Evaluation of Cytotoxic Effect of Nystatin on Yeast Cells By Electrochemical Monitoring of Intracellular NADH with Double Mediator System

Introduction Nicotinamide Adenine Dinucleotide (reduced form), NADH is a very important redox compound that is essential for metabolic reactions and ATP production in all living cells. Intracellular NADH metabolism is also important to consider the cancer treatment. Intracellular NADH has been conventionally measured by WST assay using a cell membrane-permeable redox mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulfate) and a water soluble tetrazolium to form formazan. This colorimetric method is very easy and useful for cell counting and cytotoxicity test. However, WST assay is time-consuming and then it is not able to apply for evaluation of acute cytotoxicity by chemical compounds including drugs and pollutants. On the other hand, some research groups have already shown that electrochemical method with double mediator system is useful to monitor rapidly intracellular NADH level corresponding to the cell viability [1-5].In this paper, we would like to report the usefulness of the electrochemical interface using a small screen printed carbon electrode (SPCE) and a new double mediator system combining 1-methoxy PMS and ferricyanide. It was demonstrated that our electrochemical interface was available for faster and wide range of mammalian cell counting as compared with WST assay. Furthermore our method could be applied to acute cytotoxicity test on mammalian cells. The optimization of measurement condition is mentioned in the presentation and the advantages of our new double mediator system against other double mediator system are also discussed in the presentation. Method We used PC12 cell (Rat adrenal pheochromocytoma cell line) as a mammalian cell sample. The cells were grown in culture flasks containing DMEM with 5% FBS and 10%HS at 37℃ with 5% CO2 atmosphere. For the cell counting experiment, the medium was exchanged to HBSS before experiments. Cells were removed from the bottom of culture flask by trypsin treatment and suspended in HBSS. After measurement of cell density of the original cell suspension with a hemocytometer, cell suspension was exactly diluted to prepare various density of cell suspension. Each diluted cell suspension was taken in a well of 96 well plate and final concentration of 500 μM potassium ferricyanide and 10 μM 1-metoxy PMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, a SPCE (working electrode area is 1 mm2) was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V vs. Ag/AgCl reference electrode on the SPCE. The electrochemical measurement was carried out using a potentiostat (Multi-Autolab Cabinet, Metrohm Autolab).We further tried to test the acute cytotoxicity of oxamic acid that is a famous inhibitor of anaerobic glycolysis. Acute cytotoxicity test was carried out with the same electrochemical instrument by following procedure. Each concentration of oxamic acid/HBSS was prepared and added into PC12 cell/HBSS suspension (5.5 x 105 cells /well) in a well (final concentration of oxamic acid: 0 to 20 mM) and the cell suspension was incubated for 1hour. And then potassium ferricyanide (final 500 mM) and 1-metoxy PMS (final 10 μM) solutions were added into the cell suspension. After incubation for more 10 minutes, a SPCE was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V. Results and Conclusions In the first place, it was demonstrated that our electrochemical interface with a new combination of double mediator system was very useful for very wide range of cell counting. The amperometric oxidation current showed very good linearity vs. the number of viable cells in the range from 7500 to 964000 cells/well (Data is not shown). The result strongly supported the combination of 1-methoxy PMS and ferricyanide is effective to monitor intracellular NADH level and to speculate the cell viability.Therefore, in the second place, we applied our electrochemical interface to test the acute toxicity by oxamic acid. The inset of Figure 1 shows the decrease of oxidation current of the double mediator system after 1 hour oxamic acid treatment. The decrease of oxidation current was clearly dependent on the concentration of oxamic acid. It indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by oxamic acid treatment. This result suggested that our electrochemical interface with a new double mediator system might be useful to evaluate acute cytotoxicity of drugs.

