Morphology-based optical separation of subpopulations from a heterogeneous murine breast cancer cell line.

PubMed

Tamura, Masato; Sugiura, Shinji; Takagi, Toshiyuki; Satoh, Taku; Sumaru, Kimio; Kanamori, Toshiyuki; Okada, Tomoko; Matsui, Hirofumi

2017-01-01

Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated in vitro by using a spheroid assay and in vivo by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics.

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

PubMed Central

Meshki, John; Douglas, Steven D.; Hu, Mingyue; Leeman, Susan E.; Tuluc, Florin

2011-01-01

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells. PMID:21966499

Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S2 cells through reactive oxygen species and the mitochondria-dependent pathways.

PubMed

Huang, S-H; Hsu, M-H; Hsu, S-C; Yang, J-S; Huang, W-W; Huang, A-C; Hsiao, Y-P; Yu, C-C; Chung, J-G

2014-03-01

We have reported previously that phenethyl isothiocyanate (PEITC) induces apoptosis in human osteosarcoma U-2 OS cells. Cytotoxic activity of PEITC towards other cancer cells such as human malignant melanoma and skin cancer cells has not been reported. In this study, the anticancer activity of PEITC towards human malignant melanoma cancer A375.S2 cells was investigated. To determine the mechanisms of PEITC inhibition of cell growth, the following end points were determined in A375.S2 cells: cell morphological changes, cell cycle arrest, DNA damage and fragmentation assays and morphological assessment of nuclear change, reactive oxygen species (ROS) and Ca(2+) generations, mitochondrial membrane potential disruption, and nitric oxide and 10-N-nonyl acridine orange productions, expression and activation of caspase-3 and -9, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, poly (adenosine diphosphate-ribose) polymerase, and cytochrome c release, apoptosis-inducing factor and endonuclease G. PEITC induced morphological changes in time- and dose-dependent manner. PEITC induced G2/M phase arrest and induced apoptosis via endoplasmic reticulum stress-mediated mitochondria-dependent pathway. Western blot analysis showed that PEITC promoted Bax expression and inhibited Bcl-2 expression associated with the disintegration of the outer mitochondrial membrane causing cytochrome c release, and activation of caspase-9 and -3 cascade leading to apoptosis. We conclude that PEITC-triggered apoptotic death in A375.S2 cells occurs through ROS-mediated mitochondria-dependent pathways.

Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong-Ju; He, Wen-Qi; Chen, Ling

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however,more » were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.« less

[Phloretin induces apoptosis of BEL-7402 cells in vitro].

PubMed

Luo, Hui; Wang, Ya-jun; Chen, Jie; Liu, Jiang-qin; Zhang, Hai-tao

2008-07-01

To examine the effect of phloretin on apoptosis of BEL-7402 cells. The viability changes of BEL- 7402 cells as a result of phloretin-induced toxicity were analyzed using MTT assay, and the cell morphology changes were observed with fluorescence microscope. Flow cytometry was used to analyze the cell cycle and mitochondrial membrane potential changes, and chromogenic substrate assay performed to detect caspase activity. Phloretin induced obvious cytotoxicity against BEL-7402 cells with IC50 of 89.23 microg/mL. The growth curve demonstrated decreased growth of the cells as phloretin concentration increased. Cell apoptosis occurred 24 h after treatment with 40-160 microg/mL phloretin. Morphological, the cells exposed to phloretin exhibited nuclear chromatin condensation and increased fluorescence intensity. The activity of caspase-9 reached the peak level 12 h after phloretin exposure, and leak levels of caspase-6 and caspase-3 activities occurred 18 and 24 h after the exposure, respectively. Phloretin can induce BEL-7402 cell apoptosis though the mitochondrial pathway.

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation

PubMed Central

Schlicher, Robyn K.; Hutcheson, Joshua D.; Radhakrishna, Harish; Apkarian, Robert P.; Prausnitz, Mark R.

2010-01-01

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphological changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced non-lytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure. PMID:20350691

Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

NASA Astrophysics Data System (ADS)

Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-04-01

Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

PubMed Central

Hagiwara-Chatani, Natsumi; Shirai, Kota; Kido, Takumi; Horigome, Tomoatsu; Yasue, Akihiro; Adachi, Naoki; Hirai, Yohei

2017-01-01

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche. PMID:28057922

[The mechanism of docetaxel-induced apoptosis in human lung cancer cells].

PubMed

Li, Y; Shi, T; Zhao, W

2000-05-01

To study the mechanism of docetaxel-induced apoptosis. Morphological study, DNA gel electrophoresis, flow cytometry and fluorescin labeled Annexin V to detect apoptosis, RT-PCR to detect the gene related with apoptosis. Human lung cancer A549 cells treated with docetaxel induced cell cycle arrest at G2M phase, leading to apoptosis. The morphology of A549 showed nuclear chromatine condensation and fragmentation. Typical ladder pattern of DNA fragmentation was observed. Sub-G1 peak was found by flow cytometry. Transcription of Fas gene was enhanced, while no change in c-myc and bcl-2 genes. Annexin labeling results revealed the co-existence of cell apoptosis and necrosis in docetaxel-treated A549 cells. Docetaxel induces apoptosis and necrosis of human lung cancer. The induction of apoptosis may be related to expression of Fas.

Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.

PubMed

Kume, Toshiaki; Kawato, Yuka; Osakada, Fumitaka; Izumi, Yasuhiko; Katsuki, Hiroshi; Nakagawa, Takayuki; Kaneko, Shuji; Niidome, Tetsuhiro; Takada-Takatori, Yuki; Akaike, Akinori

2008-10-10

Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

INHIBITION OF INDUCED DIFFERENTIATION OF C3H/1OT 1/2 CLONE 8 MOUSE EMBRYO CELLS BY TUMOR PROMOTERS

EPA Science Inventory

C3H/10T 1/2 cells were induced to differentiate into muscle cells by treatment with 5-azacytidine, and the effects of tumor promoters, nonpromoters, and inhibitors of tumor promotion on this induced differentiation were investigated. Cell morphology was dramatically changed withi...

Induction of apoptosis of liver cancer cells by nanosecond pulsed electric fields (nsPEFs).

PubMed

He, Ling; Xiao, Deyou; Feng, Jianguo; Yao, Chenguo; Tang, Liling

2017-02-01

The application of nanosecond pulsed electric fields (nsPEFs) is a novel method to induce the death of cancer cells. NsPEFs could directly function on the cell membrane and activate the apoptosis pathways, then induce apoptosis in various cell lines. However, the nsPEFs-inducing-apoptosis action sites and the exact pathways are not clear now. In this study, nsPEFs were applied to the human liver cancer cells HepG2 with different parameters. By apoptosis assay, morphological observation, detecting the mitochondrial membrane potential (ΔΨ m ), intracellular calcium ion concentration ([Ca 2+ ]i) and the expressions of key apoptosis factors, we demonstrated that nsPEFs could induce the morphology of cell apoptosis, the change in ΔΨ m , [Ca 2+ ]i and the upregulation of some key apoptosis factors, which revealed the responses of liver cancer cells and indicated that cells may undergo apoptosis through the mitochondria-dependent pathway after nsPEFs were applied.

