Evaluation of antifungal activity of antimycotics by automatic analyzing system

Antifungal activity of several antimycotics has been evaluated using an automatic analyzing system (AAS), which is composed of a specially designed reaction vessel, microscopic observation system, image analyzing system, and computer program for automatic tracing of hypha growth. The agar plate was prepared on the ceiling of the reaction vessel, and spore mass of a fungus (Aspergillus niger) was inoculated onto it. After the preincubation at 28 °C for 24 h the reaction vessel was set on a microscope stage and connected to the liquid flow system. An appropriate hypha was selected for the measurement of growth process during the following steps: first contact with saline for 30 min for the adaptation, the second contact with same saline for 30 min, contact with saline containing an antimycotic substance for 60 min, and contact with flushing saline for 60 min. During a sequence of these steps, the apical tip of a growing hypha displayed on a TV monitor was followed automatically. The dynamic response of hypha to an agent was analyzed by several parameters. Morphological changes of the hypha caused by respective agents were recorded on VTR for further analysis. By using this system, the antifungal activity of antimycotics could be quantitatively determined within several hours.     

Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha.

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

Automatic evaluation of antifungal volatile compounds on the basis of the dynamic growth process of a single hypha

An automatic analysing system was developed and employed for the evaluation of antifungal activity of volatile compounds in the gas phase. Aspergillus niger was inoculated on agar medium in the reaction vessel. The reaction vessel was incubated at 28° C for 24 h and then a volatile compound was introduced into the vessel either in a batch or flow manner. The antifungal activity of the respective compounds estimated in situ was expressed by the dynamic response parameters of a single hypha. All volatiles tested in the present system inhibited hyphal growth, except linalyl acetate: Limone and geraniol were the most inhibitory. In contrast, linalyl acetate promoted hyphal growth. By definition of the parameters, the fungicidal and fungistatic effects could be distinguished. Correspondence to: H. Matsuoka     

Influence of antifungal pre-treatment in preparing test mycelium on MIC values of several antifungal agents againstAspergillus niger

Antifungal activity of two imidazoles (miconazole and ketoconazole) and one polyene (amphotericin B) was evaluated using an automatic growth analysis system. Spores ofAspergillus niger were inoculated on the polylysine-coated glass bottom of a culture vessel. A colony formed in liquid medium was exposed to an antifungal agent and subsequently washed. Based on the dynamic growth rate of a test hypha selected from the colony in response to the antifungal agent, the minimum inhibitory concentration (MIC) was evaluated. The influence of time of reading (1, 2 and 3 h after washing) on the MIC determined was investigated. MICs for test hyphae subjected to antifungal pre-treatment were compared with those for hyphae without pre-treatment. Hyphae pre-treated with an antifungal agent for 1 h were found to become adapted and tolerant to that antifungal agent. Hyphae exposed and adapted to an imidazole obtained tolerance to amphotericin B as well as to the other imidazole.     

Determination of anti-Aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha

The dynamic growth rate of a single hypha of Aspergillus niger was analysed using an automatic system. A colony of A. niger was in contact with saline, saline containing an antifungal agent, and flushing saline, in sequence. The growth rate of a test hypha selected arbitrarily from the colony responded dynamically to the antifungal agent. The minimum concentration that caused the complete inhibition of hyphal growth was defined as the minimum inhibitory concentration (MIC). The MIC values obtained were compared with those determined by conventional methods based on increasing rate of colony diameter or dry matter weight.     

Studies on the synergistic effect of amphotericin B and 5-fluorocytosine on the growth rate of single hyphae ofAspergillus fumigatus by a biocell-tracer system

The biocell-tracer system is a microscopical system to measure the growth rate of a single fungal hypha. The synergistic effect of amphotericin B (AMPH) and 5-fluorocytosine (5-FC) on the growth of hyphae ofAspergillus fumigatus was studied by using this system. Although neither 2µg/ml of AMPH nor 250µg/ml of 5-FC alone showed any effect on the hyphal growth, their combination at these concentrations showed a distinct inhibitory activity. The biocell-tracer system is useful for antifungal activity testing in filamentous fungi.     

