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.     

Asymmetric incorporation of [14C]cyanate and of fluorescein isothiocyanate in mamillary body of conditioned rats

A marked decrease in overall learning capacity has been observed in rats injected with cyanate. Therefore it was of interest to test whether learning influenced carbamylation of brain proteins. Incorporation of [¹⁴C]cyanate into proteins of the mamillary body was selectively modified following operant conditioning of the rat, so that trained rats showed an asymmetric image with higher levels of incorporation in the right side than in the left side, as compared to control rats. These results were confirmed using fluorescein isothiocyanate. The asymmetry persisted once the learning had been well established.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.