The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

PubMed

Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

1993-11-01

The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells – Relevance to Capsular Opacification

PubMed Central

Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

2016-01-01

Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation. PMID:27832099

Aluminium oxide nanoparticles induced morphological changes, cytotoxicity and oxidative stress in Chinook salmon (CHSE-214) cells.

PubMed

Srikanth, Koigoora; Mahajan, Amit; Pereira, Eduarda; Duarte, Armando Costa; Venkateswara Rao, Janapala

2015-10-01

Aluminium oxide nanoparticles (Al2 O3 NPs) are increasingly used in diverse applications that has raised concern about their safety. Recent studies suggested that Al2 O3 NPs induced oxidative stress may be the cause of toxicity in algae, Ceriodaphnia dubia, Caenorhabditis elegans and Danio rerio. However, there is paucity on the toxicity of Al2 O3 NPs on fish cell lines. The current study was aimed to investigate Al2 O3 NPs induced cytotoxicity, oxidative stress and morphological abnormality of Chinnok salmon cells (CHSE-214). A dose-dependent decline in cell viability was observed in CHSE-214 cells exposed to Al2 O3 NPs. Oxidative stress induced by Al2 O3 NPs in CHSE-214 cells has resulted in the significant reduction of superoxide dismutase, catalase and glutathione in a dose-dependent manner. However, a significant increase in glutathione sulfo-transferase and lipid peroxidation was observed in CHSE-214 cells exposed to Al2 O3 NPs in a dose-dependent manner. Significant morphological changes in CHSE-214 cells were observed when exposed to Al2 O3 NPs at 6, 12 and 24 h. The cells started to detach and appear spherical at 6 h followed by loss of cellular contents resulting in the shrinking of the cells. At 24 h, the cells started to disintegrate and resulted in cell death. Our data demonstrate that Al2 O3 NPs induce cytotoxicity and oxidative stress in a dose-dependent manner in CHSE-214 cells. Thus, our current work may serve as a base-line study for future evaluation of toxicity studies using CHSE-214 cells. Copyright © 2015 John Wiley & Sons, Ltd.

Sigma Receptor Ligand, (+)-Pentazocine, Suppresses Inflammatory Responses of Retinal Microglia

PubMed Central

Zhao, Jing; Ha, Yonju; Liou, Gregory I.; Gonsalvez, Graydon B.; Smith, Sylvia B.; Bollinger, Kathryn E.

2014-01-01

Purpose. To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)–induced inflammatory changes in retinal microglia cells. Methods. Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. Results. The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine–mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine–mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release. Conclusions. Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia. PMID:24812552

Transactivation of TrkB by Sigma-1 receptor mediates cocaine-induced changes in dendritic spine density and morphology in hippocampal and cortical neurons

PubMed Central

Ka, Minhan; Kook, Yeon-Hee; Liao, Ke; Buch, Shilpa; Kim, Woo-Yang

2016-01-01

Cocaine is a highly addictive narcotic associated with dendritic spine plasticity in the striatum. However, it remains elusive whether cocaine modifies spines in a cell type-specific or region-specific manner or whether it alters different types of synapses in the brain. In addition, there is a paucity of data on the regulatory mechanism(s) involved in cocaine-induced modification of spine density. In the current study, we report that cocaine exposure differentially alters spine density, spine morphology, and the types of synapses in hippocampal and cortical neurons. Cocaine exposure in the hippocampus resulted in increased spine density, but had no significant effect on cortical neurons. Although cocaine exposure altered spine morphology in both cell types, the patterns of spine morphology were distinct for each cell type. Furthermore, we observed that cocaine selectively affects the density of excitatory synapses. Intriguingly, in hippocampal neurons cocaine-mediated effects on spine density and morphology involved sigma-1 receptor (Sig-1 R) and its downstream TrkB signaling, which were not the case in cortical neurons. Furthermore, pharmacological inhibition of Sig-1 R prevented cocaine-induced TrkB activation in hippocampal neurons. Our findings reveal a novel mechanism by which cocaine induces selective changes in spine morphology, spine density, and synapse formation, and could provide insights into the cellular basis for the cognitive impairment observed in cocaine addicts. PMID:27735948

Silencing of ATP11B by RNAi-Induced Changes in Neural Stem Cell Morphology.

PubMed

Wang, Jiao; Wang, Qian; Zhou, Fangfang; Wang, Dong; Wen, Tieqiao

2017-01-01

RNA interference (RNAi) technology is one of the main research tools in many studies of neural stem cells. This study describes effects of ATP11B on the morphology change of neural stem cells by using RNAi. ATP11B belongs to P4-ATPases family, which is preferential translocate phosphatidylserine of cell membrane. Although it exists in neural stem cells, its physiological function is poorly understood. By using RNAi technology to downregulate expression of ATP11B, we found distinct morphological changes in neural stem cells. More important, psiRNA-ATP11B-transfected cells displayed short neurite outgrowth compared to the control cells. These data strongly suggest that ATP11B plays a key role in the morphological change of neural stem cells.

Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi

2013-01-04

Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delaymore » of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.« less

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

PubMed Central

Hakansson, Anders P.; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-01-01

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells. PMID:21423701

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

PubMed

Hakansson, Anders P; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-03-10

Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

BolA inhibits cell elongation and regulates MreB expression levels.

PubMed

Freire, Patrick; Moreira, Ricardo Neves; Arraiano, Cecília Maria

2009-02-06

The morphogene bolA is a general stress response gene in Escherichia coli that induces a round morphology when overexpressed. Results presented in this report show that increased BolA levels can inhibit cell elongation mechanisms. MreB polymerization is crucial for the bacterial cell cytoskeleton, and this protein is essential for the maintenance of a cellular rod shape. In this report, we demonstrate that bolA overexpression affects the architecture of MreB filaments. An increase in BolA leads to a significant reduction in MreB protein levels and mreB transcripts. BolA affects the mreBCD operon in vivo at the level of transcription. Furthermore, our results show that BolA is a new transcriptional repressor of MreB. The alterations in cell morphology induced by bolA seem to be mediated by a complex pathway that integrates PBP5, PBP6, MreB, and probably other regulators of cell morphology/elongation.

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro.

PubMed

Tiwari, Meenakshi; Tripathi, Anima; Chaube, Shail K

2017-01-01

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H 2 O 2 )-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H 2 O 2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H 2 O 2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H 2 O 2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H 2 O 2 -induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H 2 O 2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes.

PubMed

Petrovsky, Roman; Krohne, Georg; Großhans, Jörg

2018-03-01

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope. Copyright © 2018 Elsevier GmbH. All rights reserved.

Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling.

PubMed

Zhao, Li; Li, Jing; Hao, Yan Hui; Gao, Ya Bing; Wang, Shui Ming; Zhang, Jing; Dong, Ji; Zhou, Hong Mei; Liu, Shu Chen; Peng, Rui Yun

2017-05-01

To investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms. NK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01). Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Carbon nanowall scaffold to control culturing of cervical cancer cells

NASA Astrophysics Data System (ADS)

Watanabe, Hitoshi; Kondo, Hiroki; Okamoto, Yukihiro; Hiramatsu, Mineo; Sekine, Makoto; Baba, Yoshinobu; Hori, Masaru

2014-12-01

The effect of carbon nanowalls (CNWs) on the culturing rate and morphological control of cervical cancer cells (HeLa cells) was investigated. CNWs with different densities were grown using plasma-enhanced chemical vapor deposition and subjected to post-growth plasma treatment for modification of the surface terminations. Although the surface wettability of the CNWs was not significantly dependent on the CNW densities, the cell culturing rates were significantly dependent. Morphological changes of the cells were not significantly dependent on the density of CNWs. These results indicate that plasma-induced surface morphology and chemical terminations enable nanobio applications using carbon nanomaterials.

KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

PubMed Central

Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

2015-01-01

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

Elastocapillary Instability in Mitochondrial Fission

NASA Astrophysics Data System (ADS)

Gonzalez-Rodriguez, David; Sart, Sébastien; Babataheri, Avin; Tareste, David; Barakat, Abdul I.; Clanet, Christophe; Husson, Julien

2015-08-01

Mitochondria are dynamic cell organelles that constantly undergo fission and fusion events. These dynamical processes, which tightly regulate mitochondrial morphology, are essential for cell physiology. Here we propose an elastocapillary mechanical instability as a mechanism for mitochondrial fission. We experimentally induce mitochondrial fission by rupturing the cell's plasma membrane. We present a stability analysis that successfully explains the observed fission wavelength and the role of mitochondrial morphology in the occurrence of fission events. Our results show that the laws of fluid mechanics can describe mitochondrial morphology and dynamics.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

PubMed

Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

2015-03-01

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells

PubMed Central

Shaari, Khozirah; Rosli, Rozita

2018-01-01

Reactive oxygen species are well known for induction of oxidative stress conditions through oxidation of vital biomarkers leading to cellular death via apoptosis and other process, thereby causing devastative effects on the host organs. This effect is believed to be linked with pathological alterations seen in several neurodegenerative disease conditions. Many phytochemical compounds proved to have robust antioxidant activities that deterred cells against cytotoxic stress environment, thus protect apoptotic cell death. In view of that we studied the potential of glucomoringin-isothiocyanate (GMG-ITC) or moringin to mitigate the process that lead to neurodegeneration in various ways. Neuroprotective effect of GMG-ITC was performed on retinoic acid (RA) induced differentiated neuroblastoma cells (SHSY5Y) via cell viability assay, flow cytometry analysis and fluorescence microscopy by means of acridine orange and propidium iodide double staining, to evaluate the anti-apoptotic activity and morphology conservation ability of the compound. Additionally, neurite surface integrity and ultrastructural analysis were carried out by means of scanning and transmission electron microscopy to assess the orientation of surface and internal features of the treated neuronal cells. GMG-ITC pre-treated neuron cells showed significant resistance to H2O2-induced apoptotic cell death, revealing high level of protection by the compound. Increase of intracellular oxidative stress induced by H2O2 was mitigated by GMG-ITC. Thus, pre-treatment with the compound conferred significant protection to cytoskeleton and cytoplasmic inclusion coupled with conservation of surface morphological features and general integrity of neuronal cells. Therefore, the collective findings in the presence study indicated the potentials of GMG-ITC to protect the integrity of neuron cells against induced oxidative-stress related cytotoxic processes, the hallmark of neurodegenerative diseases. PMID:29723199

Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells.

PubMed

Jaafaru, Mohammed Sani; Nordin, Norshariza; Shaari, Khozirah; Rosli, Rozita; Abdull Razis, Ahmad Faizal

2018-01-01

Reactive oxygen species are well known for induction of oxidative stress conditions through oxidation of vital biomarkers leading to cellular death via apoptosis and other process, thereby causing devastative effects on the host organs. This effect is believed to be linked with pathological alterations seen in several neurodegenerative disease conditions. Many phytochemical compounds proved to have robust antioxidant activities that deterred cells against cytotoxic stress environment, thus protect apoptotic cell death. In view of that we studied the potential of glucomoringin-isothiocyanate (GMG-ITC) or moringin to mitigate the process that lead to neurodegeneration in various ways. Neuroprotective effect of GMG-ITC was performed on retinoic acid (RA) induced differentiated neuroblastoma cells (SHSY5Y) via cell viability assay, flow cytometry analysis and fluorescence microscopy by means of acridine orange and propidium iodide double staining, to evaluate the anti-apoptotic activity and morphology conservation ability of the compound. Additionally, neurite surface integrity and ultrastructural analysis were carried out by means of scanning and transmission electron microscopy to assess the orientation of surface and internal features of the treated neuronal cells. GMG-ITC pre-treated neuron cells showed significant resistance to H2O2-induced apoptotic cell death, revealing high level of protection by the compound. Increase of intracellular oxidative stress induced by H2O2 was mitigated by GMG-ITC. Thus, pre-treatment with the compound conferred significant protection to cytoskeleton and cytoplasmic inclusion coupled with conservation of surface morphological features and general integrity of neuronal cells. Therefore, the collective findings in the presence study indicated the potentials of GMG-ITC to protect the integrity of neuron cells against induced oxidative-stress related cytotoxic processes, the hallmark of neurodegenerative diseases.

Puerarin protects against lead-induced cytotoxicity in cultured primary rat proximal tubular cells.

PubMed

Liu, Gang; Li, Zifa; Wang, Jinqiu; Wang, Hong; Wang, Zhenyong; Wang, Lin

2014-10-01

Puerarin, a potent free radicals scavenger, has been demonstrated to have protective efficacy in oxidative damage induced by nephrotoxins. In the present study, the attenuating effect of puerarin (PU) on lead (Pb)-induced apoptosis and oxidative stress was investigated in cultured primary rat proximal tubular (rPT) cells. Results showed that exposure to 0.5 µM Pb induced a decrease in cell viability accompanied with obvious cellular morphological alterations and caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, depletion of mitochondrial membrane potential (ΔΨ) and intracellular glutathione (GSH); elevation of caspase-3 activity, intracellular reactive oxygen species, and malondialdehyde levels; and inhibition of GSH peroxidase (GSH-Px) activity were revealed in the cells exposed to Pb alone. However, simultaneous supplementation with PU (50 and 100 µM) protected rPT cells from Pb-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function, and elevating the intracellular antioxidants (nonenzymatic and enzymic) levels. In conclusion, these findings suggested that PU, as a widely distributed dietary antioxidant, contributes potentially to inhibition of Pb-induced cytotoxicity in rPT cells. © The Author(s) 2014.

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

PubMed

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Low-level lasers affect Escherichia coli cultures in hyperosmotic stress

NASA Astrophysics Data System (ADS)

Pinheiro, C. C.; Barboza, L. L.; Paoli, F.; Fonseca, A. S.

2015-08-01

Physical characteristics and practical properties have made lasers of interest for biomedical applications. Effects of low-level lasers on biological tissues could occur or be measurable depending on cell type, presence of a pathologic process or whether the cells are in an adverse environment. The objective of this work was to evaluate the survival, morphology and filamentation of E. coli cells proficient and deficient in the repair of oxidative DNA lesions exposed low-level red and infrared lasers submitted to hyperosmotic stress. Wild type and endonuclease VIII deficient E. coli cells in exponential and stationary growth phase were exposed to red and infrared lasers and submitted to hyperosmotic stress. Cell viability, filamentation phenotype and cell morphology were evaluated. Cell viability was not significantly altered but previous laser exposure induced filamentation and an altered area of stressed cells depending on physiologic condition and presence of the DNA repair. Results suggest that previous exposure to low-level red and infrared lasers could not affect viability but induced morphologic changes in cells submitted to hyperosmotic stress depending on physiologic conditions and repair of oxidative DNA lesions.

