Acridine Orange Vital Staining Research Articles

Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells.

BackgroundMammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer.Materials and methodsThe cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment.ResultsAdvanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells.ConclusionOur results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

Spontaneous self-assembly of DNA fragments into nucleus-like structures from yolk granules of fertilized chicken eggs: Antoine Béchamp meets Bong Han Kim via Olga Lepeshinskaya

We found evidence that spontaneous self-assembly of DNA molecules from yolk granules occurred during the very early stage of egg fertilization. In order to find solid evidence for self-assembly of DNA molecules, we collected many available data in different stages of fertilized eggs, making a data table.At first by using acridine orange vital staining to demonstrate DNA, we noticed that some yolk granules emitted DNA signals that gradually increased with increasing incubation time from very small sizes to much larger nucleus-like structures. For convincing evidence, we also used another vital dye, Hoechst 33258 DNA-specific dye, to trace the changes in the yolk granules. The patterns of the DNA signals from yolk granules stained with Hoechst 33258 were the same as those from the yolk granules stained with acridine orange. A partial phase contrast microscopic image of the changes in the yolk granules showed some liquid-like material around the granules before the formation of the nucleus-like structures. Concomitant use of fluorescence and partial phase contrast microscopy suggested that these liquid-like materials may have been released from yolk granules in which spontaneous self-assembly of DNA molecules had occurred. Finally, in order to verify whether the DNA signals came from real DNA molecules or not, by using deoxyribonuclease I (DNAse), we confirmed that the nucleus-like structures were really assembled DNA molecules.Thus, in this article, we report evidence for the self-assembly of DNA molecules toward cell-like structures and discuss our findings, comparing them with those in the works of other pioneers, especially Antoine Béchamp, Olga Lepeshinskaya and Bong Han Kim, who insisted on the existence of a mitosis-free alternative pathway for generating new cells.

Evidence for the fusion of extracellular vesicles with/without DNA to form specific structures in fertilized chicken eggs, mice and rats

With a combination of cultivation and phase-contrast and fluorescence microscopic observation, we first found that fusion of extracellular vesicles with or without membranes occurred in fertilized chicken eggs. In order to find solid evidence for fusion, we collected many fusion data from various tissues; primo vessels and pancreases of mice and pancreases and omentums of rats. Especially, by using acridine orange vital staining to demonstrate DNA and phase-contrast and fluorescence microscopy for long real-time observation, we found that many of the extracellular vesicles involved in the fusion process contained DNAs. The fusions fall into two main patterns: pattern A characterizes a fusion of less agitated extracellular vesicles without membranes. Pattern B is a fusion of vigorously vibrating extracellular vesicles in a certain membrane. Considering all data, tables, pictures and movies, we were able to show fusions of DNA extracellular vesicles without or with membranes in several tissues of three species. Interestingly, some of the fused structures share the same morphology as normal cell's in terms of overall shape, size and DNA signals in the center. Thus, in this article we first report the evidence for the fusion of extracellular vesicles with/without DNA toward a specific structure and discuss our findings by comparing with those of other pioneer's works in search for a mitosis-free alternative pathway for generating new cells.

Cadmium accumulation and binding characteristics in intact Sertoli/germ cell units, and associated effects on stage‐specific functions in vitro: insights from a shark testis model

The increased human use of cadmium (Cd) and its increased occurrence in the environment is of concern. The testis is sensitive to Cd because of the steroid-mediated regulation of spermatogenesis, high levels of DNA synthesis and gene transcription, all of which varies in a stage-related manner. Validated techniques (acridine orange vital staining to detect apoptosis and dextran-rhodamine exclusion to assess blood-testis barrier function) were recently developed and the shark testis was proposed as an alternative model for assessing stage-specific functions in living testicular tissue and to study toxicant actions on spermatogenesis. The present paper shows that 109Cd accumulation and binding in vitro was stage-dependent (premeiotic, PrM > meiotic, M > postmeiotic, PoM), rapid and persisted in spermatocysts (intact germ cell/Sertoli cell units) 49 h after washout. In competitive binding analyses of all three spermatocyst stages, Hg, but not Zn, could replace bound 109Cd, suggesting that Cd binding was specific. These findings were associated with a biphasic apoptotic response in the PrM spermatocysts, which was maximal at 10 microm CdCl2 and 1 microm CdCl2 after 2 and 4 days in culture, respectively. Although Cd uptake in PoM cysts was more than 2-fold less than uptake in PrM cysts, the percentage dextran-rhodamine permeant PoM cysts was approximately 8-fold greater than in controls in the presence of both 10 microm CdCl2 and 30 microm CdCl2 after 4 days culture, indicating that blood-testis barrier function in PoM spermatocysts was compromised. These findings demonstrate that this model has utility for use in screening assays of environmental toxicants.

