Tomoo Homma

A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm, lambda Em = 550 nm). The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway.

Use of Congo red as a microscopic fluorescence indicator of hyphal growth

Congo red was found to be feasible as a microscopic fluorescence indicator of hyphal growth at the single-hypha level. When 1 μm Congo red was applied to mold of Aspergillus niger, the dye was found to a specific cell-wall component, chitin, without causing any inhibitory effect on hyphal growth. The bound Congo red emitted fluorescence at 614 nm. This binding reaction, however, proceeded more slowly than the growing speed of hypha. Consequently the fluorescence intensity was low at the apex where the surface area of the hypha was expanding rapidly. In contrast, as an apex where the growth was retarded, the fluorescence intensity became remarkably high. Therefore growing hyphae could be distinguished from non-growing hyphae by using Congo red.

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl2 to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.

Adaptation of Aspergillus niger to short-term salt stress

Adaptation of filamentous fungi to short-term salt stress has been analysed by a continuous measurement system. Spores of Aspergillus niger were immobilized on the polylysine-coated glass bottom of a culture vessel, which enabled the exchange of a medium containing salt (NaCl) without disturbing continuous observation. Repeated contacts with 0.75% NaCl produced hypha insensitive to this concentration of NaCl. When the NaCl concentration was increased stepwise, the tolerated concentration increased up to 1.25%. The acquisition of such a tolerance to 0.75% NaCl required about 10 min prior contact with 0.5% NaCl. Based on these results, the adaptation mechanism is discussed.

Use of plant leaf as CO2 gas sensing probe

Abstract Intracellular potential (ICP) of a spiderwort (Commelina communis) leaf drastically changed to CO 2 gas exposure. A leaf of C. communis was attached on a slide glass under a microscope. The stem of the leaf was dipped in a bathing solution composed of 0.1 M phosphate buffer (K 2 HPO 4 -KH 2 PO 4 , pH6.5) and 0.1 M NH 4 Cl. The ICP was measured with a glass capillary electrode referred to another electrode dipped in the bathing solution. When CO 2 gas was applied to the leaf under illumination, the ICP shifted sharply to the negative direction about 100mV, and then turned towards the positive direction. Finally the ICP reached a level about 50 mV higher than the initial level. When CO 2 supply was stopped, the ICP returned to the initial level immediately. When light was shielded or CO 2 concentration was decreased lower than 20%, the response curve showed a single phase. The intensity of the response was positively correlated with CO 2 concentration lower than 20% both under illumination and shielded conditions. In contrast, no response was observed, when N 2 gas or air (CO 2 content is 0.03%) was applied independent of light conditions. The responsiveness to CO 2 maintained for 10 days being set still on the slide glass. These results demonstrate the promising property of plant leaf as a CO 2 gas sensing element.

Adaptation of Aspergillus niger to several antifungal agents

Adaptation of Aspergillus niger to short-term stress induced by three antifungal agents [amphotericin B (AMPH), miconazole (MCZ), and ketoconazole (KCZ)] was observed and evaluated quantitatively using individual hyphae. Spores were inoculated onto a poly-L-lysine-coated glass plate making up the base of a culture vessel. Potato dextrose broth (PDB) was added and the vessel incubated for 24 h at 28 degrees C. The growth rate of an arbitrarily selected test hypha was measured automatically. Exposure to AMPH (0.075 micrograms ml-1) stopped the growth of the hypha. After washing with PDB, the same concentration of AMPH was applied again. The growth of the test hypha was not inhibited. This phenomenon was defined as adaptation to the short-term stress of AMPH. Similarly, adaptation was observed with MCZ (0.01 microgram ml-1) and KCZ (0.5 microgram ml-1). The time required for the test hypha to restart growth after washing with PDB depended upon the concentration of MCZ or KCZ, but not upon the concentration of AMPH.

Intracellular potential change of Tradescantia virginiana L. leaf in response to CO2 stress

Abstract The response of a Tradescantia virginiana L. leaf to CO2 stress was measured using a double-barrelled microelectrode (a potential recording electrode and an electrode carrying Lucifer yellow CH dye (LY)). After potential measurement, LY was allowed to diffuse out of the microelectrode by iontophoresis. The position of electrode tip was ascertained from the pattern of LY diffusion. The intracellular potential changed markedly in response to CO2 stress. The most typical response pattern obtained during CO2 exposure (ON response) was two-phase, initially changing in the positive direction and then in the negative direction. During the ON response, marked efflux of K+ and slight influx of Cl− occurred initially, followed by efflux of Cl− and influx of H+. On cessation of CO2 exposure, the potential showed a similar two-phase pattern (OFF response) but the ion fluxes reversed. Therefore the effect of CO2 exposure is not just decrease in intracellular pH owing to dissolution of CO2.

Analysis of electric and electrochemical connection between leaf cells of Tradescantia virginiana L. that responds to CO2 stress by changing intracellular potential

Abstract The intracellular potential of a Tradescantia virginiana L. leaf changed markedly in response to CO 2 stress. To analyse this response, the intercellular connection of the leaf was investigated. When a limited area of a leaf was exposed to CO 2 , no response was obtained at a site separate from the CO 2 -exposed region. When the leaf was cut into various-sized fragments and exposed to CO 2 , each fragment size showed remarkably similar response patterns. However, when an electrochemical impulse was applied to one epidermal cell by injecting KC1 electrophoretically, the same patterns of potential response were obtained from cells within a radius of 2 mm. Therefore, within this region, epidermal cells of T. virginiana are electrically short-circuited. In contrast, only a slight increase in the K + concentration could be observed in neighbouring cells. Therefore, the cells appear to be electrochemically isolated from each other.

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 102∼104 cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.

CO2 stress sensing using a tobacco leaf on the basis of chlorophyll fluorescence analysis

Abstract The chlorophyll fluorescence intensity of a tobacco leaf (Nicotiana tabacum ‘Xanthi nc’) increases markedly in response to more than 3% CO2. The photosynthetic activity, estimated from the O2 evolution, is also inhibited completely above 3% CO2. The fluorescence response is composed of rapid and delayed components. The rapid component is speculated to be the specific response to CO2 stress, while the delayed component is not. When isolated cells are immobilized on a membrane filter and reacted with CO2, they show only the latter component. Therefore the use of a complete tobacco leaf is necessary for the sensing of CO2 stress. The present result suggests that a plant leaf is promising as a sensing material for gaseous stress.