Introduction Nicotinamide Adenine Dinucleotide (reduced form), NADH is a very important redox compound that is essential for metabolic reactions and ATP production in all living cells. Intracellular NADH metabolism is also important to consider the cancer treatment. Intracellular NADH has been conventionally measured by WST assay using a cell membrane-permeable redox mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulfate) and a water soluble tetrazolium to form formazan. This colorimetric method is very easy and useful for cell counting and cytotoxicity test. However, WST assay is time-consuming and then it is not able to apply for evaluation of acute cytotoxicity by chemical compounds including drugs and pollutants. On the other hand, some research groups have already shown that electrochemical method with double mediator system is useful to monitor rapidly intracellular NADH level corresponding to the cell viability [1-5].In this paper, we would like to report the usefulness of the electrochemical interface using a small screen printed carbon electrode (SPCE) and a new double mediator system combining 1-methoxy PMS and ferricyanide. It was demonstrated that our electrochemical interface was available for faster and wide range of mammalian cell counting as compared with WST assay. Furthermore our method could be applied to acute cytotoxicity test on mammalian cells. The optimization of measurement condition is mentioned in the presentation and the advantages of our new double mediator system against other double mediator system are also discussed in the presentation. Experimental method We used PC12 cell (Rat adrenal pheochromocytoma cell line) as a mammalian cell sample. For the cell counting, the medium was exchanged to HBSS before experiments. After measurement of cell density of the original cell suspension with a hemocytometer, cell suspension was exactly diluted to prepare various density of cell suspension. Each diluted cell suspension was taken in a well of 96 well plate and final concentration of 500 μM potassium ferricyanide and 10 μM 1-metoxy PMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, a SPCE (working electrode area is 1 mm2) was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V vs. Ag/AgCl reference electrode on the SPCE.We further tried to test the acute cytotoxicity of oxamic acid that is a famous inhibitor of anaerobic glycolysis. Each concentration of oxamic acid/HBSS was prepared and added into PC12 cell/HBSS suspension (6 x 105 cells /well) in a well (final concentration of oxamic acid: 0 to 20 mM) and the cell suspension was incubated for 1hour. And then potassium ferricyanide (final 500 mM) and 1-metoxy PMS (final 10 μM) solutions were added into the cell suspension. After incubation for more 10 minutes, a SPCE was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V. Results and c onclusions In the first stage it was demonstrated that our electrochemical interface with a new combination of double mediator system was very useful for very wide range of cell counting. The amperometric oxidation current showed very good linearity vs. the number of viable cells in the range from 7500 to 96400 cells/well (Data is not shown). The result strongly supported the combination of 1-methoxy PMS and ferricyanide is effective to monitor intracellular NADH level and to speculate the cell viability.Therefore, in the second stage, we applied our electrochemical interface to test the acute toxicity by oxamic acid. Figure 1 shows the decrease of oxidation current of the double mediator system after 1 hour oxamic acid treatment. The decrease of oxidation current was clearly dependent on the concentration of oxamic acid as shown in Figure 2. It indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by oxamic acid treatment. This result suggested that our electrochemical interface with a new double mediator system might be useful to evaluate acute cytotoxicity of drugs.

Zinc is one of the most important transition metal of physiological importance, existing primarily as a divalent cation. A number of sensors have been developed for Zn(II) detection. Here, we present a novel fluorescent nanosensor for Zn(II) detection using a derivative of 8-aminoquinoline (N-(quinolin-8-yl)-2-(3 (triethoxysilyl)propylamino)acetamide (QTEPA) grafted on silica nanoparticles (SiNPs). These functionalized SiNPs were used to demonstrate specific detection of Zn(II) in tris-HCl buffer (pH 7.22), in yeast cell (Saccharomyces cerevisiae) suspension, and in tap water. The silane QTEPA, SiNPs and final product were characterized using solution and solid state nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption spectroscopy, transmission electron microscopy, elemental analysis, thermogravimetric techniques, and fluorescence spectroscopy. The nanosensor shows almost 2.8-fold fluorescence emission enhancement and about 55 nm red-shift upon excitation with 330 ± 5 nm wavelength in presence of 1 μM Zn(II) ions in tris-HCl (pH 7.22). The presence of other metal ions has no observable effect on the sensitivity and selectivity of nanosensor. This sensor selectively detects Zn(II) ions with submicromolar detection to a limit of 0.1 μM. The sensor shows good applicability in the determination of Zn(II) in tris-HCl buffer and yeast cell environment. Further, it shows enhancement in fluorescence intensity in tap water samples.

