Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato

Potato tuber formation starts with the stolon swelling and is regulated by jasmonates. The cascade of events leading to tuber formation is not completely understood. The aim of this study was to evaluate phospholipid composition and phospholipase activities during four stages of stolon-to-tuber transition of Solanum tuberosum L., cv. Spunta, and involvement of phosphatidic acid (PA) in stolon cell expansion during early stages. Effects of jasmonic acid (JA) treatment on phospholipid content and activation of phospholipase D (PLD) (EC 3.1.4.4) and phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP₂-PLC) (EC 3.1.4.3) were studied in the early stages (first stage, hooked apex stolon; second stage, initial swelling stolon) of tuberization. All the phospholipid species identified, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PA, and cardiolipin (CL), decreased as tuber formation progressed. PLD and PLC were activated in control tissues at an early stage. JA treatment caused a decrease of PC and PS in first stage stolons, accumulation of PA in second stage stolons, and modification of PLD and PLC activities. PA increased stolon cell area in the first and second stages. These findings indicate that phospholipid catabolism is activated from the early stages of tuber formation, and that JA treatment modifies the pattern of phospholipid (PC, PS, and PA) composition and phospholipase (PLD and PLC) activity. These phospholipids therefore may play a role in activation of an intracellular mechanism that switches the developmental fate of stolon meristem cells, causing differentiation into a tuber.     

The anti-tumor alkylphospholipid perifosine is internalized by an ATP-dependent translocase activity across the plasma membrane of human KB carcinoma cells

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER^R clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of ¹⁴C-perifosine at both 37 °C and 4 °C, and no signs for active efflux of the drug. KB PER^R clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of ¹⁴C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER^R clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.     

Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome‐wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen‐overrepresented PLDδ class. Combining live‐cell imaging, gene overexpression, lipid‐binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane‐bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.     

The anti-tumor alkylphospholipid perifosine is internalized by an ATP-dependent translocase activity across the plasma membrane of human KB carcinoma cells

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER(R) clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of (14)C-perifosine at both 37 degrees C and 4 degrees C, and no signs for active efflux of the drug. KB PER(R) clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of (14)C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER(R) clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.     

Phospholipase D and phosphatidic acid mediate heat stress induced secondary metabolism in Ganoderma lucidum

Phospholipid‐mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry‐based analysis to investigate HS‐induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld‐silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS‐activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS‐induced secondary metabolism in G. lucidum. Exogenous 1‐butanol, which decreased PLD‐mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld‐silenced strains partly blocked HS‐induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS‐induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.     

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.     

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7–20% and and HC2:11–25%) they represented the bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, fore.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation     

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml⁻¹) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml⁻¹, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.     

Encephalitozoon cuniculi: Quantitation of Parasites and Evaluation of Viability1

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment

A rapid method for the direct measurement of viable and dead adhering diatoms was developed using a fluorescent dye, TO-PRO-1 iodide. By staining the marine diatom, Nitzchia closterium, with TO-PRO-1 iodide, viable and dead cells were identified as red and yellow cells respectively, under an epifluorescence microscope employing blue excitation. Only dead cells were stained with TO-PRO-1 iodide. Viable cells were observed as red because of autofluorescence arising from intracellular chlorophyll, whereas dead cells were observed as yellow because of the fluorescence of TO-PRO-1 iodide. The percentage of TO-PRO-1-iodide-stained was correlated with the percentage of dead cells in N. closterium cells exposed to heat (60 °C, 15 min). Other microalgae containing intracellular chlorophyll could be also distinguished as viable or dead cells by this fluorometric staining method. This method was applied for the assessment of N. closterium cells killed by the electrochemical treatment and used to monitor biofouling populations and their viability directly on the electrode surface. When 1.0 V was applied against a saturated calomel electrode, 99% of the cells attached to graphite electrode were killed in 1 h. Received: 7 August 1998 / Received revision: 16 October 1998 / Accepted: 7 November 1998     

Two-step cell-death kinetics in vitro during cis-platinum, hydroxyurea and mitomycin incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). The kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. The subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Amiodarone inhibits multiple drug resistance in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.     

Two-Step Cell-Death Kinetics In Vitro During Cis-Platinum, Hydroxyurea and Mitomycin Incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). the kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. the subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water‐replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water‐limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water‐limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water‐replete and solute stress conditions. There was greater spatial stratification of dead cells under water‐limiting than water‐replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water‐limiting conditions, ROS accumulation is greater in an ΔalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration‐mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water‐replete and water‐limiting conditions.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Biogenesis of mitochondria

Summary The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria.The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

Mastoparan analogues stimulate phospholipase C- and phospholipase D-activity in Chlamydomonas: a comparative study

The G-protein activator mastoparan and its analogues are becoming popular tools for studying signalling in plants. Therefore the abilities of mastoparan, mas7, mas8, and mas17 to activate phospholipase C (PLC), PLD and to induce the deflagellation response in Chlamydomonas moewusii Gerloff were compared. The aim was to test whether their relative potencies in a plant system resemble those reported for bovine brain Go and Gi, as is generally assumed, and to determine at which concentrations cells become permeabilized, a known effect of higher concentrations. The concentrations at which 50% deflagellation was induced, were 2.0 M mastoparan, 3.0 M mas8, 3.6 M mas7, and 5.8 M mas17. Similar activities were found for the production of phosphatidic acid, which is the result of the combined activities of PLD and PLC (together with diacylglycerol kinase). PLD activity alone was measured in vivo by its ability to phosphatidylate n-butanol. Surprisingly, the concentrations that stimulated maximum activity were about 10-fold lower (1 M) than those that stimulated maximum PLC activity (10 M). Mas17 was an exception with both maxima above 10 M. All the compounds except mas17 permeabilized C. moewusii cells. The concentrations at which 50% of the cells were permeabilized to Evan's blue were 7.4 M mas8, 16.0 M mas7 and 22.4 M mastoparan. In conclusion, only mastoparan itself and the least active analogue mas17 induced maximum deflagellation, PLC and PLD activities without permeabilizing the cells.Keywords: Chlamydomonas, deflagellation, mastoparan, phospholipases C and D, phospholipid metabolism      

Isolation of d-Pimaric and Dehydroabietic Acids from Pine Oleoresin as Antinicotinic Substances

Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.