Antimicrobial effects of esters and amides of 3-(5-nitro-2-furyl)acrylic acid.

The effect of 18 newly synthesized esters and amides of 3-(5-nitro-2-furyl)acrylic acid on bacteria (Escherichia coli, Staphylococcus aureus), yeasts (Saccharomyces cerevisiae, Candida albicans), molds (Aspergillus niger, Penicillium cyclopium, Rhizopus oryzae) and algae (Chlorella pyrenoidosa, Euglena gracilis, Scenedesmus obliquus) was investigated. The MIC values revealed antimycotic, antialgal and antibacterial activity of the studied derivatives. The antimycotic activity was found to decrease with increasing the length of the alkyl chain of esters and after introduction of amino nitrogen into the furylethylene backbone. The inhibitory effect on growth is caused by blocking bioenergetic processes, glycolysis in particular.

Induction of mycelial type of development in Candida albicans by the antibiotic monorden and N-acetyl-D-glucosamine.

Two new effective methods for synchronous germ tube production in Candida albicans have been described. Both are based on the use of stationary grown cultures and their further incubation in an aerated simple mineral medium enriched with vitamins containing either high glucose concentration (100 mmol/1) and the antibiotic monorden being added, or N-acetyl-D-glucosamine (100 mmol/1) as the sole carbon source. On the other hand yeast morphology could be maintained in the medium with high glucose concentration. On the basis of the methods developed it was possible to compare respiration intensity, respiration quotients, and sensitivity against some metabolic inhibitors in both morphological forms. Labeling experiments showed slight differences in the time course of glycine incorporation. The mycelial cell walls contained more chitin than the yeastlike cells. Using light and electron microscopy the interrelationships between concentration of monorden, or N-acetyl-D-glucosamine, physiological state of inoculum and the germ tube frequency were determined. The results are discussed with regard to the induction of germ tubes by low glucose concentration in Candida albicans from the more general aspect of regulation of fungal morphogenesis.

Cytostatic activity and metabolic effect of N-trichloromethylthio-4-cyclohexane-1,2-dicarboximide on Ehrlich ascites carcinoma cells.

Effect of N-trichloromethylthio-4-cyclohexane-1,2-dicarboximide (NTCD) on energy-yielding and energy-requiring processes in Ehrlich ascites carcinoma (EAC) cells have been investigated. At concentrations higher than 10 micrograms/ml NTCD causes a rapid and practically full inhibition of both aerobic glucose uptake and lactate formation. On the other hand, at concentrations lower than 10 micrograms/ml, these metabolic parameters are stimulated. The stimulation of glycolysis, according to our previous results, suggests the interference of NTCD with mitochondrial functions. This image is supported by the marked inhibitory effect on NTCD on respiration of isolated mitochondria. The inhibition of glycolysis with higher concentrations of NTCD is the consequence of inactivation of hexokinase (EC 2.7.1.1), eventually of 6-phosphofructokinase (FC 2.7.1.11). The described effects of NTCD are given into coherence with chemical modification of appropriate functional SH groups of EAC cells by the compound studied. Proportionally to the dose and time NTCD inhibits the synthesis of macromolecules in whole EAC cells as measured by the incorporation of labeled adenine and valine into the TCA-insoluble fractions. The inhibition of biosynthetic processes followed is the consequence of exclusion of key processes in the energy metabolism and leads to the loss of EAC cells transplantability.

Induction of mycelial type of development in Candida albicans by low glucose concentration.

A new simple method for synchronous germ tube production in Candida albicans has been described, based on the further incubation of cells released from stationary grown cultures in aerated mineral medium enriched with vitamins and low glucose concentration (5 mmol/1). At higher initial glucose (e.g. 250 mmol/1)the growth proceeded in yeastlike form. At low glucose concentration germ tubes developed at 28 degrees C which is in contradiction with the results of many authors, considering 37 degrees C besides other factors to be an inevitable requirement. On the other hand the cell population from stationary growth phase was the absolute prerequisite for massive germ tube production. Its importance for other inductive techniques is assumed. The report brings comparative results concerning the physiological and biochemical properties as well as the ultrastructure of the yeastlike and mycelial forms. Neither were found any differences in respiration intensity nor in respiration quotients during the development of both growth forms. Slight dissimilarities resulted from the incorporation experiments (using (14)C labeled adenine, leucine and especially glycine). The mycelial cell walls were found to contain twice as much chitin as the yeastlike form. Some suggestion for further biochemical elucidation of dimorphism in Candida albicans and fungal morphogenesis generally are presented.