O efeito de diferentes preparações de Saccharomyces cerevisiae foi avaliado sobre o desenvolvimento das doenças do morangueiro, como mancha-de-micosferela (Mycosphaerella fragariae), mancha-de-dendrofoma (Dendrophoma obscurans) e flor-preta (Colletotrichum acutatum) além da qualidade pós-colheita dos frutos. O trabalho foi realizado entre 2004 e 2005 na Universidade Tecnológica Federal do Paraná, Campus Dois Vizinhos. Os tratamentos consistiram de pulverizações semanais de cinco diferentes preparados a partir da levedura S. cerevisiae: suspensão com fermento biológico fresco comercial, suspensão de células de levedura, suspensão autoclavada de células, filtrado de cultura em meio líquido e Agro-MOS®, produto comercial formulado a partir da levedura, além da testemunha com água destilada e do tratamento controle com fungicidas. Nenhuma das preparações apresentou efeito contra a mancha-de-micosferela; preparações com a presença de células vivas e o produto Agro-MOS® apresentaram efeito contra mancha-de-dendrofoma; preparações com suspensão do produto comercial e filtrado de cultura líquida reduziram a incidência de flor-preta em flores e frutos. Preparações de S. cerevisiae com suspensão de células, suspensão autoclavada de células e filtrado de cultura líquida promoveram aumento na produtividade dos morangueiros que variou de 589,6 a 617,8 g planta-1. Preparações de S. cerevisiae, com presença de células vivas ou não, alteraram o metabolismo do morangueiro, aumentando a atividade das enzimas quitinase e glucanase, envolvidas na resistência sistêmica adquirida. Todos os tratamentos, com exceção do tratamento com suspensão autoclavada de células, reduziram a incidência de mofo-cinzento em pós-colheita de frutos.

A vibrating membrane bioreactor (VMBR): Macromolecular transmission—influence of extracellular polymeric substances

Application of audible sound to microbial cultures has the potential to enhance growth and reduce fermentation time, but the reported effects vary between studies. Many studies have used air-to-liquid sound application, which may result in changes to the sound properties due to it passing through various media. This study investigates the effect that the particle motion component of audible sound has on beer fermentations using linear actuators (LAT) that predominantly deliver the particle motion component of sound rather than the pressure component. The fermentation experiment was conducted twice independently to account for possible batch variation, referred to as experiment #1 and experiment #2. Standardized wort with Saccharomyces cerevisiae Safale US-05 (∼10 million cells/mL) was distributed into laminated bags (2L) and subjected to either continuous 800-2000Hz stimulation as the sound treatment delivered using LATs or no sound stimulation as a control treatment. Measures of wort gravity and yeast cells in suspension were used to monitor the progress of fermentation. Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was used to measure the abundance of volatile organic compounds (VOCs). During fermentation, the LAT treatment had a lower mean wort gravity at 24h (p<0.05) (experiment #1) and at 29-92h (experiment #2) compared to the control. The LAT treatment had higher mean number of yeast cells in suspension (p<0.05) at 24, 72, 92, 120, 144h (experiment #1) compared to the control. Similar results were obtained for experiment #2 but at 36 and 78h only. The fermentation ended 21-31h earlier in the LAT treatment than the control (p<0.05) in both experiment #1 (2.0 °P) and #2 (2.1 °P) with only subtle corresponding effects on the VOC profile. These results suggest that sound stimulation with LATs could be used to increase brewery efficiency by decreasing beer fermentation time without significantly influencing flavour.

