Vital Staining Of Mitochondria Research Articles

A novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human bladder cancer cells

Background: Quinazolines, which process a wide spectrum of biological properties such as antibacterial, antifungal, antivirus, and anticancer activities, are considered one of the most important heterocycles in medicinal chemistry. Here, we described for the first time the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazoline-4-one) which emits green fluorescent in the cytosol and exhibits phototoxicity toward human bladder cancer (BC) cells under blue-light exposure. Materials and Methods: Human BC cells (5637 and T24) and immortalized uroepithelial cell (SV-HUC1) were utilized in this study. To trace the localization of MJ-56, MitoTracker and LysoTracker were applied. The cell viability with or without blue light exposure were monitored by WST-1 reagent, direct recording, and clonogenic assays. The apoptosis induction in MJ-56 treated cells was detected. Results: MJ-56 emits green fluorescent in the cytosol. Vital staining of mitochondria or lysosomes demonstrated that the MJ-56 fluorescent was not located in either organelles. MJ-56 treatment for 24 h did not cause significant loss of cell viability in BC cells. However, treatment of 0.125 μM MJ-56 for 1 h and exposed to blue light for 15 mins significantly reduced cell viability. Interestingly, our results showed that MJ-56 has minimal impact on SV-HUC1 even with the blue-light exposure. The caspase 3/7 activities in BC cells treated with MJ-56 and exposed to blue light were significantly increased 1 h post-treatment. However, the DNA fragmentation cannot be detected at 1, 6, or 24 h posttreatment due to the loss of viable cells. Conclusions: MJ-56 exhibits phototoxicity toward BC cells with minimal impact on uroepithelial cells, indicating a novel therapeutic agent against BC. The mechanism underlying MJ-56-induced cell death as well as the translational studies warrants further investigation.

Mitochondria-Specific Monoclonal Antibodies in Eggs and Embryos of the Ascidian Halocynthia roretzi.

ABSTRACTAscidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence‐labeled mitochondria

Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.

Comparative Measurement of In Vitro Paraquat and Aflatoxin B1 Cytotoxicity Using Three Different Cytotoxicity Assays in Pheochromocytoma Cells (PC-12)

ABSTRACTAmong the herbicides and mycotoxins, paraquat (PQ) and aflatoxin B1 (AFB1) are highly cytotoxic. In this study the toxicity of PQ and AFB1 in the cultured cell were determined using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], JG-B (Janus green B), and NR (neutral red) assay by multiwell scanning spectophotometry. JG-B was used not only for the vital staining of mitochondria but also for viability assay and was compared to MTT and NR assay. Various concentrations of paraquat (0.1 mM to 100 mM) and AFB1 (0.001 nM to 10 nM) on the PC-12 cells were investigated. The 50% lethal concentration of toxins (LC50) were determined for PQ (7.70 ± 2.50, 3.67 ± 1.53, 4.85 ± 2.44) and AFB1 (0.16 ± 0.01, 0.13 ± 0.04, 0.14 ± 0.02) as determined by these methods (JG-B, NR, and MTT, respectively). A significant correlation was found among the JG-B and MTT using PQ (r2 = 0.99, p < 0.05) and significant correlation was also found among the three methods (r2 = 0.95, 0.93, and 0.92, p < 0.05) using AFB1. No significant correlation was found between JG-B and MTT with NR (r2 = 0.34 and 0.35, p < 0.05, respectively) using PQ. These results suggest that both methods (MTT assay and JG-B assay) are reliable and are comparable for determining the cytotoxicity. It is concluded that the JG-B assay may be preferable to MTT assay methods because of its simplicity, low cost, sensitivity, and objectivity; in addition, this method takes little time to be done.

Comparative Measurement of Cyanide and Paraquat Mitochondrial Toxicity Using Two Different Mitochondrial Toxicity Assays

ABSTRACTCyanide (KCN) and paraquat (PQ) are very toxic to mitochondria. In this study the toxicity of KCN and PQ in the isolated rat liver mitochondria was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and JG-B (Janus green B) assay by multiwell scanning spectrophotometry. JG-B was used not only for the vital staining of mitochondria, but also for the mitochondrial viability assay and was compared to the MTT assay.The rat liver mitochondria were first isolated by centrifuge in a mixture of 0.25 M saccharose solution and 0.05 M Tris buffer. Various concentrations of paraquat (0.001 to 100 mM) and KCN (0.0001 to 100 M) on the mitochondria isolated from the liver were investigated. The 50% lethal concentration of toxins were found for PQ (4.45 ± 0.02, 4.96 ± 0.01) and KCN (0.22 ± 0.02, 0.49 ± 0.02), as determined by these assays ( JG-B and MTT, respectively ). Significant correlations were also observed among the two methods with a 95% coefficient interval (r2 = 0.84, p < 0.001; r2 = 0.91, p < 0.001; PQ and KCN, respectively). These results suggest that both methods are reliable and are comparable for determining the mitochondrial assay. It is concluded that the JG-B assay may be preferable to the MTT assay because of its simplicity, low cost, sensitivity, and objectivity; in addition, this method is not time dependent.

Rhodamine 123 as a vital stain for mitochondria of plant cells

Rhodamine 123 as a vital stain for mitochondria of plant cells

Import and processing of cytochrome b-c1 complex subunits in isolated hepatoma ascites cells. Inhibition by Rhodamine 6G.

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b-c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2-3 kDa and 5-6 kDa larger than the mature forms of apocytochrome c1 and apo-iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals. Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and particulate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10 min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m-chlorophenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing, (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing under conditions in which it appears to have little influence on coupled respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step.

Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1-10mum) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under ;nuclear' conditions and in the presence of Janus Green B and Ca(2+) is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.