A rapid and simple spectroscopic method for the determination of yeast cell viability using methylene blue.

Determination of cell viability is important in various microbiological studies. The microscopic method, counting dead cells stained by methylene blue (MB), has often been used for the determination of viability, although it is not efficient for the measurement of a large number of samples. Alternatively, some spectroscopic methods have been proposed to avoid tedious cell counting. One of these proposed methods detects the decrease in MB absorbance in the supernatant of cell suspension, because dead cells incorporate MB more efficiently than viable cells. However, at present, this spectroscopic method is rarely used due to its low throughput. Therefore, we devised a small-scale, rapid and simple method by improving several points as follows. (1) The peak wavelength of MB absorbance, 665 nm, was used to detect MB efficiently at the microtube scale. (2) The composition of the MB solution was improved by adding trisodium citrate. (3) The reaction time was shortened. And (4) the concentration ranges of both MB and cells, with which absorbance is linearly related to cell viability, were determined. The improved method enabled us to evaluate the dose-dependent toxicities of alcohols, antifungal/antimalarial quinacrine, and UV-C irradiation. The results were compatible with those of conventional microscopic counting and colony formation. The method would be applicable to automated determination and to various organisms such as bacteria and filamentous fungi which are difficult to be counted microscopically.

Escaping from the Cutoff Paradox by Accumulating Long-Chain Alcohols in the Cell Membrane.

The mechanism for the cutoff, an activity cliff at which long-chain alcohols lose their biological effects, has not been elucidated. Highly hydrophobic oleyl alcohol (C18:1) exists as a mixture of monomers and aggregated droplets in water. C18:1 did not inhibit the yeast growth but inhibited the growth of the slime mold without a cell wall. C18:1 exhibited toxicity to the yeast protoplast, which was enhanced by polyethylene glycol, a fusogen. Therefore, direct interactions of C18:1 with the membrane are crucial for the toxicity. The cutoff alcohols, C14 and C16, also exhibited strong toxicity obeying the Meyer-Overton correlation, in intact yeast cells whose membrane growth was suppressed in water. Taken together, the cutoff is avoidable by securing sufficient accumulation of the wall-permeable monomers in the membrane, which supports the lipid theory. It would be important to distinguish the effective drug structure localizing in the membrane and deal with the amount in the membrane.

Antimalarial Quinacrine and Chloroquine Lose Their Activity by Decreasing Cationic Amphiphilic Structure with a Slight Decrease in pH.

Quinacrine (QC) and chloroquine (CQ) have antimicrobial and antiviral activities as well as antimalarial activity, although the mechanisms remain unknown. QC increased the antimicrobial activity against yeast exponentially with a pH-dependent increase in the cationic amphiphilic drug (CAD) structure. CAD-QC localized in the yeast membranes and induced glucose starvation by noncompetitively inhibiting glucose uptake as antipsychotic chlorpromazine (CPZ) did. An exponential increase in antimicrobial activity with pH-dependent CAD formation was also observed for CQ, indicating that the CAD structure is crucial for its pharmacological activity. A decrease in CAD structure with a slight decrease in pH from 7.4 greatly reduced their effects; namely, these drugs would inefficiently act on falciparum malaria and COVID-19 pneumonia patients with acidosis, resulting in resistance. The decrease in CAD structure at physiological pH was not observed for quinine, primaquine, or mefloquine. Therefore, restoring the normal blood pH or using pH-insensitive quinoline drugs might be effective for these infectious diseases with acidosis.

Physicochemical Solubility of and Biological Sensitivity to Long-Chain Alcohols Determine the Cutoff Chain Length in Biological Activity.

The cutoff phenomenon associated with the effectiveness of long-chain alcohols in the induction of anesthesia is also observed for various antimicrobial activities, although the mechanism has remained unknown for over eight decades. The minimum inhibitory concentrations at 25°C for budding yeast growth exponentially decreased with increasing chain length of n-alcohols (C2-C12), whereas alcohols ≥C13 lost the inhibitory effect. Thus, growth inhibition by n-alcohols obeys the Meyer-Overton correlation up to C12 and exhibits a cutoff phenomenon. The densities of n-alcohols are low, and the melting point and hydrophobicity increase with chain length. C13 and C14 inhibited yeast growth at 39.8°C, above their melting points. Alcohols ≤C14 inhibited thermophilic bacterial growth at 50°C, whereas C16 inhibited it at 67.5°C, above their melting points. Thus, the high melting points of long-chain alcohols contribute to the cutoff phenomenon. C14 did not effectively inhibit yeast growth in a static culture at 39.8°C, in contrast to a shaking culture, in which the low density-dependent concentration gradient was eliminated. The duration of the transient growth inhibition of yeast by C12 was prolonged by sonication, which prevented hydrophobic aggregation. Therefore, a nonuniform distribution owing to low density and high hydrophobicity contributes to the cutoff. C14 inhibited the growth at 25°C of the pdr1,3,5 mutant, defective in multidrug efflux pumps, whereas C12 did not inhibit the growth of yeast overexpressing PDR5, indicating that the sensitivity to long-chain alcohols contributed to the cutoff. A balance between the physicochemical solubility of and the biological sensitivity to long-chain alcohols determines the cutoff chain length.

