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PubMed2004

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Candida albicans↗

PubMed1982

Inhibition of protein synthesis in Saccharomyces cerevisiae by the 12,13-epoxytrichothecenes trichodermol, diacetoxyscirpenol and verrucarin A. Reversibility of the effects.

Inhibition of protein synthesis by trichodermol, diacetoxyscirpenol and verrucarin A in cells and spheroplasts of Saccharomyces cerevisiae was investigated. Inhibition was reversible for trichodermol and diacetoxyscirpenol, both drugs being removed from their target site(s) by washing, but was reversible for verrucarin A. These results are interpreted in relation to variations in chemical structure between these trichothecenes.

Antineoplastic Agents↗

PubMed1998

A dual labelling method for measuring uptake of low molecular weight compounds into the pathogenic yeast Candida albicans.

In contrast to other eukaryotic cells the pathogenic yeast Candida albicans is resistant to many structurally unrelated metabolic inhibitors. Reduced permeability due to the cell wall and/or altered plasma membrane composition is thought to be at least partly responsible for this phenomenon. To study the uptake of low molecular weight compounds into C. albicans we developed a dual labelling method. Intact cells, metabolically inactivated cells, spheroplasts or membrane fragments of C. albicans were incubated with various [14C]-labelled compound in the presence of [3H]-labelled water. After separation of cells and supernatant isotope ratios [3H]/[14C] were determined. Quotients of the isotope ratios from cells and supernatant, called enrichment coefficients, were calculated under all four conditions. The enrichment coefficients indicated whether a compound can enter C. albicans cells, is trapped within the cell wall, is enriched in the lipophilic membrane compartment, is actively accumulated or actively exported by multidrug resistance carriers. We used six structurally unrelated compounds to test our method. We found no evidence for a general impermeability of C. albicans.

Anti-Bacterial Agents↗

PubMed1976

Regulation of mitochondrial protein synthesis at the polyribosomal level.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.

Chloramphenicol↗

PubMed1979

Combined action of semisynthetic penicillin and macrolide antibiotic on Pseudomonas aeruginosa in vitro and in vivo.

For the purpose of studying therapy for pseudomonas infection, the synergistic effect of carbenicillin and midecamycin or 9,3''-di-O-acetyl midecamycin on Pseudomonas aeruginosa were studied in vitro and in vivo. Midecamycin and 9,3''-di-O-acetyl midecamycin were effective against spheroplasts of P. aeruginosa induced by carbenicillin treatment. As a result of investigating treatment using a combination of both carbenicillin and macrolide antibiotics on mouse infection model caused by subcutaneous injection of carrageenan solution containing viable cells of P. aeruginosa, the combined treatment was demonstrated to be significantly more effective than the control's single treatment by carbenicillin or the macrolide antibiotic alone.

Animals↗

PubMed2002

[New aspects of the pathogenesis of lyme disease].

Morphological changes of B. burgdorferi as well as changes in expression of surface proteins caused by environmental determinants are essential in pathogenesis of Lyme disease. Cysts, spherical form (spheroplasts, L-form) and "blebs" (gemmae) can be responsible for long lasting antigenic stimulation, signs of chronic borreliosis, and even probably connected with MS and Alzheimer disease. Mechanisms to avoid elimination and persistence in the host include: expression of low heterogenic Osp A, B replaced by polymorphic in sequence and antigenic reactivity OspC, the hindrance of access to some membrane proteins by other proteins on the spirochete's surface, effects of tick saliva proteins action. Hiding of spirochetes is possible by invagination into fibrocytes membrane as well as, coating by antigens derived from lymphocytes B. Distribution of spirochetes is facilitated by binding to platelets through integrin aIIb b3, and to the endothelial cells through integrins av b3 i a5b1, recognition of decorin by lipoproteins DbpA i DbpB, receptor for NAG (N-acetyl glucosamina). Endothelial cells, toxic products of granulocytes, monocytes, macrophages as well as phagocytosis counterpart in pathogenesis. Induced cytokines are connected with activation subsets of T lymphocytes involved in inflammatory response. Cytokines produced by Th1 as cytotoxic CD8 accompany the disease. Important are also dendritic cells regarded as initiators of Th1 response with participation of IL-12. In pathogenesis of Lyme disease participation of autoimmunity is notified, especially molecular similarities between OspA and human lymphocytic antigen (hLFA-1). Neurotoxin, produced by B. burgdorferi Bbtox1 was identified. Encephalopathy signs in Lyme borreliosis could be result of releasing toxico-metabolic products, ability of spirochetes to pass the blood-brain barrier as well as, effect of lymphocytes migration. Active invasion of brain endothelium as ability to adherence to endothelial wall could be the source of focused or disseminated inflammation of brain vessels. Antiaxonal antibodies could disturb axon conduction without damaging. But damage of white matter could be connected with damage of mielin production cells, probably by antibodies, induced in cross reaction.

