The influence of tetraphenylborates (hydrophobic anions) on yeast cell electro-rotation

The action of a series of tetraphenylborate ion (TPB) derivatives on yeast cells was studied by electro-rotation of the pre-treated cells. TPB derivatives in which all four phenyl groups were substituted with fluorine, chlorine or trifluoromethyl were much more toxic than the unsubstituted compound, the effect increasing dramatically with increasing size of substituents. These observations suggest that the toxicity of these hydrophobic ions is determined mainly by their size and possibly also by the chemical inductivity of their substituent groups. The order of the toxicities of these ions was in fair agreement with literature values for their translocation rates across artificial bilayers. Incubation times of 3 h were used as standard, longer incubations (up to 48 h) showed that the number of cells affected by low doses of TPB increased with the logarithm of time after the first hour of incubation. Although measurements of the percentage of cells showing co-field rotation showed that controls were not adversely affected by incubations as long as 9 h, rotation spectra showed that some cells suffer loss of internal conductivity during extended incubations. Decrease of the pH of the incubation medium, or inclusion of high concentrations of NaCl or KCl, potentiated the effects of these hydrophobic ions. The toxicity developed slowly, and the sensitivity of the assay was only very weakly dependent on the cell suspension density.     

31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl2 or Hg(NO3)2 and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 microS/cm at 10 microS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 microS/cm) the change in the rotational spectrum was usually 'all-or-none', whereas at 200 microS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K+ release. A rapid reduction of the effects of Hg(II) was seen when 3-30 mM K+ (or Na+) or when 1 mM Ca2+ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.     

The effect of mercuric salts on the electro-rotation of yeast cells and comparison with a theoretical model

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl₂ or Hg(NO₃)₂ and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 μS/cm at 10 μS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 μS/cm) the change in the rotational spectrum was usually ‘all-or-none’, whereas at 200 μS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K⁺ release. A rapid reduction of the effects of Hg(II) was seen when 3–30 mM K⁺ (or Na⁺) or when 1 mM Ca²⁺ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.     

电旋转技术用于少根根霉孢囊孢子介电性的测定

利用电旋转技术研究了少根根霉（Rhizopus arrhizus）孢囊孢子的电旋转谱,在发现另外两个峰的同时,在场频500Hz附近又发现了1个明显的正旋转峰。同时还发现萌发孢囊孢子与休眠孢囊孢子的电旋转谱有明显的差别,其负旋转峰值明显地小于休眠孢囊孢子的负旋转峰值。借助经修改的椭圆细胞双壳模型（two-shell model) 模拟出了原生质膜和原生质的电常数,结果表明萌发孢子的原生质膜和原生质的电导率较休眠孢子的相应电导率有明显增大。说明电旋转技术能够反映活细胞的生理变化。     

Spinning response of yeast cells to rotating electric fields

The frequency-dependent rotation or spinning motion of yeast cells subjected to a fourpole rotating electric field was examined over a very wide frequency range (500 Hz to 500 MHz). In the lower frequency range (500 Hz – 700 KHz) the yeast cells were observed to spin in a direction counter to the applied field, with a small peak at about 600 Hz and a more pronounced one at 20 KHz. For frequencies above 700 KHz the spinning of the cells switched direction from counter-field to co-field, with a maximum in the rotation rate at about 70 MHz and a subpeak at 20 MHz. The rate was also observed to exhibit a square dependence on the magnitude of the applied rotating field.     

Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine

A mildly acidic pH in the lumen of the small intestine markedly enhances the transport of folate. This study investigated the relationship between pH and the affinity between folic acid and the apical membrane transporter using brush border membrane vesicles from rat jejunum and differentiated monolayer cultures of the colon carcinoma cell line, CaCo-2. Uptake studies with BBMV were conducted at folic acid concentrations of 0.1 to 50 mumol/l, conditions which were suitable for analyzing uptake data based on the Michaelis-Menten equation modified to include a nonsaturable component. These analyses yielded apparent Km values of 0.6 and 12.3 microM at pH 5.5 and pH 7.4, respectively (P less than 0.05). Values for Vmax were lower at pH 5.5 than at pH 7.4 (0.8 vs. 1.6 pmol/mg protein per 10 s, P less than 0.05). The studies with CaCo-2 cells employed folic acid concentrations of 0.1 to 5 mumol/l. Under these conditions the apparent Km for folic uptake was lowest at pH 6.0, where the Km was 0.7 mumol/l. The apparent Km increased sharply as a neutral pH was approached; reaching a value of 13.9 mumol/l at pH 7.1. These data suggest that the prominent pH effect on intestinal folate transport is, in part, explained by an increased affinity of the folate substrate for its membrane transporter.     

Electrorotation measurements of diamide-induced platelet activation changes.

Electrorotation is a special dielectric spectroscopic technique capable of measuring the polarizability of single platelets. The rotational speed of the particles is recorded as a function of the frequency of the applied rotating electric field. As previously shown, the speed of electrorotation in the range of the first characteristic frequency (anti-field rotation) decreased upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide incubation induced morphological changes in control platelets. These changes were accompanied by a shift of the first characteristic frequency of electrorotation toward higher values and a parallel increase of the anti-field rotation. This was explained by a decrease of membrane conductivity and by the changed polarizability of platelet interior due to the observed internal platelet structure changes. Diamide inhibited activation assessed by both electrorotation and TMA-DPH fluorescence in the case of all activators except the ionophore A 23187. Because diamide largely inhibited the A 23187-induced serotonin release, it was concluded that, despite the diamide treatment, the direct increase of cytoplasmic Ca2+ was still able to induce membrane conductivity changes accessible by electrorotation, but this did not complete the final release step of the activation process.     

HERBICIDES THE RELATIVE TOXICITY OF NITROPHENOLS TO VARIOUS ORGANISMS

The effect of increasing degrees of nitration on the toxicity of phenols was assessed by measuring the toxicity of phenol, o - and p -nitro phenols and 2:4-dinitro phenol with several different test organisms. Toxicity levels were determined by the concentration required in each case to bring about the following responses: (i) to halve the rate of radial growth of Trichoderma viride at pH 3, (ii) to halve the respiration rate of yeast at pH 3, (iii) to halve the rate of frond multiplication of Lemna minor at pH 5·4, (iv) to bring about 50% mortality of seedlings of Brassica alba in a spraying test and finally (v) to halve the rate of respiration of leaf disks of B. alba vacuum-infiltrated with the toxic solution at pH 3.     

Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

The stoicheiometry of the absorption of protons with phosphate and L-glutamate by yeasts of the genus Saccharomyces.

1. A study was made of the pH changes occurring when 0.1-4 mumol of glutamate, phosphate and certain phosphate esters was added at about pH 4.8 to washed cell preparations (50 mg dry wt.) of strains of Saccharomyces. The system also contained deoxyglucose and antimycin to inhibit energy metabolism and so prevent proton ejection from the yeast. 2. A strain of Sacc. carlsbergensis was grown in a chemostat with a limiting supply of phosphate in order to enhance the subsequent rate of phosphate transfer into the yeast. These preparations absorbed 0.2 mumol of phosphate with about 3 equiv. of protons/mol of phosphate. The charge balance was maintained by the efflux of 2 equiv. of K-+ from the yeast. 3. Larger amounts of phosphate were absorbed with fewer proton equivalents. 4. Arsenate and phosphate caused similar pH changes. 5. Glucose 6-phosphate, ATP and certain order phosphate esters each initiated a rise in pH, possibly because hydrolytic extracellular enzymes released phosphate that was subsequently absorbed. 6. Four strains of yeast were grown with glutamate as principal source of nitrogen. Each absorbed extra protons in the presence of L-glutamate. 7. One of them, a strain of Sacc. cerevisiae, absorbed 0.2 mumol of glutamate with 3equiv. of protons/mol of glutamate, and in these circumstances 1-2 equiv. of K-+ left the yeast cells. 8. The role of ionic gradients in the transport of these anions is discussed.     