The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spincemaille, Pieter; Pham, Duc-Hung; Chandhok, Gursimran

2014-10-15

Background: Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cu-toxicity zebrafish larvae model applicable to WD. Methods: The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on livermore » morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. Results: OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7B{sup H1069Q}, but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cu-induced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. Conclusions: OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. General significance: All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment. - Highlights: • Wilson disease (WD) is characterized by accumulation of toxic copper (Cu). • OSIP108 increases viability of Cu-treated cellular models applicable to WD. • OSIP108 injections preserve liver morphology of Cu-treated zebrafish larvae. • OSIP108 injections into zebrafish larvae abrogates Cu-induced oxidative stress.« less

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death.

PubMed

Garcia-Belinchón, Mercè; Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Casanelles, Elisenda; Ribas, Judit; Yuste, Victor J

2015-08-21

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death*

PubMed Central

Garcia-Belinchón, Mercè; Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Casanelles, Elisenda; Ribas, Judit; Yuste, Victor J.

2015-01-01

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as “apoptosis-necrosis continuum.” To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death. PMID:26124276

[Differentiation of human periodontal ligament stem cells into neuron-like cells in vitro].

PubMed

Zhen, Lei; Liu, Hong-Wei

2009-02-01

To isolate and purify the human periodontal ligament stem cells (PDLSC) and investigate the differentiation potentials of PDLSC into neuron-like cells in vitro. PDLSC were isolated and cultivated. PDLSC of passage 2 was plated at a density of 5 x 10(3) per mL. At 80% confluence, the PDLSC were preinduced for 24 hours, and were subsequently replaced with an inducing medium containing certain concentration of 13-mercaptoethanal (beta-ME). After 6 hours of induction, the results were evaluated by morphological observation, immunocytochemical staining for neuron specific enolase (NSE), neurofilament (NF) and glial fibrillary acid protein (GFAP) expression and RT-PCR for NSE, NF, GFAP mRNA. Meanwhile, the uninduced PDLSC were used as a negative control. PDLSC could be differentiate into cells with typical neuronal morphology. Immunohisto-chemistry and RT-PCR confirmed that the induced cells expressed NSE and NF, two marked enzymes of neuron cell. PDLSC can be induced into neuron-like cells in vitro. PDLSC have the capability of multilineage differentiations.

Cannabinoids induce incomplete maturation of cultured human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murison, G.; Chubb, C.B.H.; Maeda, S.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. Themore » THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.« less

Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1.

PubMed

Mai, Anja; Muharram, Ghaffar; Barrow-McGee, Rachel; Baghirov, Habib; Rantala, Juha; Kermorgant, Stéphanie; Ivaska, Johanna

2014-05-01

Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in β1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.

Lipophilic organic pollutants induce changes in phospholipid and membrane protein composition leading to Vero cell morphological change.

PubMed

Liao, Ting T; Wang, Lei; Jia, Ru W; Fu, Xiao H; Chua, Hong

2014-01-01

Membrane damage related to morphological change in Vero cells is a sensitive index of the composite biotoxicity of trace lipophilic chemicals. However, judging whether the morphological change in Vero cells happens and its ratio are difficult because it is not a quantitative characteristic. To find biomarkers of cell morphological change for quantitatively representing the ratio of morphological changed cell, the mechanism of cell membrane damage driven by typical lipophilic chemicals, such as trichlorophenol (TCP) and perfluorooctanesulphonate (PFOS), was explored. The ratio of morphologically changed cells generally increased with increased TCP or PFOS concentrations, and the level of four major components of phospholipids varied with concentrations of TCP or PFOS, but only the ratio of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) decreased regularly as TCP or PFOS concentrations increased. Analysis of membrane proteins showed that the level of vimentin in normal cell membranes is high, while it decreases or vanishes after TCP exposure. These variations in phospholipid and membrane protein components may result in membrane leakage and variation in rigid structure, which leads to changes in cell morphology. Therefore, the ratio of PC/PE and amount of vimentin may be potential biomarkers for representing the ratio of morphological changed Vero cell introduced by trace lipophilic compounds, thus their composite bio-toxicity.

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

PubMed Central

Sorokin, Dmitry V; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, Pavel; Kozubek, Stanislav; Matula, Pavel; Skalníková, Magdalena; Raška, Ivan; Bártová, Eva

2015-01-01

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase. PMID:26208041

[Apoptosis and activity changes of telomerase induced by essential oil from pine needles in HepG2 cell line].

PubMed

Wei, Feng-xiang; Li, Mei-yu; Song, Yu-hong; Li, Hong-zhi

2008-08-01

To study the effects of essential oil extracted from pine needles on HepG2 cell line. HepG2 cells were treated with essential oil extracted from pine needles. Cell growth rate was determined with MTF assay, cell morphologic changes were examined under transmission electromicroscope and HE straining. Flow cytometry was used to exmine apoptotic cells. Bcl-2 gene expression was determined by flow cytometry and telomerase activity by TRAP assay. Essential oils from pine needles could not only repress the growth of HepG2 cells significantly, but also induce apoptosis to them. Both dose-effect and time-effect relationship could be confirmed. Typical morphology changes of apoptosis such as nuclear enrichment and karyorrhexis were observed through transmission electromicroscope and HE straining. Telomerase activity was down regulated in the essential oil extracted from pine needles induced apoptotic cells. The expression of bcl-2 gene was suppressed after the essential oil from pine needles treatement. The essential oil extracted from pine needles can inhibit cell growth of HepG2 cell line and induce apoptosis, which may associate with inhibition of telomerase activity and bcl-2 may be involved in the regulation of telomerase activity.

Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2015-02-01

In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

Metformin inhibits the radiation-induced invasive phenotype of esophageal squamous cell carcinoma.

PubMed

Nakayama, Akira; Ninomiya, Itasu; Harada, Shinichi; Tsukada, Tomoya; Okamoto, Koichi; Nakanuma, Shinichi; Sakai, Seisho; Makino, Isamu; Kinoshita, Jun; Hayashi, Hironori; Oyama, Katsunobu; Miyashita, Tomoharu; Tajima, Hidehiro; Takamura, Hiroyuki; Fushida, Sachio; Ohta, Tetsuo

2016-11-01

Esophageal cancer is one of the most aggressive tumor types because of its invasiveness and metastatic potential. Several reports have described an association between increased invasiveness after ionizing radiation (IR) treatment and epithelial-to-mesenchymal transition (EMT). The biguanide metformin is reported to prevent transforming growth factor-β (TGF-β)-induced EMT and proliferation of cancer. This study examined whether IR induces EMT and promotes the invasive potential of TE-9 esophageal squamous cell carcinoma cells and the effect of metformin on IR-induced EMT. After IR exposure, TE-9 cells showed a spindle-shaped morphology and lost cell-cell adhesion. Immunoblotting showed that IR induced expression of mesenchymal markers (vimentin and N-cadherin), transcription factors (Slug, Snail, and Twist), and matrix metalloproteinases. A scratch wound assay and Matrigel invasion assay showed that IR enhanced the invasive potential and migratory capacity of TE-9 cells. Expression of hypoxia-related factor-1α and TGF-β was increased after IR. IR also induced phosphorylation of Smad2 and Smad3. Metformin inhibited radiation-induced EMT-like morphological changes, and enhanced invasion and migration of TE-9 cells. Metformin inhibited IR-induced phosphorylation of Smad2 and Smad3. Although phosphorylation of AMP-activated protein kinase was enhanced by IR and metformin, phosphorylation of mammalian target of rapamycin was enhanced by IR and suppressed by metformin. These results indicated that metformin suppressed IR-induced EMT via suppression of the TGF-β-Smad phosphorylation pathway, and a part of the non-Smad pathway. Metformin might be useful to prevent IR-induced invasion and metastasis of esophageal squamous cell carcinoma.