Induction of Germline Cell Cycle Arrest and Apoptosis by Sodium Arsenite in Caenorhabditis elegans

The nematode Caenorhabditis elegans has been shown to be a model organism in studying aquatic toxicity. Although epidemiological studies have shown that arsenic is teratogenic and carcinogenic to humans, the lethality assay indicated that C. elegans is less sensitive to inorganic arsenic than any other organisms that have been tested thus far. In the present study, we used the more malleable germline of C. elegans as an in vivo system to investigate the genotoxic effects of arsenite. After animals were exposed to sodium arsenite at concentrations ranging from 1 microM to 0.5 mM, mitotic germ cells and germline apoptosis were scored after DAPI staining and acridine orange vital staining, respectively. DMSO rescue experiments were performed by exposing C. elegans to 0.01 mM arsenite in the presence of DMSO (0.1%) for 24 h, and reactive oxygen species (ROS) were semiquantified by CM-H(2)DCFDA vital staining. The results indicated that arsenic exposure reduced the brood size of C. elegans and caused mitotic cell cycle arrest and germline apoptosis, which, to some extent, exhibited a concentration- and time-dependent manner. The addition of 0.1% DMSO completely rescued arsenic-induced cell cycle arrest and partially suppressed germline apoptosis. Furthermore, treatment of animals with arsenite at a dose of 0.01 mM significantly increased ROS production in the intestine, which could be reduced by DMSO treatment. The present study also indicated that C. elegans might be used as an in vivo model system to study the mechanisms of arsenic-induced genotoxic effects.

Biphosphinic palladacycle complex mediates lysosomal-membrane permeabilization and cell death in K562 leukaemia cells

The cell death mechanism of cytotoxicity induced by the Biphosphinic Palladacycle Complex (BPC) was studied using a K562 leukaemia cell line. The IC 50 values obtained for K562 cells post-72 h of BPC were less than 5.0 μM by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue assays. Using the Acridine Orange vital staining combining fluorescence microscopy it was observed that the complex triggers apoptosis in K562 cells, inducing DNA fragmentation, as analysed through electrophoresis. Lysosomal-membrane permeabilization was also observed in K562 cells post-5 h of BPC, which suggests intralysossomal accumulation by proton-trapping, since its p K a value ranged from 5.1 to 6.5. Caspase-3, and -6 activity induced by BPC in K562 cells was prevented by the cathepsin-B inhibitor [ N-( l-3- trans-propylcarbamoyl-oxirane-2-carbonyl)- l-isoleucyl- l-proline] (CA074). These events occurred in the presence of endogenous bcl-2 and bax expression. Acute toxicological studies demonstrated that BPC produces no lesions for liver and kidney fourteen-days after drug administration (100 mg/kg — i.p.). White and red blood cells of BPC-treated mice presented normal morphological characteristics. Taken together, these data suggest a novel lysosomal pathway for BPC-induced apoptosis, in which lysosomes are the primary target and cathepsin B acts as death mediator.

Non-apoptotic concentrations of prodigiosin (H +/Cl − symporter) inhibit the acidification of lysosomes and induce cell cycle blockage in colon cancer cells

Prodigiosin (PG) is a bacterial red pigment with interesting immunosuppressive and apoptotic properties that have been partly attributed to its ability to uncouple V-ATPase through the promotion of the H +/Cl − symporter. In the present study, we investigate the effect of non-apoptotic concentrations of PG on the lysosomal-pH and on cell cycle progression in colon cancer cells. Lysosomal-pH was tested in DLD-1 cells using acridine orange vital staining. Orange granules, indicative of acidified lysosomes, decreased significantly in cells treated with 25 nM of PG for 1 / 2 h, and disappeared completely at 100 nM. This suggests that PG can induce lysosomal alkalinization without any apparent cytotoxic effect. Cell cycle progression was analysed in HT29 cells and we found that PG induces a blockage in the G1 phase. This blockage correlates with p21 WAF1/CIP1 induction, and it can be triggered either dependently or independently of p53. In conclusion, the reversible increase in lysosomal-pH and cytosol acidification induced by non-apoptotic concentrations of PG in colon cancer cells, suggests that the apoptotic process induced by PG can not be solely explained by changes in intracellular pH. The effect of intracellular acidification on cell cycle arrest must be analysed more exactly.

