Effect of the Hsp90 modulators on the heat-shock response in eukaryotic cells.

The possible role of the heat-shock protein 90 (Hsp90) complex on the heat-shock (HS) response in yeast using the Hsp90 inhibitors geldanamycin (GA) and 17-allylamino-17-demethoxygeldanamycin (AAG), and prednisolone and 17beta-estradiol as modulators was investigated. Following long- or short-term administration of the drugs, either alone or in combination, the response was determined as cell viability and growth after exposure to HS. Upon short-term preconditioning, both Hsp90 inhibitors conferred cycloheximide-dependent thermal resistance to the yeast cultures, while upon long-term treatment the induction of thermotolerance was confined only to AAG. Co-administration of prednisolone or 17beta-estradiol failed to significantly alter the response to Hsp90 inhibitors. However, since short-term incubation with prednisolone alone induced thermotolerance, increased the budding cell fraction and tended to reduce the adaptive response to GA, its effect on GA-induced thermotolerance is not yet explained. Generally, GA and AAG showed a comparable short-term action but a different long-term effect on the HS response in yeast; this response was not related to any regulation by prednisolone or 17beta-estradiol (while 17beta-estradiol was unable to modify the response, the action of prednisolone in both the stress response and the cell cycle was equivocal).

The heat shock response is dependent on the external environment and on rapid ionic balancing by pharmacological agents in Saccharomyces cerevisiae.

AIMS: To investigate whether non-preconditioned yeast cells survive under heat shock, when placed in growth medium originated from protected cells and to provide insights into the ionic contribution in the response. METHODS AND RESULTS: The heat shock response was investigated by determining cell viability following exposure of yeast cells to 53 degrees C for 30 min, either in the absence or presence of drugs. Preconditioning was performed by incubating the cultures at 37 degrees C for 2 h. Under heat shock, non-preconditioned cell survival was significantly enhanced by the presence of the cell-free supernatant of resistant cultures. Addition of omeprazole or tetraethylammonium ions during the heat shock resulted in similar increases. Neither amiodarone nor mepivacaine showed any analogous effect. Omeprazole enhanced survival when added before the heat shock, while amiodarone exhibited a cytocidic action. CONCLUSIONS: Rapid balancing of ions may contribute to cell survival during heat shock, while survival under mild stress could probably be co-ordinated by additional events. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence is provided for the implication of the external environment and ionic homeostasis in the survival of yeast cells under unfavourable environmental conditions. This knowledge may be of importance in controlling both fermentation and therapeutic approaches.

Induction of morphological alterations by antineoplastic agents in yeast.

Saccharomyces cerevisiae was used as an alternative experimental model in order to investigate the effects of antineoplastic agents on eukaryotic cells. After being exposed to the most common clinically used antineoplastic agents, yeast cells were examined under the light microscope. Folate and pyrimidine antagonists, platinum derivatives, mitomycin C, actinomycin D and bleomycin induced alterations in yeast cellular morphology, which were not observed following treatment with drugs belonging to any category other than the antineoplastics, leading to the suggestion that these alterations could potentially be used as an experimental tool in pre-screening for new chemotherapeutic leads.

Cross-talk between cellular stress, cell cycle and anticancer agents: mechanistic aspects.

Environmental conditions such as temperature, radiation, hypoxia, nutrients and drugs stimulate the adaptive sensory and signaling machinery of the cell. This stress response may influence cell cycle regulation, cellular differentiation, oncogenic transforma-tion, cell survival and apoptosis. The impact of the cytoprotective reprogramming in cancer pharmacology is presented by the recent extensive literature regarding the interplay between stress tolerance and anticancer drug effectiveness and resistance, relying on the dominating intrinsic pathways, which are simultaneously activated and regulate the death process either positively or negatively. This review presents the data that argue for the emergence of either common or specific mechanisms depending on the type, duration and severity of stress in all eukaryotic organisms from yeast to mammals. The understanding of the complexity and the balance between noxious and protective signal transduction pathways would contribute to a more explicit evaluation of the current therapeutic regiments and to the development of new leads targeting malignancy.

Changes in histamine content following pharmacologically-induced mast cell degranulation in the rat conjunctiva.

Compound 48/80 was applied into one eye of male Wistar rats and a drop of vehicle into the contralateral eye. Another group of rats received sodium cromoglycate in both eyes every 6 h for a period of 48 h. One eye was challenged with compound 48/80 30 min after the end of treatment with sodium cromoglycate. The eyes were monitored clinically and the histamine content of the conjunctiva was determined fluorometrically. The basal histamine levels in rat conjunctival homogenates were quantified. Pharmacologically-induced mast cell degranulation by a single application of 0.1 g ml(-1)of compound 48/80 resulted in significant decreases of conjunctival histamine levels 1, 12 and 24 h after challenge. Sodium cromoglycate prevented the effect of compound 48/80 when administered into the eye prior to the challenge with the non-immunogenic histamine releaser. Upon termination of the application, the membrane stabilizer was unable to reverse the reduced histamine levels in the conjunctival homogenates.