Morphological Changes and Antibiotic-Induced Thermal Resistance in Vegetative Cells of Bacillus subtilis

PubMed Central

Dul, Michael J.; McDonald, William C.

1971-01-01

The morphology and thermal resistance of vegetative cells of Bacillus subtilis W168 were examined after growth at 37 and 53 C. Vegetative cells grown at 37 C exhibited a typical trilaminar morphology, whereas cells grown at 53 C exhibited a cell wall which was apparently thicker and more loosely organized and had a poorly defined periphery. A concurrent increase in thermal resistance to a heat shock of 60 C occurs with the change in cell wall morphology. The change to the aberrant cell wall form, or its reversal to the normal form, is always accompanied by the gain or the loss of thermal resistance, respectively. The inhibition of protein synthesis by chloramphenicol has little effect upon the acquisition of thermal resistance at 53 C. Addition of the disaccharide pentapeptide subunit to the cell wall peptidoglycan is apparently essential to growth at 53 C and the acquisition of thermal resistance, since both growth and thermal resistance are inhibited by bacitracin. Two antibiotics, penicillin and cycloserine, which inhibit the final cross-linking of the cell wall peptidoglycan at two separate points, do not affect the acquisition of thermal resistance at 53 C. These same antibiotics induce a high degree of thermal resistance at 37 C. It is proposed that a change in the cell wall structure is related to an increased thermal resistance. Images PMID:4995654

Evidence for a role of MRCK in mediating HeLa cell elongation induced by the C1 domain ligand HMI-1a3.

PubMed

Talman, Virpi; Gateva, Gergana; Ahti, Marja; Ekokoski, Elina; Lappalainen, Pekka; Tuominen, Raimo K

2014-05-13

Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of β2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology. Copyright © 2014 Elsevier B.V. All rights reserved.

Dichromatic and monochromatic laser radiation effects on survival and morphology of Pantoea agglomerans

NASA Astrophysics Data System (ADS)

Thomé, A. M. C.; Souza, B. P.; Mendes, J. P. M.; Soares, L. C.; Trajano, E. T. L.; Fonseca, A. S.

2017-05-01

Despite the beneficial effects of low-level lasers on wound healing, their application for treatment of infected injuries is controversial because low-level lasers could stimulate bacterial growth exacerbating the infectious process. Thus, the aim of this work was to evaluate in vitro effects of low-level lasers on survival, morphology and cell aggregation of Pantoea agglomerans. P. agglomerans samples were isolated from human pressure injuries and cultures were exposed to low-level monochromatic and simultaneous dichromatic laser radiation to study the survival, cell aggregation, filamentation and morphology of bacterial cells in exponential and stationary growth phases. Fluence, wavelength and emission mode were those used in therapeutic protocols for wound healing. Data show no changes in morphology and cell aggregation, but dichromatic laser radiation decreased bacterial survival in exponential growth phase and monochromatic red and infrared lasers increased bacterial survival at the same fluence. Simultaneous dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation and simultaneous dichromatic laser could be the option for treatment of infected pressure injuries by Pantoea agglomerans.

The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells.

PubMed

Maldonado, Maricela; Wong, Lauren Y; Echeverria, Cristina; Ico, Gerardo; Low, Karen; Fujimoto, Taylor; Johnson, Jed K; Nam, Jin

2015-05-01

The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e., proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Juri; Department of Pediatrics, Nippon Medical School, Tokyo; E-mail: juri-f@nms.ac.jp

2005-07-22

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate intomore » neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.« less

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Xionggao; Department of Ophthalmology, Hainan Medical College, Haikou; Wei, Yantao

2012-03-09

Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells inducedmore » by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGF{beta}-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.« less

Selenium suppresses glutamate-induced cell death and prevents mitochondrial morphological dynamic alterations in hippocampal HT22 neuronal cells.

PubMed

Ma, Yan-Mei; Ibeanu, Gordon; Wang, Li-Yao; Zhang, Jian-Zhong; Chang, Yue; Dong, Jian-Da; Li, P Andy; Jing, Li

2017-01-19

Previous studies have indicated that selenium supplementation may be beneficial in neuroprotection against glutamate-induced cell damage, in which mitochondrial dysfunction is considered a major pathogenic feature. However, the exact mechanisms by which selenium protects against glutamate-provoked mitochondrial perturbation remain ambiguous. In this study glutamate exposed murine hippocampal neuronal HT22 cell was used as a model to investigate the underlying mechanisms of selenium-dependent protection against mitochondria damage. We find that glutamate-induced cytotoxicity was associated with enhancement of superoxide production, activation of caspase-9 and -3, increases of mitochondrial fission marker and mitochondrial morphological changes. Selenium significantly resolved the glutamate-induced mitochondria structural damage, alleviated oxidative stress, decreased Apaf-1, caspases-9 and -3 contents, and altered the autophagy process as observed by a decline in the ratio of the autophagy markers LC3-I and LC3-II. These findings suggest that the protection of selenium against glutamate stimulated cell damage of HT22 cells is associated with amelioration of mitochondrial dynamic imbalance.

Emerging roles of apoptotic microtubules during the execution phase of apoptosis.

PubMed

Oropesa Ávila, Manuel; Fernández Vega, Alejandro; Garrido Maraver, Juan; Villanueva Paz, Marina; De Lavera, Isabel; De La Mata, Mario; Cordero, Mario D; Alcocer Gómez, Elizabet; Delgado Pavón, Ana; Álvarez Córdoba, Mónica; Cotán, David; Sánchez-Alcázar, José Antonio

2015-09-01

Apoptosis is a genetically programmed energy-dependent process of cell demise, characterized by specific morphological and biochemical events in which the activation of caspases has an essential role. During apoptosis the cytoskeleton participates actively in characteristic morphological rearrangements of the dying cell. This reorganisation has been assigned mainly to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent reports have showed that microtubules are reformed during the execution phase of apoptosis organizing an apoptotic microtubule network (AMN). AMN is organized behind plasma membrane, forming a cortical structure. Apoptotic microtubules repolymerization takes place in many cell types and under different apoptotic inducers. It has been hypothesized that AMN is critical for maintaining plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disorganization leads apoptotic cells to secondary necrosis and the release of potential toxic molecules which can damage neighbor cells and promotes inflammation. Therefore, AMN formation during physiological apoptosis or in pathological apoptosis induced by anti-cancer treatments is essential for tissue homeostasis and the prevention of additional cell damage and inflammation. © 2015 Wiley Periodicals, Inc.

Therapeutic effect comparison of hepatocyte-like cells and bone marrow mesenchymal stem cells in acute liver failure of rats.

