Quercetin-induced yeast apoptosis through mitochondrial dysfunction under the accumulation of magnesium in Candida albicans

Latterly, the upsurge in use of antifungal drugs has brought about the emergence of several drug-resistance strains, making it skeptical to continue relying on current therapeutic regime. In the necessity of resistance-free antifungal agent, flavonoids presented possibilities of replacing existing drugs, displaying antifungal activity against pathogenic fungi. Among them, quercetin, one of the most representative flavonoids, exhibited antifungal activity against Candida albicans. To inspect the further understanding regarding quercetin, the antifungal mode of action of quercetin was investigated. In the initial step, the apoptosis was monitored after quercetin treatment. Moreover, intracellular levels of Mg²⁺ was assessed and was determined that Mg²⁺ increase occurred under the influence of quercetin. In addition, several features of mitochondrial dysfunction were monitored. Mitochondrial dysfunction triggers decrease in mitochondrial redox levels and leads to disruption in mitochondrial antioxidant system. Increased intracellular ROS and decreased intracellular redox levels were also displayed, indicating the occurrence of overall disruption in antioxidant systems. Sequentially, DNA fragmentation was observed and this DNA damage in turn induces apoptosis. In analyses, hexaamminecobalt(III) chloride (Cohex) was applied to inhibit Mg²⁺ transport between cytosol and mitochondria. Cohex attenuated the effects induced by quercetin, which demonstrates that the presence of Mg²⁺ is essential in quercetin-induced apoptosis.     

Calcium-dependent binding of EDTA-extractable proteins to calf lens fiber membrane structures

Calf lens fiber membranes and fractions enriched in junction-like structures have been isolated in the absence and presence of EDTA. Their biochemical features have been studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting experiments have provided evidence that a distinct group of EDTA-extractable proteins, being one of the main protein components of calf lens fiber membranes and very likely also of junction-like structures, is bound to these membranes via calcium ions. In addition to these proteins, four polypeptides with apparent molecular weights between 14 000 and 17 000 are characteristic for detergent-insoluble lens fiber structures prepared in calcium-rich medium. The absence of EDTA-extractable proteins in the urea-soluble calcium-containing fraction implies that they are not components of the cytoskeleton and that the calcium-dependent binding of these proteins to the membrane is urea-resistant. The use of EDTA throughout the whole membrane isolation procedure results in their complete removal from the membranes which already starts during buffer washing. This indicates that EDTA-extractable proteins exclusively consist of extrinsic membrane proteins which probably are not involved in cytoskeleton binding.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

A mutant of Halobacterium halobium with constitutive production of bacteriorhodopsin

Abstract A purple mutant of Halobacterium halobium was isolated in a previous study. The 'in vitro' absorption spectra of the cells gave a broad shoulder around 570 nm. The amounts of bacteriorhodopsin were high under any growth condition (including aerobic) inhibitory for the wild-type strain. The mutant grew faster under illuminated microaerophilic conditions and showed faster proton extrusion than the wild-type strain. This evidence shows that the mutant has a constitutive bacteriorhodopsin production not influenced by the oxygen concentration in the medium. However, some stimulation by light was found.     

In vivo and in vitro sugar transport in frog intestine

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.     

Ultrastructure of Nitrosospira sp. X101 with crystalline outer membrane protein (COMP)

Abstract The outer membrane (OM) structure of Nitrosospira sp. X101 was studied by different electron microscopic techniques and SDS-PAGE. A crystalline outer membrane protein was visible in freeze-etched cells, occasionally seen also in the thin sectioned cells, but was difficult to see in a negatively-stained preparation. The lattice probably consists of large globular protein subunits with a hexagonal arrangement. The molecular weights of the major proteins in the cell envelope are 35 kDa, 40 kDa and 42 kDa.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.