Blister↗

PubMed1992

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.

Cytochrome b Group↗

PubMed1990

Quantification of physical and cyto-physiological conditions for the electrofusion of Saccharomyces cerevisiae.

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 Vp/cm and a DC field of 2 square pulses (7 kV/cm; 60 microsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or 1.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 M. The presence of CaCl2 (up to 0.4 mM) or 0.1 mM CaCl2 with 0.1 mM MgCl2 enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28 degrees C.

Buffers↗

PubMedDate not supplied

[Changes in the electrical characteristics of the internal surface of cytoplasmic membrane of bacteria Escherichia coli after the binding of microdoses of copper ions by its external surface].

The effect of microdoses of copper ions bound with the external surface of the cytoplasmic membrane of Escherichia coli spheroplasts on the charge density of its superficial structures was investigated by the ESR method. Positively charged spin probes of KAT(n)-type and the newly synthesized KAT15 were used. The experimental data were analyzed using the formalism of Gouy-Chapman theory. The binding of microdoses of copper ions by the external surface of the cytoplasmic membrane changed the value of charge density on the membrane internal surface.

Cell Membrane↗

PubMedDate not supplied

[Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood].

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.

Antibiosis↗

PubMedDate not supplied

Effects of the bacteriocin PsVP-10 produced by Pseudomonas sp. on sensitive bacterial strains.

The bacteriocin PsVP-10 is a 2.6 Kda peptide which was isolated and purified from Pseudomonas sp. This bacteriocin possesses lethal activity over Enterococcus faecalis, Salmonella typhimurium and Shigella flexneri. The experimental assays showed that the bacteriocin is able to be adsorbed by all cells of these bacterial species and also by their isolated cell walls. It was observed that the resistant mutants and their respective cell walls are unable to adsorb the bacteriocin. Assays performed with spheroplasts obtained from sensitive bacterial species and their resistant mutants show a rapid lethal effect of the bacteriocin PsVP-10. This results indicated furthermore, it is also shown that the optimal pH and temperature for the adsorption were 7.2 and 37 degrees C, respectively. The study carried out with organic solvents like methanol, ethanol, isopropanol and the detergents sodium dodecyl sulfate and triton X-100 showed a moderate inhibition of the bacteriocin lethal action for the Gram negative cells. The enzymes lysozime, protease XIV and trypsine type III-S did not present any effect over the adsorption capacity of the bacteriocin with any of the bacterial species studied.

Adsorption↗

PubMed1990

Pharmacodynamics of beta-lactam antibiotics on gram-negative bacteria: initial killing, morphology and postantibiotic effect.

The aim of this study was to investigate the pharmacodynamics of beta-lactam antibiotics on Gram-negative bacteria by studying the in vitro postantibiotic effect (PAE), initial killing and morphology. The PAE of aztreonam, cefotaxime, ceftazidime, imipenem, mecillinam and piperacillin on Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens was studied by use of bioluminescence assay of bacterial ATP. The strains were exposed for 2 h to the beta-lactam antibiotics which were then eliminated by 10(-4) dilution. With E. coli, spheroplasts were seen during incubation with cefotaxime, ceftazidime, imipenem and mecillinam and a long (greater than 1 h) PAE was noted. In contrast, aztreonam and piperacillin produced a short (less than or equal to 1 h) PAE. This was seen concomitantly with the production of filaments and weak initial killing.

Amdinocillin↗

PubMed1989

Action of mycosubtilin, an antifungal antibiotic of Bacillus subtilis, on the cell membrane of Saccharomyces cerevisiae.

Mycosubtilin, an antibiotic of the iturin group, inhibits the growth of Saccharomyces cerevisiae by a fungicidal action. Increasing concentrations of mycosubtilin decrease the incorporation of radioactive precursors into proteins, RNA and polysaccharides without specificity. Yeast spheroplasts are lysed by mycosubtilin. Its action on the cytoplasmic membrane induces important modifications of the membrane permeability: nucleotides, proteins and lipids are released from the cells. These releases increase with increasing concentrations of mycosubtilin.

