Wasabi Compound 6-(Methylsulfinyl) Hexyl Isothiocyanate Induces Cell Death with Coexisting Mitotic Arrest and Autophagy in Human Chronic Myelogenous Leukemia K562 Cells

A natural compound from Wasabia japonica, 6-(methylsulfinyl) hexyl isothiocyanate (6-MITC) was investigated for its anti-leukemia activity and mechanism of action. It was found that 6-MITC inhibited the viability of human chronic myelogenous leukemia K562 cells along with extensive mitotic arrest, spindle multipolarity, and cytoplasmic vacuole accumulation. The evidence of autophagy included the validation of autophagosomes with double-layered membranes under transmission electron microscopy, LC3I/II conversion, and the induction of G2/M phase arrest observed with acridine orange staining of treated cells, as well as the elevation of phosphorylated-histone H3 expression at the M phase. With regard to the expression of proteins related to mitosis, the down regulation of p-CHK1, p-CHK2, p-cdc25c, and p-cdc2, as well as the upregulation of cyclin B1, p-cdc20, cdc23, BubR1, Mad2, and p-plk-1 was observed. The knockdown of cdc20 was unable to block the effect of 6-MITC. The differentiation of k562 cells into monocytes, granulocytes, and megakaryocytes was not affected by 6-MITC. The 6-MITC-induced unique mode of cell death through the concurrent induction of mitosis and autophagy may have therapeutic potential. Further studies are required to elucidate the pathways associated with the counteracting occurrence of mitosis and autophagy.

Ultrastructural Imaging ofCandida albicansAdhesion to Rat Genital Epithelium through Scanning and Transmission Electron Microscopy

The adhesion ofCandida albicansto the genital epithelium has not been fully investigatedin vivo. The objective of this study was to evaluate the ultrastructural aspects ofC. albicansadhesion in the lower genital system of female Wistar rats through scanning and transmission electron microscopy. The genital infection persisted until the end of the experiment, and all rats showed the same adhesion aspects. Various associated yeast/hyphae were observed in the lumen and adhered both at the vaginal and endocervical levels where the fungal filamentation process occurred. In the vaginal epithelium, closely adhered yeasts were observed as stretched strands bridging between yeasts and the epithelium surface. Different stages of the adhesion, where yeasts internalized into the epithelial cell inside a cytoplasmic vacuole, resembling endocytosis, and a wide fibrillar-floccular, glycocalyx-like layer on the yeasts were observed. On the endocervix, the adhesion occurred between the cilia. In the uterine body, only a yeast-like form was observed with superficial contact. This study reached the initial goal of demonstrating an experimental model forin vivostudies. Continuation of this line of research is important for studies of vulvovaginal candidiasis.

Autophagy in health and disease. 1. Regulation and significance of autophagy: an overview

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment after formation of a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The identification of ATG (autophagy-related) genes that are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of macroautophagy, and its roles in cell function, which extend far beyond degradation and quality control of the cytoplasm. Macroautophagy, which plays a major role in tissue homeostasis, is now recognized as contributing to innate and adaptive immune responses. Recently, several mediators of apoptosis have been shown to control macroautophagy. Deciphering the cross talk between macroautophagy and apoptosis probably should help increase understanding of the role of macroautophagy in human disease and is likely to be of therapeutic importance.

Excess copper induces structural changes in cultured photosynthetic soybean cells

Soybean [Glycine max (L.) Merr.] cell suspensions have the capacity to develop tolerance to excess copper, constituting a convenient system for studies on the mechanisms of copper tolerance. The functional cell organisation changes observed in these cell cultures after both short-term (stressed cells) and long-term (acclimated cells) exposure to 10 μm CuSO4 are reported from structural, cytochemical and microanalytical approaches. Cells grown in the presence of 10 μm CuSO4 shared some structural features with untreated cells, such as: (i) a large cytoplasmic vacuole, (ii) chloroplasts along the thin layer of cytoplasm, (iii) nucleus in a peripheral location exhibiting circular-shaped nucleolus and a decondensed chromatin pattern, and (iv) presence of Cajal bodies in the cell nuclei. In addition, cells exposed to 10 μm CuSO4 exhibited important differences compared with untreated cells: (i) chloroplasts displayed rounded shape and smaller size with denser-structured internal membranes, especially in copper-acclimated cells; (ii) no starch granules were found within chloroplasts; (iii) the cytoplasmic vacuole was larger, especially after long-term copper exposure; (iv) the levels of citrate and malate increased. Extracellular dark-coloured deposits with high copper content attached at the outer surface of the cell wall were observed only in cells exposed to a short-term copper stress. Structural cell modifications, mainly affecting chloroplasts, accompanied the short-term copper-induced response and were maintained as stable characters during the period of adaptation to excess copper. Vacuolar changes accompanied the long-term copper response. The results indicate that the first response of soybean cells to excess copper prevents its entry into the cell by immobilising it in the cell wall, and after an adaptive period, acclimation to excess copper may be mainly due to vacuolar sequestration.

