PubMed Health⌕ Search

Biomedical subjects

K Sigler

Publications and source records attributed to K Sigler.

At least 37 records · Page 2Linked to original sources
PubMed2001

Disinfectant activity against different morphological forms of Helicobacter pylori: first results.

The pathogenic bacterium Helicobacter pylori, which has infected more than one-half of the world's human population, exists in two morphological forms; the viable helical form and the disputed viable-but-not-culturable coccoid form. Infection by the helical form proceeds through the oral-oral route, while that by the coccoid form, if possible at all, is by the faecal-oral and/or the oral-oral route. The present pilot study addresses the question of disinfectant efficacy against both forms of the bacterium.

Chemistry, Pharmaceutical↗
PubMed2001

Mechanisms of Saccharomyces cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents.

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H+-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H2O2, Fe2+, Fe- and Cu-Fenton reagents. Inactivation by Fe2+ required the presence of oxygen and hence involved auto-oxidation of Fe2+ to Fe3+. The highest Fe2- (100 microM) and H2O2 (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe2+ content than on H2O2 concentration, occurred only when Fe2+ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN3, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDS, bipyridine, 1,10-phenanthroline). Inactivation by Fe- and Cu-Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH*. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H+-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe2+ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe2+, blocked H+-ATPase inactivation by Fe2+ and the Fenton reagent but not that caused by H2O2, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H+-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.

Adenosine Triphosphate↗
PubMed2001

Fluorescence behavior of the pH-sensitive probe carboxy SNARF-1 in suspension of liposomes.

When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5'(and 6')-carboxy-10-dimethylamino-3-hydroxy- spiro[7H-benzo[c]xanthene-7,1'(3H)-isobenzofuran]-3'-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.

Benzopyrans↗
PubMed2000

Spontaneous and radical-induced plasma membrane lipid peroxidation in differently oxidant-sensitive yeast species and its suppression by antioxidants.

Formation of thiobarbituric acid-reactive substances (TBRS; nmol/mg lipids) indicative of lipid peroxidation was measured in whole cells and in isolated plasma membrane lipids from three yeast species differing in oxidant sensitivity (Schizosaccharomyces pombe, Saccharomyces cerevisiae and Rhodotorula glutinis) after exposure to the Fenton reagent, FeII, H2O2, tert-butyl hydroperoxide (TBHP) and azo compounds (AAPH, ACHN). In whole cells, spontaneous TBRS formation rose in the sequence S. pombe < S. cerevisiae < R. glutinis (1:approximately 5:approximately 7). Oxidants increased the TBRS production 13-18 fold in the sequence FeII approximately TBHP > AAPH approximately ACHN approximately Fe-Fenton > H2O2. This increase need not be solely due to increased lipid peroxidation. In isolated plasma membrane lipids from all three species, the spontaneous TBRS production referred to 1 mg lipids was 9-13-fold higher than in whole cells. In S. pombe lipids, only TBHP increased the TBRS production. In lipids from S. cerevisiae and R. glutinis, all added oxidants increased the spontaneous TBRS production 2-3 times in the sequence TBHP > ACHN > AAPH > FeII > Fe-Fenton > H2O2. Oxidant-induced TBRS production in both whole cells and isolated membrane lipids was partially suppressed by the lipid peroxidation inhibitors 2,6-di-tert-butyl-4-methylphenol ("butylated hydroxytoluene"; BHT) and the newly synthesized PYA12 compound. Both agents were more effective in isolated lipids than in whole cells and against OH.-producing than against ROO.- or RO.-producing oxidants. Yeast membrane lipids, which are generally poor in polyunsaturated fatty acids, are thus subject to perceptible lipid peroxidation.

Antioxidants↗
PubMed2000

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells.

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.

Carbocyanines↗
PubMed2000

Osmosis: membranes impermeable and permeable for solutes, mechanism of osmosis across porous membranes.

A mathematical description is presented of osmotic flows across both ideally semipermeable membranes and membranes permeable not only for the solvent but also for the solute. The principles of thermodynamics of irreversible processes used for the description are given and illustrated on the example of electroosmosis. Modern ideas about the physical basis of osmotic pressure on porous membranes are discussed and an experiment is described that models the processes of osmosis on a macroscopic level.

Membranes↗
PubMed1999

Effect of killer toxin K1 on yeast membrane potential reported by the diS-C3(3) probe reflects strain- and physiological state-dependent variations.

The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells, i.e. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum lambda max indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in lambda max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.

Carbocyanines↗
PubMed1999

Oxidative stress in microorganisms--I. Microbial vs. higher cells--damage and defenses in relation to cell aging and death.