Anticancer drugs as inducers of thermotolerance in yeast.

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53 degrees C. The cells were previously grown in the presence of potential stressors (anticancer drugs; e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.

Molybdate induces thermotolerance in yeast.

Application of a mild heat pretreatment, performed by shifting cells from 27 degrees C to 37 degrees C led to the protection of yeast cells from death due to a subsequent extreme heat shock at 53 degrees C. The presence of cycloheximide inhibited this induction of thermotolerance, indicating the involvement of de novo protein. The phosphatase inhibitor sodium molybdate induced thermotolerance to the non-pretreated yeast cells. This induction of thermotolerance did not seem to depend upon de novo protein synthesis. Thus, acquisition of thermotolerance in yeast may involve a number of cellular mechanisms depending on the conditions the organism encounters at any particular time.

Effects of chromatin function inhibitors on yeast whole cells and spheroplasts.

Saccharomyces cerevisiae has long been used as an alternative experimental model in the study of cancer and anticancer drug action. Although this simple eukaryote has provided useful information, its value as an experimental model is often controversial due to the presence of the cell wall -- a cellular structure which is absent in higher eukaryotes. The aim of this study was to investigate the possible involvement of the cell wall in the ineffectiveness of some anticancer drugs in yeast, by enzymatic removal of the cell wall. The effects of exposing whole-cell cultures and spheroplasts to chromatin function inhibitors for 22 hours were investigated. Vinblastine, etoposide and paclitaxel had no cytotoxic effects on whole-cell cultures either with or without the addition of verapamil. The growth profiles of yeast spheroplasts following drug exposure were similar to those observed in whole cells. These data demonstrate that the resistance of the lower eukaryote to these drugs was not overcome by the enzymatic removal of the cell wall.

Effect of antineoplastic agents on non proliferating yeast cells: a possible membrane effect of doxorubicin.

The effect of antineoplastic agents on non proliferating cells of Saccharomyces cerevisiae was investigated. Non growing populations were obtained by suspending cells in saline or H2O and survival rates were determined after exposing the cells to various commercially available agents for five hours. The only agent found to be effective was Doxorubicin which reduced survival rates to less than 5% (p < 0.001). The action of this drug could be detected in only 4 minutes and was not concentration dependent, therefore it is probable that DNA damage is caused mostly by oxygen free drug radicals. Furthermore, our observations strongly imply the damage of cellular membranes is an alternative reason for cell death, with phosphatidyl-inositol being the most probable candidate target for the drug.

Antiphage activity in extracts of plants growing in Greece.

Crude aqueous extracts of 255 plant taxa growing in various areas of Greece were screened for antiphage activity against 6 bacteriophages (T1, T2, T4, T7, MS 2 and ФX 174). This laboratory model has been used for the initial detection of antiviral and/or antineoplastic activity. Thirty eight extracts showed antiphage activity against one or more of the bacterial viruses used. When the active extracts were tested in the presence of Tryptone Soya Broth, part of the antiphage activity remained in 7 extracts, whereas when tested in the presence of fresh human plasma, the antiphage activity was abolished, possibly due to the precipitation of the active compounds by tannins or related substances.

Effect of calcium channel blockers on the action of various antitumour agents in the yeast Saccharomyces cerevisiae.

The cytotoxic effects of a 22 h treatment with four antineoplastic agents in the yeast Saccharomyces cerevisiae ATCC 2366 were investigated. Two agents, doxorubicin and 5-fluorouracil (5-FU), were effective in decreasing the colony-forming ability of yeast cells. Following examination under the light microscope, the effect of doxorubicin appeared to be, at least partially, due to killing of yeast cells whereas the effect of 5-FU was rather due to changes in cell structure leading to abnormal bud formation. For amsacrine (AMSA) and melphalan, cytotoxicity was totally absent. In the presence of diltiazem the above described effects were not significantly changed. When verapamil was added in the culture medium the cytotoxic activity of doxorubicin and 5-FU did not change. However, following treatment with AMSA in combination with verapamil, cell survival was significantly decreased whereas the presence of verapamil increased the yeast survival which was observed after melphalan treatment.

Response of Saccharomyces cerevisiae strains to antineoplastic agents.