PubMed

Li, Dongliang; Fan, Jingjing; He, Xiuhua; Zhang, Xia; Zhang, Zhiqiang; Zeng, Zhiyu; Ruan, Mei; Cai, Lirong

2015-01-01

To evaluate the therapeutic efficacy of rat bone marrow mesenchymal stem cells (BMSCs) induced into hepatocyte-like cells and of un-induced BMSCs in acute liver failure rats. BMSCs in highly homogenous passage 3 were cultured using the whole bone marrow adherent culture method. Hepatic-related characters were confirmed with morphology, RT-PCR analysis, glycogen staining and albumin (ALB) immunofluorescence assay. Carbon tetrachloride (CCl4) was injected intraperitoneally to establish an acute rat liver failure model. Hepatocyte-like cells or un-induced BMSCs were respectively injected into the models to examine rats' appearance, liver function assay and liver tissue pathology. Hepatocyte-like morphology, higher expression of cytokeratin 18 (CK18) mRNA and ALB protein, and glycogen accumulation were confirmed in the induced BMSCs. The transplanted DAPI-labeled BMSCs were localized in the liver tissue 3-14 days after transplantation. The levels of liver function indicators (AST, ALT, ALP, and TBIL) from transplanted rats were significant decreased and pathology was improved, indicating the recovery of liver function. However, the differences were statistically insignificant. Both hepatocyte-like cells and un-induced BMSCs had a similarly positively therapeutic efficacy on liver regeneration in rat liver failure model.

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

PubMed Central

Deakin, Nicholas O.; Turner, Christopher E.

2011-01-01

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin, and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly. PMID:21148292

Microenvironment Induced Spheroid to Sheeting Transition of Immortalized Human Keratinocytes (HaCaT) Cultured in Microbubbles Formed in Polydimethylsiloxane

PubMed Central

Chandrasekaran, Siddarth; Giang, Ut-Binh; King, Michael R.; DeLouise, Lisa A

2011-01-01

The in vivo cellular microenvironment is regulated by a complex interplay of soluble factors and signaling molecules secreted by cells and it plays a critical role in the growth and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, and disease development and progression. In this study we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100–300 µm in diameter formed in hydrophobic polymer polydimethylsiloxane (PDMS) with ~60–100 µm circular openings and aspect ratio ~3.5. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized keratinocyte cell line. We observe that HaCaT cells, seeded in microbubbles (15–20 cells / MB) and cultured under standard conditions, adopt a compact 3-D spheroidal morphology. Within 2–3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow factors secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not transition to a sheeting morphology even after several weeks of culture. The importance of soluble factor accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96 well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the growth media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca2+ concentration and changes in Ecadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy. PMID:21724250

Effect of nagilactone E on cell morphology and glucan biosynthesis in budding yeast Saccharomyces cerevisiae.

PubMed

Hayashi, Kengo; Yamaguchi, Yoshihiro; Ogita, Akira; Tanaka, Toshio; Kubo, Isao; Fujita, Ken-Ichi

2018-05-14

Nagilactones are norditerpene dilactones isolated from the root bark of Podocarpus nagi. Although nagilactone E has been reported to show antifungal activities, its activity is weaker than that of antifungals on the market. Nagilactone E enhances the antifungal activity of phenylpropanoids such as anethole and isosafrole against nonpathogenic Saccharomyces cerevisiae and pathogenic Candida albicans. However, the detailed mechanisms underlying the antifungal activity of nagilactone E itself have not yet been elucidated. Therefore, we investigated the antifungal mechanisms of nagilactone E using S. cerevisiae. Although nagilactone E induced lethality in vegetatively growing cells, it did not affect cell viability in non-growing cells. Nagilactone E-induced morphological changes in the cells, such as inhomogeneous thickness of the glucan layer and leakage of cytoplasm. Furthermore, a dose-dependent decrease in the amount of newly synthesized (1, 3)-β-glucan was detected in the membrane fractions of the yeast incubated with nagilactone E. These results suggest that nagilactone E exhibits an antifungal activity against S. cerevisiae by depending on cell wall fragility via the inhibition of (1, 3)-β-glucan biosynthesis. Additionally, we confirmed nagilactone E-induced morphological changes of a human pathogenic fungus Aspergillus fumigatus. Therefore, nagilactone E is a potential antifungal drug candidate with fewer adverse effects. Copyright © 2018 Elsevier B.V. All rights reserved.

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

PubMed

Tamminen, Jenni A; Myllärniemi, Marjukka; Hyytiäinen, Marko; Keski-Oja, Jorma; Koli, Katri

2012-07-01

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity. Copyright © 2012 Wiley Periodicals, Inc.

Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

PubMed

Chang, Chih-Wei; Su, Yu-Chin; Her, Guor-Mour; Ken, Chuian-Fu; Hong, Jiann-Ruey

2011-01-01

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12-24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24-48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.

Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

PubMed

Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

2017-06-01

Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca 2+ , mitochondria membrane potential (ΔΨ m ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca 2+ production, and decreased the level of ΔΨ m and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways. © 2017 Wiley Periodicals, Inc.

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

PubMed

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

Changes in neutrophil morphology and morphometry following exposure to cigarette smoke.

PubMed Central

Lannan, S.; McLean, A.; Drost, E.; Gillooly, M.; Donaldson, K.; Lamb, D.; MacNee, W.

1992-01-01

Acute cigarette smoking delays neutrophils within the pulmonary circulation in some smokers. Evidence from an in-vitro Micropore filter model of the pulmonary capillaries indicates that this may be due to a smoke induced decrease in cell deformability. In order to determine whether changes in cell shape are associated with the observed decrease in neutrophil deformability following smoke exposure, cell morphology, using scanning electron microscopy, and morphometric measurements, made using transmission electron microscopy, were performed on aliquots of neutrophils harvested from whole blood in non-smoking subjects before and after exposure in vitro to cigarette smoke. Smoke exposure increased the maximum diameter and circumference of neutrophils, without changing their area. There was also a change in the maximum to minimum cell diameter ratio, which indicated that the cells had become less spherical. Scanning electron microscopy showed that smoke exposed cells had developed blebbing of their surface membranes, suggestive of an oxidative injury to the cell membrane rather than the shape changes associated with cell activation. These changes in the morphology and morphometry of smoke exposed neutrophils may contribute to the reduction in cell deformability induced by cigarette smoke. Images Fig. 3 Fig. 4 Fig. 5 PMID:1571278

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis

PubMed Central

Wang, Yan; Xu, Yingqiong; Liu, Qian; Zhang, Yuanyuan; Gao, Zhen; Yin, Mingzhu; Jiang, Nan; Cao, Guosheng; Yu, Boyang; Cao, Zhengyu; Kou, Junping

2017-01-01

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases. PMID:28352215

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

PubMed Central

Cao, X; Zhao, Y; Yu, Y; Wang, Y; Zhang, M; Zhang, W; Wang, J

1998-01-01

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia. Images Figure 3 Figure 4 PMID:9767469

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells.

PubMed

Torshabi, Maryam; Esfahrood, Zeinab Rezaei; Gholamin, Parisan; Karami, Elahe

2016-11-01

Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells. We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein. The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy. In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

Artesunate induces AIF-dependent apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Zhou, Chen-juan; Chen, Tong-Sheng

2012-03-01

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, Part 1: early and late spectral changes are indicative of the presence of apoptosis in cell cultures

NASA Astrophysics Data System (ADS)

Mulvey, Christine S.; Zhang, Kexiong; Liu, Wei-Han Bobby; Waxman, David J.; Bigio, Irving J.