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differ in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of Reactive Oxygen Species (ROS) on cell constituents, secondary lipid- or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast or Podospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationary-phase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway--apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillary sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such as bak or bax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.

Animals↗
PubMed1999

New phenolic antioxidants of PYA and PYE class increase the resistance S. cerevisiae strain SP4, its SOD- and catalase-deficient mutants to lipophilic oxidants.

Was demonstrate the protection ability against reactive oxygen species afforded to S. cerevisiae (wild-type strain SP4 and its mutants deficient in major antioxidant enzymes--catalase T and A, CuZnSOD) by PYA and PYE, new groups of phenolic antioxidants (quaternary ammonium salts of dihydrocinnamic acid amino esters with different alkyl chains; synthesized in this laboratory). The survival of strains exposed to the lipophilic oxidation inducers tert-butyl hydroperoxide (TBHP) and 1,1'-azobis(4-cyclohexane carbonitrile) (ACCN) with or without antioxidant pretreatment was determined by plating. S. cerevisiae mutant deficient in SOD was found to be hypersensitive to TBHP and ACCN while the sensitivity of the strain deficient in catalase T and A was about the same as in the wild-type strain. A 1-h preincubation of cells of both the wild-type and the mutant strains with the phenolic antioxidants prior to exposure to TBHP or ACCN substantially increased the cell survival. The magnitude of protection depended on the strain and the length of the alkyl chain of the antioxidant; the best average protection against TBHP was provided by PYE and PYA compounds with 12- and 16-membered alkyl chains whereas PYE-8 and PYA-12 derivatives afforded the best average protection against ACCN.

Antioxidants↗
PubMed1999

Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay.

No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1998

Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae.

Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Carbocyanines↗
PubMed1998

Inactivation of the plasma membrane ATPase of Schizosaccharomyces pombe by hydrogen peroxide and by the Fenton reagent (Fe2+/H2O2): nonradical vs. radical-induced oxidation.

In the absence of added Fe2+, the ATPase activity of isolated Schizosaccharomyces pombe plasma membranes (5-7 mumol P(i) per mg protein per min) is moderately inhibited by H2O2 in a concentration-dependent manner. Sizable inactivation occurs only at 50-80 mmol/L H2O2. The process, probably a direct oxidative action of H2O2 on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both the K(m) of the enzyme for ATP and the V of ATP splitting. On exposing the membranes to the Fenton reagent (50 mumol/L Fe2+ + 20 mmol/L H2O2), which causes a fast production of HO. radicals, the ATPase is 50-60% inactivated and 90% of added Fe2+ is oxidized to Fe3+ within 1 min. The inactivation occurs only when Fe2+ is added before H2O2 and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO. radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe2+ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe2+ for H2O2, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO. production.

Adenosine Triphosphatases↗
PubMed1998

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeast Schizosaccharomyces pombe.

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grown Schizosaccharomyces pombe cells was assessed and compared with that of Saccharomyces cerevisiae in order to determine the catalase activity regulation in S. pombe. In contrast to S. cerevisiae, the total catalase activity of permeabilized S. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3-5%) of it is contributed by de novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7-8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due to de novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.

Aerobiosis↗
PubMed1997

Measurement of membrane potential in Saccharomyces cerevisiae by the electrochromic probe di-4-ANEPPS: effect of intracellular probe distribution.

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

Cell Membrane↗
PubMed1997

Biotechnological aspects of membrane function.

An overview is given of the biotechnological utilizability of various features of cell membranes. Techniques are given that describe how to make use of the barrier and transport functions of membranes for biotechnological purposes, ranging from cell permeabilization and construction of immobilized biocatalysts to manipulating excretion and uptake properties of the membranes by various methods. Glucose transporters, iron-transporting membrane systems, and pumps engaged in pleiotropic drug resistance are treated in more detail as particularly biotechnologically important membrane proteins.

Biological Transport↗
PubMed1997

Animal membrane receptors and adhesive molecules.

This review summarizes some important data, principles, opinions, commentaries, and modern methodology concerning the receptor structure, interactions, signaling and receptor-mediated cell functions. Three sections give a brief overview of the signaling synergy, multivariant signaling, and some reactions in phosphorylation networks. A concise report about the cytotoxic reaction of NK cells represents an example of multistage recognition reaction, involving differently acting receptors, changes in affinities of cell-cell interactions, and secretion of regulatory and cytotoxic molecules. Some interesting trends in receptor engineering, including antibody molecules as a special receptor phenomenon are mentioned in the final section.

Amino Acid Sequence↗