Extracellular reduction of menadione and ferricyanide in yeast cell suspension

Discrimination of intra- and extracellular 23Na + signals in yeast cell suspensions using longitudinal magnetic resonance relaxography

Different characteristics between menadione and menadione sodium bisulfite as redox mediator in yeast cell suspension

Single cell studies and simulation of cell–cell interactions using oscillating glycolysis in yeast cells

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

Microfiltration of protein mixtures and the effects of yeast on membrane fouling

To develop a real-time in situ method to quantify loss of viability of Aureobasidium pullulans PRAFS8 cells attached to plasticized polyvinyl chloride (pPVC) with incorporated biocides, and to use the method to compare biocide efficacy in situ. A. pullulans PRAFS8, transformed with green fluorescent protein (GFP), was used to quantify the efficacy of a range of biocides incorporated into pPVC. Experimentally, it was found that a density of 1.53 x 10(6) yeast cells per cm(2) of pPVC was optimal as increasing the density of the yeast cells to 6.12 x 10(6) cm(-2) attached to pPVC containing the biocide 2-n-octyl-4-isothiazolin-3-one (OIT) decreased the rate of fluorescence loss. A strong positive correlation between fluorescence and viable yeast cell number was observed and fluorescence was used as a direct indicator of cell viability. The effectiveness of five commercial biocides, commonly incorporated into pPVC at their in-use concentrations, was tested against yeast cells attached to the pPVC surface. The loss of fluorescence and hence viability in situ was quantified using image analysis. The biocides N-(trichloromethylthio) phthalimide (NCMP), 10,10'-oxybisphenoxarsine (OBPA), OIT and 2,3,5,6-tetrachloro-4-(methylsulphonyl) pyridine (TCMP) caused complete loss of fluorescence within 30-50 h. In contrast the biocide dichloro-octyl-isothiazoline caused only 55 +/- 15% fluorescence loss after 50 h. Starvation of the yeast cells in suspension for 24 h prior to attachment reduced their initial sensitivity to OBPA, NCMP, OIT and TCMP by 15-20%, but eventually the fluorescence was also completely lost. The use of A. pullulans expressing cytosolic GFP enables the in situ quantification of loss of viability when cells are attached to pPVC with incorporated biocides. GFP fluorescence was used as a real-time indicator of cell viability and thus can be applied for direct quantification of the effectiveness of a broad range of biocides, incorporated into the polymer mass and used to protect a variety of plastics or other materials from microbial growth.

The surface of polytetrafluoroethylene (PTFE) was modified using plasma immersion ion implantation (PIII) with the aim of improving its ability to immobilize yeast. The density of immobilized cells on PIII-treated and -untreated PTFE was compared as a function of incubation time over 24 h. Rehydrated yeast cells attached to the PIII-treated PTFE surface more rapidly, with higher density, and greater attachment strength than on the untreated surface. The immobilized yeast cells were removed mechanically or chemically with sodium hydroxide and the residues left on the surfaces were analysed with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). The results revealed that the mechanism of cell attachment on both surfaces differs and a model is presented for each. Rapid attachment on the PIII-treated surface occurs through covalent bonds of cell wall proteins and the radicals on the treated surface. In contrast, on the untreated surface, only physisorbed molecules were found in the residue and lipids were more highly concentrated than proteins. The presence of lipids in the residue was found to be a consequence of damage to the plasma membrane during the rehydration process and the increased cell stress was also apparent by the amount of Hsp12 in the protein residue. The immobilized yeast cells on PIII-treated PTFE were found to be as active as yeast cells in suspension.

Yeast cells are capable of accumulation of various heavy metals, preferentially accumulating those of potential toxicity and also those of value. They retain their ability to accumulate heavy metals under a wide range of ambient conditions. In the present study it was shown that yeast cells in suspension accumulate heavy metal cations such as Cu2+, Co2+. The level of copper accumulation was dependent on the ambient metal concentration and was markedly inhibited by extremes of ambient pH. Temperature (5–40°C) and the presence of the alkali metal sodium had much smaller effects on the level of copper accumulation. This suggests that in waste-waters of pH 5.0–9.0, yeast biomass could provide an effective bioaccumlator for removal and/or recovery of the metal. During bioaccumulation and subsequent processes it is necessary to retain the biomass. It was shown in the present study that this could be achieved by cell immobilization. Immobilization allowed for complete removal of Cu2+, Co2+, and Cd2+ from synthetic metal solutions. The immobilized material could be freed of metals by use of the chelating agent ethylenediamine tetraacetic acid (EDTA) and recycled for further bioaccumulation events with little loss of accumulation capacity.