Local Anesthetics and Antipsychotic Phenothiazines Interact Nonspecifically with Membranes and Inhibit Hexose Transporters in Yeast.

Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake, but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt(0) strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not the HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby mitigating the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects based on lipid theory.

Tetracaine, a local anesthetic, preferentially induces translational inhibition with processing body formation rather than phosphorylation of eIF2α in yeast.

It is unclear whether local anesthetics, such as tetracaine, and antipsychotics, such as phenothiazines, act on lipids or proteins. In Saccharomyces cerevisiae, these drugs inhibit growth, translation initiation, and actin polarization, and induce cell lysis at high concentrations. These activities are likely due to the cationic amphiphilic structure common to these agents. Although drug-induced translational inhibition is conserved in mammalian cells, other mechanisms, including the phosphorylation of eIF2α, a eukaryotic translational initiation factor, remain poorly understood. At a concentration of 10 mM, tetracaine rapidly inhibited translation initiation and lysed cells, whereas, at 2.5 mM, it slowly induced inhibition without lysis. The pat1 disruptant defective in mRNA decapping and the xrn1 disruptant defective in 5'-3' exoribonuclease were partially resistant to translational inhibition by tetracaine at each concentration, but the gcn2 disruptant defective in the eIF2α kinase was not. Phosphorylation of eIF2α was induced by 10 mM but not by 2.5 mM tetracaine, whereas processing bodies (P-bodies) were formed at 2.5 mM in Pat1-dependent and -independent manners. Therefore, administration of tetracaine inhibits translation initiation with P-body formation at both concentrations but acts via the Gcn2-eIF2α system only at the higher concentration. Because other local anesthetics and phenothiazines induced Pat1-dependent P-body formation, the mechanisms involved in translational inhibition by these cationic amphiphiles are similar. These results suggest that this dose-dependent biphasic translational inhibition by tetracaine results from an increase in membrane proteins that are indirectly inhibited by nonspecific interactions of cationic amphiphiles with membrane lipids.

Effects of elevated CO2 on levels of primary metabolites and transcripts of genes encoding respiratory enzymes and their diurnal patterns in Arabidopsis thaliana: possible relationships with respiratory rates.

Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates.

Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces cerevisiae.

Mitotic cyclin-dependent kinase (CDK) is activated by Cdc25 phosphatase through dephosphorylation at the Wee1-mediated phosphorylation site. In Saccharomyces cerevisiae, regulation of Mih1 (Cdc25 homologue) remains unclear because inactivation/degradation of Swe1 (Wee1 homologue) is the main trigger for G2/M transition. By deleting all mitotic cyclins except Clb2, a strain was created where Mih1 became essential for mitotic entry at high temperatures. Using this novel assay, the essential domain of Mih1 was identified and Mih1 regulation was characterized. Mih1(3E1D) with phosphomimetic substitutions of four putative PKC target residues in Mih1 had a reduced complementation activity, whereas Mih1(4A) with those nonphosphorylatable substitutions was active. The band pattern of Mih1 by SDS-PAGE was similar to that of Mih1(4A) only after inactivation of Pkc1 in a pkc1(ts) mutant. Over-expression of GFP-tagged Mih1 or GFP-Mih1(4A) accumulated as dot-like structures in the nucleus, whereas GFP-Mih1(3E1D) was localized in the cytoplasm. Over-expression of an active form of Pkc1 excluded GFP-Mih1 from the nucleus, but had minimal effect on GFP-Mih1(4A) localization. Furthermore, addition of ectopic nuclear localization signal to the Mih1(3E1D) sequence recovered complementation activity and nuclear localization. These results suggest that Mih1 is negatively regulated by Pkc1-mediated phosphorylation, which excludes it from the nucleus under certain conditions.

Nitrate addition alleviates ammonium toxicity without lessening ammonium accumulation, organic acid depletion and inorganic cation depletion in Arabidopsis thaliana shoots.

When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.

Structural analysis of compounds with actions similar to local anesthetics and antipsychotic phenothiazines in yeast.