Study on the hyphal responses of Aspergillus fumigatus

The hyphal responses of an A. fumigatus isolate to a trizolederivative-fluconazole (FCZ) were studied with a Bio-Cell Tracer system. The numerical data were recorded as the original growth rate (Pre-GR), the time needed for FCZ reaching to its target in hypha (τ_on), the growth rate under the FCZ effect (Exp-GR) and the growth rate after FCZ was removed (Post-GR). Based on above numerical data, the inhibitory rates in the exposure and post exposure periods were calculated as the Exp-I% and Post-I% values. It was found there were variable inhibitory rate values (I%) in individual hyphae corresponding to different FCZ concentrations. It was shown by correlation analysis of the numerical data that the Pre-GR values were negatively correlated with the τ_on values and positively correlated with both the Exp-I% and Post-I% values. Additionally, the τ_on values are negatively correlated with the Exp-I% and Post-I% values. Those results suggested that the hyphal growth rate and the susceptibility of the FCZ target be the important factors to determine the hyphal responses to the FCZ effect. Serial morphological alternations were captured while the hyphal growth curves were changing under the FCZ effects. Of the morphological data, the interesting alternations were visualized when the hyphae were affected by 16 μg/ml FCZ. As shifting of the hyphal growth curves, the hyphae were repeatedly seen as swollen tips and germination from the swollen sites. It is indicated that the hyphal tips are the most sensitive parts of this mycelia fungus to the FCZ affects. Additionally, because the hyphal regrowth was observed as germination from the swollen tips before FCZ was removed, an adaptation phenomenon could be proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transmission of the eect of an antifungal agent within a single hypha

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occured during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.     

Characterization of antifungal glycoprotein in red-light-irradiated broadbean leaflets

 Red-light treatment of broadbean leaflets resulted in the production of antifungal substance(s) against Botrytis cinerea. The antifungal substance(s) was positively charged, as the antifungal constituent was removed by the cation exchanger CM cellulose. Treatment of infection droplets with glycosidases (α-mannosidase, β-galactosidase, β-glucosidase), glycol-specific reagent periodate (NaIO₄), and proteinase K completely eliminated antifungal activity, suggesting that both protein and carbohydrate are active components. The protein content of infection droplets was 0.148 mg/ml. The HPLC gel column analysis of infection droplets resulted in four fractions; all the fractions showed antifungal activity. Received: June 14, 2002 / Accepted: August 12, 2002 Correspondence to:Y. Honda     

Development of a forced ventilation micropropagation system for large-scale photoautotrophic culture and its utilization in sweet potato

Summary A forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO₂ inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato (Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development.     

Determination of antibiotic EC50 using a zero‐flow microfluidic chip based growth phenotype assay

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC₅₀ values and confirmed the validity of the present system by comparing the EC₅₀ values obtained through conventional test tube method. We demonstrated that the EC₅₀ values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.     

Evaluation of <Emphasis Type="Italic">Fusarium solani</Emphasis> Hyphae and Conidia Susceptibility to Amphotericin B and Itraconazole: Study of a Clinical Case

Fusarium species are hyaline moulds belonging to the hyalohyphomycosis group that are usually found in the soil and plants. This organism has emerged as a cause of disseminated invasive disease. The correlation between in vitro value and clinical efficacy is low and many patients remain unresponsive to treatment despite in vitro susceptibility. We determined growth control for Fusarium solani using the BioCell-Tracer system that measures the growth rate of a single fungal hypha, and the effect of different concentrations of amphotericin B and itraconazole. The MIC for these two drugs was also determined by a broth microdilution technique, using RPMI 1640. Different MICs for amphotericin B were obtained by the two different methods. This paper describes a case of infection due to Fusarium solani in an allogeneic bone marrow transplanted patient, the microbiological diagnostic, antifungal susceptibility tests for conidia and hypha and clinical correlation.     

Metabolism of Maleic Hydrazide in Tea, Camellia sinensis

The results obtained with tea plants treated with ¹⁴C-maleic hydrazide indicate that tea plants can metabolize maleic hydrazide to some extent. According to autoradiographic studies, two radioactive spots were found on X-ray films after exposure and development. Based on two-dimensional paper chromatography, possible ring cleavage, and infrared spectroscopic techniques, the possible metabolic products are lactic acid, succinic acid, maleimide and hydrazine. The biological activity of the metabolic products was tested by utilizing the Avena first internode test. Based on this investigation, it was noted that each radioactive area contained compound(s) that possess(es) some growth promoting properties. However, the results obtained with unknown number one (U₁) indicate that it possesses greater growth-promoting properties than unknown two (U₂). finally, the approximate concentrations of U₁ and U₂ were determined by comparison with known concentrations of IAA and in relation to the amount of growth produced during the 19½-hour period of incubation.     