Interaction of cytotoxic antibiotic dactylarin with glycolytic thiol enzymes in Ehrlich ascites carcinoma cells.

Cytotoxic effect of dactylarin on Ehrlich ascites carcinoma cells is caused by the inhibition of some SH-dependent glycolytic enzymes, especially of hexokinase (EC 2.7.1.1), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 6-phosphofructokinase (EC 2.7.1.11). Dactylarin interacts with thiols, which explains its inhibitory effectiveness on the above glycolytic enzymes.

The inhibitory effect of vinylfurans on the glycolysis in tumor and yeast cells.

Most of the eighteen vinylfurane derivatives studied fully inhibit the glycolysis of both Ehrlich ascites carcinoma (EAC) cells and respiratory deficient yeast Saccharomyces cerevisiae at concentrations lower than 0.5 mmol/l. The inhibition of glycolysis is a consequence of some thiol enzymes inactivation. This concerns namely hexokinase (EC 2.7.1.1), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and especially 6-phosphofructokinase (EC 2.7.1.11). Interference of vinylfurans with energy metabolism resulted in the depression of biosynthetic processes followed (14C-precursors incorporation into proteins and nucleic acids) and finally in the loss of EAC cell transplantability.

Effect of 2,3-dinitrilo-1,4-dithia-9,10-antraquinone on Ehrlich ascites carcinoma and yeast cells.

The inhibition of glycolysis by 2,3-dinitrilo-1,4-dithia-9,10-antraquinone (DDA) in Ehrlich ascites carcinoma (EAC) cells as well as in the investigated respiratory and fermentative strains of yeasts was found to be the result of inactivation of thiol enzymes of this pathway. Increasing concentration of DDA caused, in EAC cells, marked inhibition of hexokinase (HK), phosphofructokinase (PFK) and practically total inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These three enzymes, as well as alcohol dehydrogenase (ADH) were also inactivated by DDA in yeasts. DDA inhibited the biosynthetic processes as measured by following the rate of [14C]adenine and [14C)]valine incorporation into TCA-precipitable fractions proportionally to the degree of glucose consumption by EAC or the yeast cells.

Antimicrobial activity of 2-vinylfuran derivatives.

2-Vinylfuran derivatives were found to inhibit algal and yeast growth. Experiments with a respiratory type of Saccharomyces cerevisiae DT XII, its respiration-deficient mutant DT XII A, and Candida albicans showed that all 2-vinylfurans are inhibitors of key processes of energy metabolism (especially glycolysis). The properties determining the inhibitory activity are chemical reactivity and lipophilicity. The reactivity of the studied derivatives was characterized by second-order rate constants kappa (L. mol-1.2-1) for reaction with mercaptoacetic acid (as a model thiol), and the lipophilicity by calculated sigma pi i-constants. An equation correlating the structure and the activity of 2-vinylfurans was derived. The significance of reactions of 2-vinylfurans with thiols or other nucleophilic groups of cell components is stressed. The reaction centre of 2-vinylfurans in these reactions is the electrophilic exocyclic double bond. The presence of a nitro group in position 5 of the furan ring is not indispensable for biological activity of 2-vinylfurans.

Effect of 2,3-dinitrilo-1,4-dithia-9,10-anthraquinone on Mycobacterium smegmatis.

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells of Mycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associated with this cyclic (glutamate, asparagine). DDA inhibits the incorporation of both 14C-leucine and 14C-adenine into appropriate macromolecules of M. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity of U-14C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth of M. smegmatis probably through an interference with the cell energy-carbon metabolism.

Dynamics of growth of atypical mycobacteria in different liquid cultivation media.

The dynamics of growth of Mycobacterium smegmatis, M. fortuitum and M. phlei in liquid media used also for cultivation of typical mycobacteria (Sauton, Youmans, Kirchner, Sula) was compared with that in Davis and Merrill media. In the Merrill medium glucose (as the only organic component) was replaced with another carbon source and the effect of this modification was investigated. The results obtained show that the Merrill medium, its modification in particular, is suitable for cultivation of M. smegmatis and M. fortuitum. 2-Oxoglutarate and succinate are important as the sole carbon sources in the case of M. fortuitum and M. phlei respectively.

The reaction of carbonyl cyanide phenylhydrazones with thiols.