Effects of Inhibitors of Δ24(25)-Sterol Methyl Transferase on the Ultrastructure of Epimastigotes ofTrypanosoma cruzi

Trypanosoma cruziis the ethiological agent of Chagas disease. New compounds are being developed based on the biosynthesis and function of sterols, becauseT. cruzihas a requirement for specific endogenous sterols for growth and survival. Sterol biosynthesis inhibitors (SBIs) are drugs commonly used against fungal diseases. These drugs act by depleting essential and specific membrane components and/or inducing the accumulation of toxic intermediary or lateral products of the biosynthetic pathway. In this work we present the effects of WSP488, WSP501, and WSP561, specific inhibitors of Δ24(25)-sterol methyl transferase, on the ultrastructure ofT. cruziepimastigotes. All three drugs inhibited parasite multiplication at low concentrations, with IC50values of 0.48, 0.44, and 0.48 μM, respectively, and induced marked morphological changes including (a) blockage of cell division; (b) swelling of the mitochondrion, with several projections and depressions; (c) swelling of the perinuclear space; (d) presence of autophagosomes and myelin-like figures; (e) enlargement of the flagellar pocket and of a cytoplasmic vacuole located in close association with the flagellar pocket; (f) detachment of the membrane of the cell body; and (g) formation of a vesicle at the surface of the parasite between the flagellar pocket and the cytostome. Our results show that these drugs are potentin vitroinhibitors of growth ofT. cruzi.

Effects of chronic ingestion of sodium fluoride on myocardium in a second generation of rats

Possible effects of long term exposure (6 months) to sodium fluoride (NaF) through drinking water on the morphology and biochemistry of myocardial tissue in second generation adult male rats were investigated. Wistar strain female and male rats were reared until the second generation of rats obtained, during which they were given 1, 10, 50 and 100 mg/L NaF in drinking water. Of the second generation, 28 male rats were divided into four groups and had the same treatment. All the second generation rats were sacrificed and autopsied at the end of the 6 months. In the samples of myocardial tissues, the levels of serum fluoride and the activities of principal antioxidant enzymes were determined, and a histopathological examination was conducted. Significant histopathological changes were found in the myocardial tissue of rats treated with 50 and 100 mg/L NaF. These were myocardial cell necrosis, extensive cytoplasmic vacuole formation, nucleus dissolution in myosits, swollen and clumped myocardial fibers, fibrillolysis, interstitial oedema, small hemorrhagic areas and hyperaemic vessels. Additionally, the increased activities of superoxide dismutase (SOD), glutathione peroxidase (GSH–Px), catalase (CAT) and thiobarbituric acid–reactive substance (TBARS) levels were observed in the myocardial tissues of rats treated with 10 and 50 mg/L NaF. On the other hand, the activities of SOD, GSH–Px, and CAT decreased, but the TBARS levels increased in the myocardial tissues of rats treated with 100 mg/L. The present results revealed that prolonged ingestion of fluoride through drinking water, particularly with high doses, induced significant histopathological and biochemical changes leading to myocardial tissue damage.

Cleavage of Host Keratin 8 by a Chlamydia-Secreted Protease

ABSTRACT Chlamydiae have to replicate within a cytoplasmic vacuole in eukaryotic cells. Expansion of the chlamydia-laden vacuole is essential for chlamydial intravacuolar replication, which inevitably causes host cell cytoskeleton rearrangements. A cleavage fragment of keratin 8 corresponding to the central rod region was detected in the soluble fraction of chlamydia-infected cells. Since keratin 8 is a major component of the intermediate filaments in simple epithelial cells, cleavage of keratin 8 may increase the solubility of the host cell cytoskeleton and thus permit vacuole expansion in chlamydia-infected cells. A chlamydia-secreted protease designated CPAF (chlamydial protease/proteasome-like activity factor) was both necessary and sufficient for keratin 8 cleavage in chlamydia-infected cells, suggesting that chlamydiae have evolved specific mechanisms for modifying the host cell cytoskeleton.