Antifungal Agents↗

PubMed1988

[Comparative action of 8 azole derivatives against Candida albicans: fungistatic action and cytologic study by scanning electron microscopy].

The authors compared the in vitro antifungal activity of eight imidazole derivatives (clotrimazole, econazole, isoconazole, ketoconazole, miconazole, oxiconazole, terconazole, tioconazole) against 42 strains of Candida albicans by the agar dilution method using casitone medium. The geometric (G) mean MIC values, the MIC 90 and the MIC 50 values and the corresponding standard deviations of each antifungal agent were determined. The G-MIC values were found to be in the range of 0.008-0.390 micrograms ml-1. The effects of these eight antifungal agents on the ultrastructure of C. albicans yeast cells and spheroplasts were studied by scanning electron microscopy (SEM). The results showed a good correlation between the lesions observed and the structure of the imidazole derivatives tested. On the basis of the SEM results, the compounds could be divided into three groups: (1) ketoconazole and terconazole; (2) econazole, isoconazole, miconazole, oxiconazole and tioconazole; (3) clotrimazole.

Antifungal Agents↗

PubMed1986

[Cellular ultrastructure of the meningococcus when incubated in a continuous culture of human FL-line amnion].

Some details of the ultrastructure of several meningococcal strains having had contacts with cells in continuous human amnion cell culture FL for 6 hours to 2 days have been defined with greater precision by means of electron microscopy. The study has shown that the contact of meningococci with the tissue culture is accompanied by the appearance of meningococcal forms with the defective cell wall, similar to L-forms: spheroplast, protoplast, gigantic cells and microcells, as well as budding variants. The meningococcal variants with the defective cell wall, appearing in the cell culture, and the forms occurring (in different proportions) in "ripe" meningococcal populations developing in the culture media for a long time and isolated from a human body have been found to have no essential differences in their fine structure. These data indicate that any external influences (meningococci are highly sensitive to such influences) produce sufficiently rapid changes, similar to L-transformation, in the fine structure of these microorganisms.

Amnion↗

PubMed1987

Effects of microwave irradiation on some membrane-related processes in bacteria.

In a series of experiments performed on intact cells or spheroplasts of E. coli and Bac. subtilis a possibility of non-thermal effects induced by continuous microwave irradiation of a low power density (at wave length range from 0.0 to 7.8 mm) was studied. Thymidine and thymine uptake, leakage of potassium and hydrogen ions as well as the uptake of the transforming DNA by the component cells of Bac. subtilis were shown to be affected in a way typical of that due to heating of a sample. No specific dependence of the effects observed on wavelength was found.

Bacillus subtilis↗

PubMed1979

[Ampicillin-resistant "Haemophilus": their detection and occurrence in Brussels area (author's transl)].

Three hundred and five strains of Haemophilus (129 H. influenzae, 55 H. parainfluenzae, 97 H. parahaemolyticus, 5 H. haemolyticus, 10 H. paraphrophilus and 9 H. paraphrohaemolyticus) isolated from pathological material over the year 1976, were systematically tested for beta-lactamase production. Only 2 strains of H. parainfluenzae produced this enzyme. Both were able to transfer ampicillin resistance to Escherichia coli K12. All strains but the two beta-lactamase producers were susceptible to penicillin G, ampicillin and cephalotin. However, the correct interpretation of the susceptibility tests needed the microscopic observation of prints of the inhibition zones surrounding the disks: all sensitive strains presented a hazy growth around the disks which corresponded to the presence of spheroplasts; this phenomenon was not observed with the 2 beta-lactamase producing strains of H. parainfluenzae which grew up to the disks but presented a typical bacillary form.

Ampicillin↗

PubMedDate not supplied

[Interaction between a fluorescent probe and the surface structures of Escherichia coli].

The interaction of ANS with Escherichia coli cells, isolated cell walls, total cell envelopes and inner membranes was investigated in the presence and absence of Ca2+ ions. The addition of Ca2+ to intact cells at room temperature did not result in enhancement of fluorescence intensity. On the contrary the addition of Ca2+ to intact cells at 2 degrees C resulted in a progressive increase of fluorescence intensity with the maximum reached approximately by the 20th minute. The addition of Ca2+ to different membrane preparations showed a two-fold increase. Addition of Ca2+ to spheroplast membrane preparations did not result in any increase of the number of binding sites for ANS. There was a four-fold increase in quantum yields. All membrane preparations in titrations with Ca2+ showed saturation kinetics. The obtained results are discussed in terms of structural alterations in E. coli membranes and in relation with biological effects.

Anilino Naphthalenesulfonates↗