Local anesthetics and antipsychotic phenothiazines cause a rapid shutdown of both actin polarization and translation initiation in yeast cells, like some environmental stresses. These compounds all have an amphiphilic structure, surfactant activity and the ability to lyse yeast cells. To elucidate the structures responsible for the shutdown activity and cell lysis, we investigated a variety of amphiphiles. In the hydrophobic region, the straight alkyl structure was sufficient for the shutdown of actin polarization and translational initiation. In the hydrophilic region of the straight alkyl compounds, cationic trimethyl ammonium (TMA) and non-ionic hydroxyl structure (alcohols) shut down both reactions, while an anionic structure, sulphate, with a long alkyl chain (≥C6) shut down actin polarization only. On the compounds that shut down both reactions, including the clinical drugs, TMA compounds and alcohols, the potencies of shutdown and lysis exponentially increased with increasing the number of carbons in the hydrophobic region, whereas safety was affected by the structures of both hydrophilic and hydrophobic regions. These results indicate that the yeast system can easily evaluate clinical drugs, and provide a structural basis for designing compounds to shut down intracellular reactions.

Ammonium-dependent respiratory increase is dependent on the cytochrome pathway in Arabidopsis thaliana shoots.

Oxygen uptake rates are increased when concentrated ammonium instead of nitrate is used as sole N source. Several explanations for this increased respiration have been suggested, but the underlying mechanisms are still unclear. To investigate possible factors responsible for this respiratory increase, we measured the O2 uptake rate, activity and transcript level of respiratory components, and concentration of adenylates using Arabidopsis thaliana shoots grown in media containing various N sources. The O2 uptake rate was correlated with concentrations of ammonium and ATP in shoots, but not related to the ammonium assimilation. The capacity of the ATP-coupling cytochrome pathway (CP) and its related genes were up-regulated when concentrated ammonium was sole N source, whereas the ATP-uncoupling alternative oxidase did not influence the extent of the respiratory increase. Our results suggest that the ammonium-dependent increase of the O2 uptake rate can be explained by the up-regulation of the CP, which may be related to the ATP consumption by the plasma-membrane H+ -ATPase.

Effects of AOX1a deficiency on plant growth, gene expression of respiratory components and metabolic profile under low-nitrogen stress in Arabidopsis thaliana.

Expression of alternative oxidase (AOX) and cyanide (CN)-resistant respiration are often highly enhanced in plants exposed to low-nitrogen (N) stress. Here, we examined the effects of AOX deficiency on plant growth, gene expression of respiratory components and metabolic profiles under low-N stress, using an aox1a knockout transgenic line (aox1a) of Arabidopsis thaliana. We exposed wild-type (WT) and aox1a plants to low-N stress for 7 d and analyzed their shoots and roots. In WT plants, the AOX1a mRNA levels and AOX capacity increased in proportion to low-N stress. Expression of the genes of the components for non-phosphorylating pathways and antioxidant enzymes was enhanced, but differences between WT and aox1a plants were small. Metabolome analyses revealed that AOX deficiency altered the levels of certain metabolites, such as sugars and sugar phosphates, in the shoots under low-N stress. However, the carbon (C)/N ratios and carbohydrate levels in aox1a plants were similar to those in the WT under low-N stress. Our results indicated that the N-limited stress induced AOX expression in A. thaliana plants, but the induced AOX may not play essential roles under stress due to low-N alone, and the C/N balance under low-N stress may be tightly regulated by systems other than AOX.

Environmental stresses and clinical drugs paralyze a cell.

Cells respond and adapt to various extracellular changes. Environmental stresses, such as high osmolarity and acute glucose deprivation, rapidly and transiently shut down translation initiation and actin polarization in the yeast Saccharomyces cerevisiae. Certain clinical drugs, such as local anesthetics and antipsychotic phenothiazines, and cationic surfactants also cause shutdowns similar to those triggered by environmental stresses. These compounds all have an amphiphilic structure, a cationic hydrophilic region, surfactant activity, and the ability to lyse yeast cells. Since low concentrations of these compounds shut down intracellular reactions in the absence of cell lysis, the compounds might change the state of the cell's membrane by intercalating into the membrane and thus generate signals for the shutdown, as do environmental stresses. The intracellular shutdowns caused by stresses might essentially be the same as the paralysis caused by clinical drugs at the cellular level.

Local anesthetics, antipsychotic phenothiazines, and cationic surfactants shut down intracellular reactions through membrane perturbation in yeast.