The effect of extracellular pH on radiosensitization by misonidazole and acidic or basic analogues

The effect of extracellular pH (pHe) on the radiosensitization of hypoxic Chinese hamster V79 cells in vitro by the 2-nitroimidazole, misonidazole, and analogues substituted with basic or acid functions has been studied. Misonidazole (1 mmol dm-3) gave an enhancement ratio (e.r.) of 1.6 which remained unchanged over the pHe range of 3.8-9.5. Control hypoxic survival curves in the absence of sensitizer also remained essentially unchanged over this pHe range. These results contrast with those seen for 0.1 mmol dm-3 Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol), a base with pKa = 8.9): the ER increased from 1.4 to 2.1 as pHe increased from 5.6 to 8.4. However, with the weaker bases, Ro 03-8800 and nimorazole (morpholino derivatives with pKa = 6.3 and 5.2 respectively) the e.r. remained constant over a wide pHe range. Nitroimidazoles substituted with acidic functions gave decreasing sensitization with increasing pHe. For azomycin (pKa = 7.2) at 1 mmol dm-3 the e.r. decreased from 1.9 at pHe 4 to 1.0 at pHe 9. The effect of the proton conductor carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 10 mumol dm-3) on radiosensitization by Ro 03-8799 (0.1 mmol dm-3) and misonidazole (1.0 mmol dm-3) was also studied. At pHe 6.67 the e.r. for Ro 03-8799 was increased from 1.36 to 1.76 by the presence of CCCP, whereas at pHe 7.33 the e.r. was unchanged. In contrast the e.r. for misonidazole was unchanged at pHe 6.65 and 7.33. These results are consistent with pH differentials across the cell membrane creating intracellular:extracellular concentrations gradients for radiosensitizers with acidic or basic functions.     

Inhibition of (Na,K)-ATPase by tetravalent vanadium

Vanadyl, the tetravalent state of vanadium and a divalent cation, VO2+, was a relatively powerful inhibitor of highly purified membrane-bound sodium and potassium ion transport adenosine triphosphatase. The sensitivity of the ATPase activity to vanadyl characteristically correlated positively with the specific activity of the enzyme preparation. Inhibition ranged from nearly complete inhibition at less than 5 microM vanadyl for some of the purest fractions (specific activity approximately 45 mumol/min/mg of protein) to no observable inhibition at 300 microM vanadyl in one crude preparation of the enzyme with a specific activity of 10 mumol/min/mg of protein. The level of free vanadyl was reduced by incubation with these membranes, but this reduction was not sufficient to account for the low sensitivity to vanadyl observed in crude preparations. A reduction in specific activity by partial inactivation of a sensitive preparation by treatment with FeCl3 and ascorbate reduced its sensitivity to vanadyl. Anionic ligands of the enzyme, vanadate or ATP, increased the rate of recovery from inhibition after chelation of free vanadyl. At pH 6.1, the inhibition was characteristically fully reversible (t1/2 approximately 10 min), whereas at pH 8.1 it was stable for hours. The degree and stability of enzyme inhibition by vanadyl increased for several hours during incubation of the vanadyl-enzyme mixture, and at pH 6.1 the properties of the inhibitor itself also changed with time. Preincubation of the ion at that pH for 5 h before addition of the enzyme produced a more stable inhibition. The time- and pH-dependent changes in the degree and stability of enzyme inhibition probably relate to the complex chemistry of the vanadyl ion in solution.     