2011-11-01

Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the detailed time-course of scattering changes in a Chinese hamster ovary (CHO) and PC-3 prostate cancer cells treated with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally, blocking caspase activity and downstream apoptotic morphology changes prevented late scattering changes. These observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

PubMed Central

Pajak, B.; De Smedt, T.; Moulin, V.; De Trez, C.; Maldonado-Lopez, R.; Vansanten, G.; Briend, E.; Urbain, J.; Leo, O.; Moser, M.

2000-01-01

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels. Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur. Key Words: dendritic cell • Escherichia coli • immunohistochemistry PMID:10961175

HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal {alpha} helix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mallick, Jaideep; Datta, Kasturi

2005-10-01

Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domainmore » of HABP1, its N- and C-terminal truncated variants ({delta}N.HABP1 and {delta}C.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, {delta}C.HABP1 too directly interacts with CDC 25 while {delta}N.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal {alpha}-helix.« less

High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways

NASA Technical Reports Server (NTRS)

Wang, Minli; Hada, Megumi; Huff, Janice; Pluth, Janice M.; Anderson, Janniffer; ONeill, Peter; Cucinotta, Francis A.

2010-01-01

The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.

Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cote, Marceline; Miller, A. Dusty; Liu, Shan-Lu

2007-08-17

The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here, we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylatedmore » in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induces cell cycle arrest and senescence, leading to impaired cell proliferation.« less

Short-term cadmium exposure induces gas exchanges, morphological and ultrastructural disturbances in mangrove Avicennia schaueriana young plants.

PubMed

Garcia, Janaina S; Dalmolin, Ândrea C; Cortez, Priscila A; Barbeira, Paulo S; Mangabeira, Pedro A O; França, Marcel G C

2018-06-01

Mangroves have been subject to more metal contamination, including cadmium (Cd). This study evaluated if a relatively short Cd exposure may induce metabolic, morphological and ultrastructural cell disturbance in Avicennia schaueriana. Cd induced evident constraints to seedlings since there was reduction in leaf gas exchanges and the plants did not survive for more than 10 days at a higher Cd exposure in controlled conditions. The highest Cd accumulation was observed in roots and gradually less in stem and leaves. Cadmium induced lignin deposition was observed in xylem cells of all vegetative organs. Intense sclerification in xylem cells, endoderm and change in the hypoderm organization were also detected. Cadmium clearly induced chloroplast deformities with ruptures of its membranes, thylakoids and core and provoked cytoplasm disorganization. These metal constraints under natural conditions for long term can lead to the accumulation of cellular and metabolic damages and jeopardize seedlings establishment and local biodiversity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Induction of a gradual, reversible morphogenesis of its host's epithelial brush border by Vibrio fischeri.

PubMed

Lamarcq, L H; McFall-Ngai, M J

1998-02-01

Bacteria exert a variety of influences on the morphology and physiology of animal cells whether they are pathogens or cooperative partners. The association between the luminous bacterium Vibrio fischeri and the sepiolid squid Euprymna scolopes provides an experimental model for the study of the influence of extracellular bacteria on the development of host epithelia. In this study, we analyzed bacterium-induced changes in the brush borders of the light organ crypt epithelia during the initial hours following colonization of this tissue. Transmission electron microscopy of the brush border morphology in colonized and uncolonized hosts revealed that the bacteria effect a fourfold increase in microvillar density over the first 4 days of the association. Estimates of the proportions of bacterial cells in contact with host microvilli showed that the intimacy of the bacterial cells with animal cell surfaces increases significantly during this time. Antibiotic curing of the organ following colonization showed that sustained interaction with bacteria is essential for the retention of the induced morphological changes. Bacteria that are defective in either light production or colonization efficiency produced changes similar to those by the parent strain. Conventional fluorescence and confocal scanning laser microscopy revealed that the brush border is supported by abundant filamentous actin. However, in situ hybridization with beta-actin probes did not show marked bacterium-induced increases in beta-actin gene expression. These experiments demonstrate that the E. scolopes-V. fischeri system is a viable model for the experimental study of bacterium-induced changes in host brush border morphology.

Induction of a Gradual, Reversible Morphogenesis of Its Host’s Epithelial Brush Border by Vibrio fischeri

PubMed Central

Lamarcq, Laurence H.; McFall-Ngai, Margaret J.

1998-01-01

Bacteria exert a variety of influences on the morphology and physiology of animal cells whether they are pathogens or cooperative partners. The association between the luminous bacterium Vibrio fischeri and the sepiolid squid Euprymna scolopes provides an experimental model for the study of the influence of extracellular bacteria on the development of host epithelia. In this study, we analyzed bacterium-induced changes in the brush borders of the light organ crypt epithelia during the initial hours following colonization of this tissue. Transmission electron microscopy of the brush border morphology in colonized and uncolonized hosts revealed that the bacteria effect a fourfold increase in microvillar density over the first 4 days of the association. Estimates of the proportions of bacterial cells in contact with host microvilli showed that the intimacy of the bacterial cells with animal cell surfaces increases significantly during this time. Antibiotic curing of the organ following colonization showed that sustained interaction with bacteria is essential for the retention of the induced morphological changes. Bacteria that are defective in either light production or colonization efficiency produced changes similar to those by the parent strain. Conventional fluorescence and confocal scanning laser microscopy revealed that the brush border is supported by abundant filamentous actin. However, in situ hybridization with β-actin probes did not show marked bacterium-induced increases in β-actin gene expression. These experiments demonstrate that the E. scolopes-V. fischeri system is a viable model for the experimental study of bacterium-induced changes in host brush border morphology. PMID:9453641

Relative biological effectiveness of accelerated heavy ions for induction of morphological transformation in Syrian hamster embryo cells.

PubMed

Han, Z B; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

1998-09-01

Syrian hamster embryo cells were used to study the morphological transformation induced by accelerated heavy ions with different linear energy transfer (LET) ranging from 13 to 400 keV/micron. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), then inoculated to culture dishes. Morphologically altered colonies were scored as transformants. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to X-rays first increased with LET, reached a maximum value of about 7 at 100 keV/micron, then decreased with the further increase of LET. Our findings confirmed that high LET heavy ions are much more effective than X-rays for the induction of in vitro cell transformation.

Morphological and genetical changes of endothelial progenitor cells after in-vitro conversion into photoreceptors.