A novel spectrofluorimetric method based on a reaction with an azoisoxazoles–benzenesulfonamide derivative for determination of uranium (VI) ions in water samples

Selective fluorometric detection and determination of uranium ions is provided here using a novel fluorescent reagent, namely (E)-4-([4-hydroxynaphthalen-1-yl]diazenyl)-N-(5-methyleisoxazol-3-yl) benzenesulfonamide (U^VI reagent). The U^VI reagent offers a selective fluorescence enhancement behaviour at emission wavelength = 557 nm. The parameters affecting fluorometric detection of uranium ions, such as the pH, solvent type, ligand concentration, interaction time, and interfering ions, were investigated and adjusted. The proposed U^VI reagent can detect and determine uranium ions even at low concentrations, for which the obtained limit of detection was 0.1 ppm. Additionally, this proposed determination protocol was successfully used to detect, monitor, and determine uranium ions in actual water samples.     

Synthesis and antifungal activity of novel s-substituted 6-fluoro-4-alkyl(aryl)thioquinazoline derivatives

6-Fluoro-4-quinazolinol is prepared by the cyclization reaction of 2-amino-5-fluorobenzoic acid and formamide. The resulting thiol obtained by treatment of hydroxyl group with phosphorus (V) sulfide is converted under phase transfer condition to 4-substituted 4-alkylthio-6-fluoroquinazoline derivatives by reaction with halide. The structures of the compounds are confirmed by elemental analysis, IR, and (1)H NMR. Title compounds 3a, 3g, and 3h are found to possess good antifungal activities. Using the mycelial growth rate method in the laboratory, the mechanism of action of 3g against Fusarium oxysporum in vitro is studied. The results indicate that 3a, 3g, and 3h have high inhibitory effect on the growth of most of the fungi with EC(50) values ranging from 8.3 to 64.2 microg/mL. After treating F. oxysporum with compound 3g at 100 microg/mL, only 6.5% of its spore bourgeoned. The permeability of the cell membrane increases along with the malformation of the hypha and condensation of its endosome. After treatment with compound 3g at 100 microg/mL within 12h, the mycelial reducing sugar, D-GlcNAc, content and chitinase activity decline, but the soluble protein content shows no obvious change.     

Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease A chain folding

The kinetics of folding of the two forms of unfolded ribonuclease A have been measured as a function of solvent viscosity by adding either glycerol or sucrose. The aim is to find out if either reaction is rate limited by segmental motion whose rate depends on external friction. The fast folding reaction (U₂ ? N) is known to be the direct folding process, and the slow folding reaction (U₁ ? N) is known to be rate limited by an interconversion between two forms (U₁ ? U₂) which are present after unfolding in strongly denaturing conditions. No dependence on solvent viscosity is found, either for the direct folding reaction or for the interconversion reaction. Each folding reaction has also been tested to see if its rate depends on the concentration of one or more partly folded intermediates, by adding denaturants destabilize any partly folded structures. Different guanidine salts are used as denaturants to vary the denaturing effectiveness of the salt while holding the guanidinium ion concentration constant. The rates both of the direct folding reaction and of the interconversion reaction decrease in relation to the denaturing effectiveness of the salt. However, there is a basic difference between the responses of the fast and slow folding reactions to low concentrations of denaturants. Although each folding reaction produces native protein, there is an 800-fold decrease in the rate of the fast folding reaction in 1M guanidine thiocyanate and only a 13-fold decrease in the rate of the slow folding reaction. This is consistent with the fast reaction being the direct folding process and the slow reaction being rate limited by the initial conversion of the slowrefolding to the fast-refolding form. Both the lack of viscosity dependence and the effects of denaturants indicate that the formation of structure is rate limiting in the direct folding reaction, U₂ ? N. The failure to find a viscosity dependence for the interconversion reaction, U₁ ? U₂, indicates that in this reaction also friction-limited segmental motion is not the rate-limiting process. Since the U₁ ? U₂ interconversion still occurs when the polypeptide chain is completely unfolded, the surprising result is that its rate in refolding conditions depends significantly on a reaction intermediate which is “denatured” by guanidine salts.     

