus expression vector systems

Summary Lactate can be used as a rapid indicator, for semi-continuous determination of the optimal harvest time, when using the insect cell (Sf9) culture baculovirus expression vector system (ICC-BEVS) for production of a protein. The method is based on the lactic acid production of ICC-BEVS and the viable cell density. It shows, that when lactate/viable cells reached 1 mg/(cells × 10^–6), no further product was produced, and the optimal terminating time was reached. Delay of harvest may cause degradation of product and may be a waste of time.     

Assembly of anion-controlled cadmium(II) coordination polymers based on flexible bis(imidazole) derivative

Four Cd(II) metal-organic complexes, namely, [Cd(Cl)₂(bbdmbm)] (1), [Cd(NO₃)(N₃)(bbdmbm)_1.5] (2), [Cd(BBA)₂(bbdmbm)(H₂O)] (3), [Cd(DNBA)₂(bbdmbm)] (4), (bbdmbm = 1,1-(1,4-butanediyl)bis(5,6-dimethylbenzimidazole), HBBA = 4-bromobenzoic acid, and HDNBA = 3,5-dinitrobenzoic acid) have been obtained from hydrothermal reactions of different Cd(II) salts with the mixed ligands of bbdmbm and five anions (Cl⁻, NO₃⁻, N₃⁻, BBA and DNBA). Single crystal X-ray diffraction analyses reveal that the four complexes exhibit different structures. Complex 1 possesses a one-dimensional (1D) helical chain, which is finally extended into a two-dimensional (2D) supramolecular structure through π-π stacking interactions. Complex 2 shows a 1D ladderlike chain bridged by bbdmbm ligands with two kinds of coordination conformations. Complex 3 is a 1D coordination polymer and is ultimately extended into a 2D supramolecular network through H-bonding interactions. Complex 4 displays a dinuclear cluster, which is finally packed into a three-dimensional (3D) supramolecular framework through three kinds of π-π stacking interactions. The Cd(II) exhibits four different coordination modes in complexes 1-4, respectively. The results indicate that the anion ligands with different steric hindrance and size play important roles in the coordination modes of Cd(II) and construction of the title complexes, leading to the structural diversity. In addition, the conformations of bbdmbm ligand also show some effect on the final structures. Fluorescence properties of complexes 1-4 are reported in this paper.     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.