High osmolarity and glucose deprivation cause rapid shutdowns of both actin polarization and translation initiation in yeast. Like these stresses, administration of local anesthetics and of antipsychotic phenothiazines caused similar responses. All these drugs have amphiphilic structures and formed emulsions and permeabilized the cell membrane, indicating that they have the same features as a surfactant. Consistently with this, surfactants induced responses similar to those of local anesthetics and phenothiazines. Benzethonium chloride, a cationic surfactant, showed a more potent shutdown activity than phenothiazines, whereas SDS, an anionic surfactant, transiently depolarized actin without inhibiting translation initiation, suggesting that a cationic charge in the amphiphile is important to the shutdown of both reactions. The clinical drugs and the cationic surfactants at low concentrations caused shutdown without membrane permeabilization, suggesting that these compounds and stresses activate shutdown, via perturbation rather than disruption of the cell membrane.

LAS24/KOG1, a component of the TOR complex 1 (TORC1), is needed for resistance to local anesthetic tetracaine and normal distribution of actin cytoskeleton in yeast.

It is known that some local anesthetics inhibit the growth of budding yeast cells. To investigate the pathway of local anesthetics' action, we isolated and characterized mutants that were hyper-sensitive to tetracaine, and at the same time, temperature-sensitive for growth. They were collectively called las (local anesthetic sensitive) mutants. One of the LAS genes, LAS24, was found to be identical to KOG1, which had been independently discovered as a member of the TOR complex 1 (TORC1). Las24p/Kog1p is a widely conserved TOR binding protein containing the NRC domain, HEAT repeats and WD-40 repeats, but its function remains unknown. Like the tor mutants, the las24 mutants were found to have a defect in cell wall integrity and to show sensitivity to rapamycin. Furthermore, Las24p is required not only in TORC1-mediated (rapamycin-sensitive) pathways such as translation initiation control and phosphorylation of Npr1p and Gln3p, but also for the normal distribution of the actin cytoskeleton, which has been regarded as a TORC2-mediated event. Intriguingly, the temperature-sensitivity of the las24 mutant was suppressed by either activation of Tap42/PPase or by down-regulation of the RAS/cAMP pathway. Suppressors of the temperature-sensitivity of the las24-1 mutant were found not to be effective for suppression of the tetracaine-sensitivity of the same mutant. These observations along with the facts that tetracaine and high temperature differentially affected the las24-1 mutant suggest that Las24p/Kog1p is not a target of tetracaine and that the tetracaine-sensitive step may be one of downstream branches of the TORC1 pathway. Consistent with the broad cellular functions exerted by the TOR pathway, we found that Las24p was associated with membranes and was localized at vacuoles, the plasma membrane and small vesicles.

Simultaneous yet independent regulation of actin cytoskeletal organization and translation initiation by glucose in Saccharomyces cerevisiae.

Acute glucose deprivation rapidly but transiently depolarizes the actin cytoskeleton and inhibits translation initiation in Saccharomyces cerevisiae. Neither rapid actin depolarization nor translation inhibition upon glucose removal occurs in a reg1 disruptant, which is defective in glucose repression, or in the tpk1(w) mutant, which has weak cAPK activity. In the absence of additional glucose, recovery of either actin polarization or translation initiation relies upon respiration, the Snf1p protein kinase, and the transcription factors Msn2p and Msn4p. The readdition of glucose to glucose-starved cells causes a rapid recovery of actin polarization as well as translation initiation without respiration. These results indicate that the simultaneous regulation of actin polarization and translation initiation is divided into three reactions: 1) rapid shutdown depending on Reg1p and cAPK after glucose removal, 2) slow adaptation depending on Snf1p and Msn2p/4p in the absence of glucose, and 3) rapid recovery upon readdition of glucose. On glucose removal, translation initiation is rapidly inhibited in a rom2 disruptant, which is defective in rapid actin depolarization, whereas rapid actin depolarization occurs in a pop2/caf1 disruptant, which is defective in rapid inhibition of translation initiation. Thus, translation initiation and actin polarization seem to be simultaneously but independently regulated by glucose deprivation.

Transient inhibition of translation initiation by osmotic stress.

Cells respond and adapt to changes in the environment. In this study, we examined the effect of environmental stresses on protein synthesis in the yeast Saccharomyces cerevisiae. We found that osmotic stress causes irreversible inhibition of methionine uptake, transient inhibition of uracil uptake, transient stimulation of glucose uptake, transient repression of ribosomal protein (RP) genes such as CYH2 and RPS27, and the transient inhibition of translation initiation. Rapid inhibition of translation initiation by osmotic stress requires a novel pathway, different from the amino acid-sensing pathway, the glucose-sensing pathway, and the TOR pathway. The Hog1 MAP kinase pathway is not involved in the inhibition of either methionine uptake or translation initiation but is required for the adaptation of translation initiation after inhibition and the repression of RP genes by osmotic stress. These results suggest that the transient inhibition of translation initiation occurs as a result of a combination of both acute inhibition of translation and the long-term activation of translation by the Hog1 pathway.