Electrorotation of axolotl embryos

The frequency dependent dielectric properties of individual axolotl embryos (Ambystoma mexicanum) were investigated experimentally utilizing the technique of electrorotation. Individual axolotl embryos, immersed in low conductivity media, were subjected to a known frequency and fixed amplitude rotating AC electric field and the ensuing rotational motion of the embryo was monitored using a conventional optical microscope. None of the embryos in the pregastrulation or neurulation stages of development exhibited any rotational motion over the field frequency range (10 Hz-5 MHz). Over the same frequency range, the embryos in the gastrulation stage of development exhibited both co-field and counterfield rotation over different ranges of the applied field frequency. Typically, the counterfield rotation exhibited a peak in the rotation spectrum at similar 1 KHz while the co-field peak was located at similar 1-2 MHz. The rotational spectral data was analyzed using a multishelled spherical embryo model to determine the electrical character of embryos during the early development stages (Stages 5-16; i.e., 16 cell through open neural plate stages).     

Production of fructo-oligosaccharides from molasses by Aureobasidium pullulans cells

Three different strains of Aureobasidium pullulans were grown in batch cultures to compare their abilities for the production of fructo-oligosaccharides. Specific intracellular enzyme activity was the highest with strain KCCM 12017 and enzyme production was closely coupled to growth. Using A. pullulans cells, 166 g/l fructo-oligosaccharides was produced from 360 g/l molasses sugar as sucrose equivalent at 55 degrees C and pH 5.5 after 24 h incubation.     

Determination of buffering capacity of rat myocardium during ischemia

To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.     

Lipid peroxides and atherosclerosis.

Plasma lipid peroxide concentrations were measured in 100 patients with occlusive arterial disease proved angiographically (50 patients with ischaemic heart disease, 50 with peripheral arterial disease) and compared with values in 75 control patients with no clinical evidence of atherosclerosis. Lipid peroxide concentrations were significantly higher in patients both with ischaemic heart disease (median 4.37 mumol/l (interquartile range 3.85-5.75 mumol/l); p less than 0.001) and with peripheral arterial disease (median 4.37 mumol/l (3.88-5.21 mumol/l); p less than 0.001) than in controls (median 3.65 mumol/l (interquartile range 3.29-3.89 mumol/l). Overall there was a significant but weak correlation between plasma lipid peroxide and plasma triglyceride concentrations (rs = 0.25; p less than 0.001) but not between plasma lipid peroxide and plasma total cholesterol concentrations. Furthermore, hypertension, obesity, diabetes, smoking, positive family history, and intake of beta blockers and thiazide diuretics were not associated with significant differences in lipid peroxide values. This study provides clinical support to experimental data indicating that peroxidised lipids may be important in atherogenesis and its complications and also suggests that peroxidised lipids may provide an index of the severity of atherosclerosis.     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

The liver is an organ site for the release of inosine metabolized by non-glycolytic pig red cells

The metabolic energy source used by the pig red cell, which is unable to metabolize blood-borne glucose, was examined. Potential physiological substrates include adenosine, inosine, ribose, deoxyribose, dihydroxyacetone and glyceraldehyde, of which inosine was previously implicated. A net ATP synthesis by red cells occurs during in situ perfusion through the adult miniature pig liver. HPLC analysis of the perfusate revealed the presence primarily of inosine and hypoxanthine. Inosine production by the liver was 0.015 mumol/g per min. Moreover, red cells maintain ATP when suspended in a balanced salt medium during a 6 h incubation at 38 degrees C, in which inosine is continuously infused to give an external concentration of no more than 3 mumol/l, mimicking its plasma level. Inosine consumption under these infusion conditions was 56 nmol/ml cell per h, which is two orders of magnitude lower than when inosine is present in millimolar concentration. The total red cell inosine consumption of 9.63 mumol/h is much less than the total liver inosine production of 212 mumol/h. These findings suggest that the liver is an organ site elaborating inosine, and that maintenance of a 3 mumol/l inosine in plasma is sufficient to meet the energy requirements of the pig red cells.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.