The effect of several antineoplastic agents on Saccharomyces cerevisiae strains has been investigated. Minimum inhibitory concentration (MIC), minimum cytotoxic concentration (MCC) and median effective concentration (EC50) were determined to identify strains with inherent sensitivity to the agents tested. Several strains proved to be sensitive to the antimetabolites 5-fluorouracil and methotrexate as well as to doxorubicin and cis-platine. On the contrary m-amsacrine, procarbazine, vinca alcaloids, melphalan and hydroxyurea were inactive at concentrations up to 400 micrograms ml-1. The strain ATCC 2366, the most relatively sensitive to the agents tested, was used for studying the effect of treatment duration and of drug concentration on cell survival. Methotrexate and cis-platine, which according to MIC and MCC tests seemed ineffective for this strain, reduced survival significantly after 6 h of treatment. A correlation of the shape of the survival curves with MIC and MCC values was attempted.

Involvement of potassium ions in the action of various antineoplastic drugs on the growth of Saccharomyces cerevisiae.

The involvement of potassium ions in the action of some antineoplastic drugs on the growth of Saccharomyces cerevisiae was studied by incubating yeast cells in the presence of drugs at various concentrations and KCl at concentrations of 50 mmol l-1 and 100 mmol l-1. The presence of 6.25-50 micrograms ml-1 amsacrine or melphalan alone in the culture medium had no significant effect on yeast growth. Addition of KCl significantly increased the sensitivity to these drugs. On the contrary, incubation of yeast cells with KCl had no effect on the cytotoxic action of doxorubicin, methotrexate or 5-fluorouracil.

Synthesis and antimicrobial properties of 2H-pyran-3(6H)-one derivatives and related compounds.

The synthesis of several derivatives of 2H-pyran-3(6H)-ones and their Michael adducts is described. Phenylthio, benzenesulfonyl, p-acetylaminobenzenesulfonyl, and p-bromophenyl substituents are beneficial for activity against gram-positive bacteria. 2-[4-(Phenylthio)phenyl]-2-methyl-6-methoxy-2H-pyran-3(6H)-one (8a) showed a minimum inhibitory concentration of 1.56 micrograms/mL against Staphylococcus aureus ATCC 2593, and 2-[4-(phenylthio)phenyl]-2-methyl-6-[(p-nitrobenzoyl)oxy]-2H-pyran-3 (6H)-one (9) showed a minimum inhibitory concentration of 0.75 microgram/mL against Streptococcus sp. C203M. In general, derivatives of 6-hydroxy-2H-pyran-3(6H)-ones with substituents at C-2 and C-6 showed significant activity against gram-positive bacteria. More specifically, the bulkier the C-2 substituent, the greater the antibacterial activity. Michael adducts of thiols (13) showed activity, which may be due to a retro-Michael reaction. In conclusion, the alpha,beta-enone system is essential for the activity of 6-hydroxy-2H-pyran-3(6H)-ones, and the size and nature of substituents at C-2 are associated with antimicrobial activity.

[Chemical and pharmacological study of Phragmites communis Trin]. / Contribution à l'étude chimique et pharmacologique de Phragmites communis Trin.

A chemical study of the aerial parts of Phragmites communis Trin. concerning free amino-acids, fatty acids, sterols, tocopherols and polyphenols has been carried out. The amino-acids content was studied at two different periods (winter, summer) in order to elucidate differences. The isolated polyphenols were investigated for antibacteriophage properties.

Immunosuppressive activity of concanavalin A.

Daily intraperitoneal doses of concanavalin A (Con A) produced a dose-related inhibition of adjuvant-induced arthritis in rats. Con A was also effective on established arthritis, markedly relieving the disease after only three doses. The inhibitory effect of Con A was neutralised by pre-incubation with ovalbumin, although this treatment did not modify the delayed phlogistic action of Con A in rat paws.

A comparison of the anaphylactoid actions of a synthetic linear dextran and a natural branched dextran.

Synthetic linear dextran of molecular weight 40,000 produces the systemic anaphylactoid reaction in rats although it is about 4 times less active than the natural branched dextran of similar molecular weight. Similarly, it is less active on local injection into the foot or skin. However, in rats which have been bred for non-reactivity to systemic dextran, it is more active on local injection, resembling the activity of a branched natural dextran of much lower molecular weight (6,000). In human skin, the synthetic linear 40,000 sample is also more active than the natural branched sample of similar molecular weight in producing a wheal and erythema. The results suggest that the dextran receptor in the skin of man may be similar to that in rats genetically resistant to systemic dextran.

Inhibition of streptolysin O-induced haemolysis by theobromine sodium salicylate.

Theobromine sodium salicylate exerts an inhibitory effect on lysis of rabbit red cells by streptolysin O. No similar action of related compounds has been demonstrated.