Functional competence of peritoneal macrophages in murine Lyme borreliosis

In mice infected with the Lyme disease spirochete, Borrelia burgdorferi, spirochetes can be cultured from the uninflamed peritoneal cavity despite their proximity to resident macrophages. Here we have evaluated the phagocytic and killing capacity of peritoneal macrophages ex vivo from such mice, and compared them to cells from uninfected controls. We noted no significant differences in the numbers of cells recovered from lavage fluid nor in the proportion of lymphocytes in that fluid or their type or state of activation; B cells vs. T cells, CD4 vs. CD8, T cell receptor display (TCRαβ vs. TCRγδ), or B cell activation markers (CD24 vs. CD44). In addition, in studies conducted with coded samples, macrophages from infected animals were similar to controls in their ability (i) to bind and ingest spirochetes as shown by immunofluorescent kinetic studies imaged by confocal microscopy, (ii) to kill spirochetes as detected by acridine orange vital staining in live confocal microscopy, and (iii) to generate H2O2, a measure of their activation. Finally, we observed no difference in the ability of cells isolated from infected mice to kill the parasite, Toxoplasma gondii, a sensitive functional measure of the activation of killing capacity. Thus, despite the failure of immune clearance that results in persistent or chronic disease in vivo, there does not appear to be a global impairment of macrophage function in the infected host. It remains to be determined whether specific sites of inflammation, e.g., skin, joints, heart, are associated with a downregulation of phagocyte function locally.

Rapid, exponential growth and increased biomass yield of someFrankia strains in buffered and stirred mineral medium (BAP) with phosphatidyl choline

Rapid, exponential growth ofFrankia sp. BR (ORS 020608) isolated fromCasuarina equisetifolia was found to require stirring (magnetic bar) and adequate buffering (MES or MOPS buffers). We used a mineral medium (BAP) at 28°C with propionate and NH4+ as the sole carbon and nitrogen source respectively. Sporulation was enhanced by stirring, but the addition of phosphatidyl choline was found to impair sporulation and slightly improve growth. In these conditions a generation time of 14–16 hours was observed with a maximal biomass yield of 108 μg of protein × mL-1 (Bicinchoninic acid) at day 4 when growth ceased. In stirred conditionFrankia growth as microcolonies (30–200 μm diameter) showing an ‘open mesh’ type of mycelium and also a random distribution of growing hyphae inside and in the periphery of the microcolonies (acridine orange vital staining). In static conditions macrocolonies and only peripheric growth can be observed. Optimal growth in buffered and stirred conditions in mineral medium has been also observed with Cj and CeFFrankia isolates. The reasons for rapid and balanced hyphal growth in stirred conditions are discussed.

Cellular changes attending mengovirus-induced cytolysis of mouse L-cells.

Abstract 1. 1. Mengovirus-induced cytopathology in L-929 cell is shown to be moderated by the host-cell's stage. Late S and G2-M cells resist both artificial lysis with deoxycholate and virus-induced cytolysis. 2. 2. Nuclear lesions are found to precede lysosomal damage as measured by acridine orange vital staining and direct measurement of enzyme release. 3. 3. Nuclear protein alterations whic occur during development of mengovirus-induced cytopathology include leakage to the cytoplasm and extracellular fluids and an oxidation-polymerization of certain non-histone proteins. The synthetic rate of major acid-soluble proteins does not increase during the early hours of infection. 4. 4. These results are considered with respect to the question of the primary event leading to mengovirus-induced cytolysis. The primary role of lysosomes is questioned.

Fluorescence Microscopic Studies on the Trematodes Eggs

In this report it was tried to discriminate the dead from the live eggs of trematodes (Paragonimus westermani and Clonorchis sinensis) by means of fluorescence microscopy.In the beginning, the attitude of the eggs of the trematodes toward the acridine orange vital staining was examined comparing with that of nematodes eggs (Ascaris, Ancylostoma etc.). As the result, it was recognized that the eggs of the both trematodes had two characters seemed to be inconsistent each orther: (1) They were liable to be injured by acridine orange solution in contrast to the eggs of nematodes, because the contents (germs and yolk cells or miracidium) of the live eggs, immersed in aqueous solution of acridine orange for several days, emitted secondary fluorescence in yellowish green. (2) On the other hand, the trematodes eggs were more resistant to the staining with acridine orange than the nematodes eggs, because by the treatment with acridine orange in short time the contents of the dead eggs did not emit secondary fluorescence. These characters, differed from that of nematodes, were more remarkable in the eggs of Paragonimus westermani than Clonorchis sinensis.As a result of the experiments, the condition of the acridine orange vital staining, suitable for the discrimination of the dead from the live eggs of the trematodes, was decided as follows:In case of acridine orange concentration in 1:5, 000 and temperature at 26°C, the time for the staining was in need of 5∼6 hours for the Paragonimus eggs, and (10∼) 60 minutes for the Clonorchis eggs.When the trematodes eggs were killed by various methods (boiling, drying, action of chemicals etc.) and then observed under the fluorescence microscope after the treatment with acridine orange according to the above mentioned condition, the contents of the live eggs did not fluoresce, but the most of the dead ones emitted secondary fluorescence in yellowish green.Therefore, it was proved that on the eggs of the trematodes it was able to discriminate the dead from the live ones according to the observation whether their contents fluoresce or not. And the observation of autofluorescence in the contents of eggs was also helpful for the discrimination.