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and aminothiols (cysteine, glutathione) to give corresponding N-(substituted phenyl)-N'-(alkylthiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular AdN type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS- ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group). The reactivitiy of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone less than carbonyl cyanide m-chlorophenylhyrazone less than carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pKa constants. On the other hand, the reactivity of thiols increases with their basicity. The reactivity of carbonyl cyanide phenylhydrazone with thiols is comparable with the reactivity of phenyl isothiocyanate and N-ethylmaleimide. It was demonstrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.

Selective and reversible modification of essential thiol groups of D-glyceraldehyde-3-phosphate dehydrogenase by isothiocyanates.

Aralkyl isothiocyanates, like aryl isothiocyanates, undergo a selective and reversible reaction with essential thiol groups of D-glyceraldehyde-3-phosphate dehydrogenase; the former 2 substances require for a reversible reaction course of more alkaline medium and presence of a thiol.

Effect of glycerol on viability and other properties of starved Mycobacterium fortuitum.

Cells of Mycobacterium fortuitum kept in 0.85% saline solution containing 0.1% Tween 80 (without glycerol) survive for a long time. In glycerol-enriched medium, they continue to lose their viability at a high rate; after 71 days of exposure the percentage of survival as indicated by colony formation and respiratory and dehydrogenase activities is lower than 1%. Surviving cells starved in medium without glycerol revealed unchanged sensitivity to streptomycin, p-aminosalicylic acid, and isoniazid.

Reactions of cysteine, its derivatives, glutathione coenzyme A, and dihydrolipoic acid with isothiocyanates.

Isothiocyanates react with deprotonated SH groups of investigated compounds to give the esters of N-monosubstituted dithiocarbamic acid. In the presence of the SH and NH2 groups (Cys, GSH), isothiocyanates react primarily with the SH groups. The reactions are dependent on the pKa SH values.

Effect of chloro- and bromo-derivatives of isocrotonic acid of bioenergetic processes in Ehrlich ascites cells and isolated mitochondria.

The effect of nine chloro- and bromoderivatives of isocrotonic acid on some bioenergetic processes in both Ehrlich ascites cells and isolated rat liver mitochondria has been investigated. Substances studied in the concentration range 25--200 microM significantly inhibited incorporation of both 14C-adenine and 14C-valine into acid-insoluble material of Ehrlich ascites cells. The rate of 14C-precursors incorporation being directly related to the concentration of the inhibitor. Gamma,gamma-bis-4-ethylphenyl-alpha,beta-dichloroisocrotonic acid fully inhibits both aerobic glucose utilization and lactic acid formation at 200 microM concentration. At lower concentrations, however, glycolysis is stimulated. Maximal stimulation of rat liver mitochondrial respiration in state 4 with succinate as substrate was reached at concentrations as low as 10 microM. On the other hand, these substances were able to release by the oligomycin inhibited respiration of rat liver mitochondria. Our data suggest that cytotoxic and cancerostatic action of isocrotonic acid derivatives lies primarily in the exclusion of key processes in the energy metabolism of Ehrlich ascites cells and isolated mitochondria.

Cytotoxic and cancerostatic effect of 1,4-dithiaanthraquinone-2,3-dicarbonitrile.

1,4-Dithiaanthraquinone-2,3-dicarbonitrile (DTA) has been found to exert a considerable cytostatic effect especially on some of the investigated types of eukaryotic cells, concretely on the HeLa cells, moulds, yeasts, protozoa and algae. In cells of the Ehrlich ascites carcinoma (EAC) DTA after a short exposition causes a parallel inhibition of incorporation of 14C-adenine and 14C-valine, in proportion to its rising concentration. The inhibition of biosynthetic processes thus made manifest, is probably a consequence of the primary DTA intervention into the energy metabolism of EAC cells, particularly in glycolysis. The effect of DTA in concentrations capable of bringing about full inhibition of glucose consumption or lactate formation in EAC cells also results in a loss of their transplantability. On the other hand, DTA also exerts a cancerostatic effect on the Ehrlich ascites carcinoma in mice.

Reactions of cellulose isothiocyanates with thiol and amino compounds.

A cellulose isothiocyanate has been prepared by treatment of cellulose with 2,4-di-isocyanatotoluene followed by hydrolysis and reaction of the resulting amine with thiophosgene. The cellulose isothiocyanate was characterized by its binding capacity with respect to [14C]-glycine, [131 I]-human serum albumin, and 2-mercaptoethanol. An analytical method for binding capacity, based on reaction with [35 S]-alpha-toluenethiol, was developed. Because of the aromatic character of the NCS group of the cellulose isothiocyanate, the covalently bonded thiol can be quantitatively liberated.