Design,Synthesis and Bioassay of 2-Phenylglycine Derivatives as Potential Pesticide Candidates

Plant diseases can seriously affect the growth of food crops and economic crops. To date, pesticides are still among the most effective methods to prevent and control plant diseases worldwide. Consequently, to develop potential pesticide molecules, a series of novel 2-phenylglycine derivatives containing 1,3,4-oxadiazole-2-thioethers were designed and synthesized. The bioassay results revealed that G₁₉ exhibited great in vitro antifungal activity against Thanatephorus cucumeris with an EC₅₀ value of 32.4 μg/mL, and in vivo antifungal activity against T. cucumeris on rice leaves at a concentration of 200.0 μg/mL (66.9 %) which was close that of azoxystrobin (73.2 %). Compounds G₂₄ (80.2 %), G₂₅ (89.4 %), and G₂₇ (83.3 %) exhibited impressive in vivo inactivation activity against tobacco mosaic virus (TMV) at a concentration of 500.0 μg/mL, which was comparable to that of ningnanmycin (96.3 %) and markedly higher than that of ribavirin (55.6 %). The antibacterial activity of G₁₆ (63.1 %), G₂₆ (89.9 %), G₂₇ (78.0 %), and G₂₈ (68.0 %) against Xoo at a concentration of 50.0 μg/mL was higher than that of thiadiazole copper (18.0 %) and bismerthiazol (38.9 %). Preliminary mechanism studies on the antifungal activity against T. cucumeris demonstrated that G₁₉ can affect the growth of mycelia by disrupting the integrity of the cell membrane and altering the permeability of the cell. These studies revealed that the amino acid derivatives containing a 1,3,4-oxadiazole moiety exhibited certain antifungal, antibacterial, and anti-TMV activities, and these derivatives can be further modified and developed as potential pesticide molecules.     

Mannan changes induced by 3-methyl-5-aminoisoxazole-4-thiocyanate,a new azole derivative,on Epidermophyton floccosum

The antifungal activity of 3-methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative, was studied on the dermatophyte Epidermophyton floccosum. The compound strongly inhibited the in vitro growth of two different strains of the fungus and even induced profound morphogenetic anomalies. Optical and electron microscopy showed that such treatment targets the endomembrane system, particularly the plasmalemma, causing abnormal extrusion of the wall mannans. This results in improper arrangement of the different parietal materials; the walls are thus weak and subject to subapical rupture which terminates cell growth and elongation of the hypha. The morphological results and the preliminary biochemical data on fungal sterols suggest that this compound employs an action mechanism similar to that of other azoles used in therapy.     

Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup

Summary An instrumental setup is described for the measurement of enzyme kinetics and morphometry in tissue sections. It consists of a Vickers M85 microdensitometer and computer-assisted Kontron Videoplan system. The Videoplan system consists of a minicomputer with two mini-floppy disks, a keyboard, a graphic tablet, a TV monitor and a printer/plotter. The measuring component of the M85 is linked to the minicomputer via a BCD interface, and the optical system of the M85 is coupled to a TV camera for display on the monitor screen. The enzyme-kinetic data obtained with the M85 in a specified area of the tissue section (density values as a function of reaction time) are stored in the minicomputer. The measurement process is controlled by a corresponding measuring program. Through correlation analysis (a component of the commercial software) between density values and reaction time, the initial and thus maximum enzyme activity is determined. Upon completion of the kinetic measurements, the measured area of tissue is transferred by the TV camera to the monitor, and the reaction area is described and measured with the graphic tablet in video dialogue and related to the initial enzyme activity. With the setup described, it is possible to make microdensitometric measurements of enzyme activities in a specified tissue area while morphometrically analyzing the associated reaction area. To illustrate the use of the system, enzyme-kinetic (succinate dehydrogenase) and morphometric measurements are performed in tissue sections from the proximal tubule of the rat nephron. Additional applications of the system are discussed.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)