PubMed

Qiang, Shi; Alsaeedi, Hiba Amer; Yuhong, Cheng; Yang, Hao; Tong, Li; Kumar, Suresh; Higuchi, Akon; Alarfaj, Abdullah A; Munisvaradass, Rusheni; Ling, Mok Pooi; Cheng, Pei

2018-06-01

Retinal degeneration is a condition ensued by various ocular disorders such as artery occlusion, diabetic retinopathy, retrolental fibroplasia and retinitis pigmentosa which cause abnormal loss of photoreceptor cells and lead to eventual vision impairment. No efficient treatment has yet been found, however, the use of stem cell therapy such as bone marrow and embryonic stem cells has opened a new treatment modality for retinal degenerative diseases. The major goal of this study is to analyze the potential of endothelial progenitor cells derived from bone marrow to differentiate into retinal neural cells for regenerative medicine purposes. In this study, endothelial progenitor cells were induced in-vitro with photoreceptor growth factor (taurine) for 21 days. Subsequently, the morphology and gene expression of CRX and RHO of the photoreceptors-induced EPCs were examined through immunostaining assay. The results indicated that the induced endothelial progenitor cells demonstrated positive gene expression of CRX and RHO. Our findings suggested that EPC cells may have a high advantage in cell replacement therapy for treating eye disease, in addition to other neural diseases, and may be a suitable cell source in regenerative medicine for eye disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

PubMed Central

van Leeuwen, Frank N.; Kain, Hendrie E.T.; van der Kammen, Rob A.; Michiels, Frits; Kranenburg, Onno W.; Collard, John G.

1997-01-01

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process. PMID:9348295

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels.

PubMed

Unemori, E N; Werb, Z

1986-09-01

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.

Automated Deep Learning-Based System to Identify Endothelial Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Kusumoto, Dai; Lachmann, Mark; Kunihiro, Takeshi; Yuasa, Shinsuke; Kishino, Yoshikazu; Kimura, Mai; Katsuki, Toshiomi; Itoh, Shogo; Seki, Tomohisa; Fukuda, Keiichi

2018-06-05

Deep learning technology is rapidly advancing and is now used to solve complex problems. Here, we used deep learning in convolutional neural networks to establish an automated method to identify endothelial cells derived from induced pluripotent stem cells (iPSCs), without the need for immunostaining or lineage tracing. Networks were trained to predict whether phase-contrast images contain endothelial cells based on morphology only. Predictions were validated by comparison to immunofluorescence staining for CD31, a marker of endothelial cells. Method parameters were then automatically and iteratively optimized to increase prediction accuracy. We found that prediction accuracy was correlated with network depth and pixel size of images to be analyzed. Finally, K-fold cross-validation confirmed that optimized convolutional neural networks can identify endothelial cells with high performance, based only on morphology. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Effect of hydrocortisone on cell morphology in C6 cells: the role of microfilaments in the inductive process. [Cytochalasin B; glycerol phosphate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berliner, J.A.; Bennett, K.; de Vellis, J.

Hydrocortisone induced cell spreading and the formation of microfilaments in C6 cells with a time course similar to that found for the induction of GPDH. Cytochalasin B caused a rapid and reversible cell rounding and microfilament breakdown. Breakdown of both sheath and network microfilaments occurred within 1 hour; after removal of cytochalasin B, reversal of morphological effects began within 30 min. High calcium was found to block reversal of the cytochalasin B induced alteration, but did not affect shape or microfilaments in non-cytochalasin B treated cells. It is concluded that since microfilament induction occurs late and the block in synthesismore » of GPDH by cytochalasin B occurs within hours after addition, microfilaments induced by hydrocortisone are probably not important in the sequence of events leading to GPDH induction. But, however, microfilaments present in uninduced cells may be important in the sequence of GPDH induction, since treatment with cytochalasin B rapidly breaks these down and also inhibits GPDH induction by hydrocortisone.« less

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

PubMed

Shu, Benshui; Wang, Wenxiang; Hu, Qingbo; Huang, Jingfei; Hu, Meiying; Zhong, Guohua

2015-07-01

The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds. © 2015 Wiley Periodicals, Inc.

The regulatory effect of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-l] benzenesulfonamide) on stem cell factor induced migration of mast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Su-Jin; College of Oriental Medicine, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701; Jeong, Hyun-Ja

SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1-pyrazol-1-]benzenesulfonamide; C{sub 16}H{sub 11}ClF{sub 3}N{sub 3}O{sub 2}S), is a highly selective cyclooxygenase (COX)-2 inhibitor. Recently, there have been reports that SC-236 protects against cartilage damage in addition to reducing inflammation and pain in osteoarthritis. However, the mechanism involved in the inflammatory allergic reaction has not been examined. Mast cells accumulation can be related to inflammatory conditions, including allergic rhinitis, asthma, and rheumatoid arthritis. The aim of the present study is to investigate the effects of SC-236 on stem cell factor (SCF)-induced migration, morphological alteration, and cytokine production of rat peritoneal mast cells (RPMCs). We observed that SCF significantly inducedmore » the migration and morphological alteration. The ability of SCF to enhance migration and morphological alteration was abolished by treatment with SC-236. In addition, production of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{beta}, and vascular endothelial growth factor (VEGF) production induced by SCF was significantly inhibited by treatment with SC-236. Previous work has demonstrated that SCF-induced migration and cytokine production of mast cells require p38 MAPK activation. We also showed that SC-236 suppresses the SCF-induced p38 MAPK activation in RPMCs. These data suggest that SC-236 inhibits migration and cytokine production through suppression of p38 MAPK activation. These results provided new insight into the pharmacological actions of SC-236 and its potential therapeutic role in the treatment of inflammatory allergic diseases.« less

Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth.

PubMed

Dreon, Marcos S; Fernández, Patricia E; Gimeno, Eduardo J; Heras, Horacio

2014-06-01

The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology. Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days. Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Shock wave trauma leads to inflammatory response and morphological activation in macrophage cell lines, but does not induce iNOS or NO synthesis.

PubMed

Günther, Mattias; Plantman, Stefan; Gahm, Caroline; Sondén, Anders; Risling, Mårten; Mathiesen, Tiit

2014-12-01

Experimental CNS trauma results in post-traumatic inflammation for which microglia and macrophages are vital. Experimental brain contusion entails iNOS synthesis and formation of free radicals, NO and peroxynitrite. Shock wave trauma can be used as a model of high-energy trauma in cell culture. It is known that shock wave trauma causes sub-lytic injury and inflammatory activation in endothelial cells. Mechanical disruption of red blood cells can induce iNOS synthesis in experimental systems. However, it is not known whether trauma can induce activation and iNOS synthesis in inflammatory cell lines with microglial or macrophage lineage. We studied the response and activation in two macrophage cell lines and the consequence for iNOS and NO formation after shock wave trauma. Two macrophage cell lines from rat (NR8383) and mouse (RAW264.7) were exposed to shock wave trauma by the Flyer Plate method. The cellular response was investigated by Affymetrix gene arrays. Cell survival and morphological activation was monitored for 24 h in a Cell-IQ live cell imaging system. iNOS induction and NO synthesis were analyzed by Western blot, in cell Western IR-immunofluorescence, and Griess nitrite assay. Morphological signs of activation were detected in both macrophage cell lines. The activation of RAW264.7 was statistically significant (p < 0.05), but activation of NR8383 did not pass the threshold of statistical significance alpha (p > 0.05). The growth rate of idle cells was unaffected and growth arrest was not seen. Trauma did not result in iNOS synthesis or NO induction. Gene array analyses showed high enrichment for inflammatory response, G-protein coupled signaling, detection of stimulus and chemotaxis. Shock wave trauma combined with low LPS stimulation instead led to high enrichment in apoptosis, IL-8 signaling, mitosis and DNA-related activities. LPS/IFN-ɣ stimulation caused iNOS and NO induction and morphological activation in both cell lines. Shock wave trauma by the Flyer Plate method caused an inflammatory response and morphological signs of activation in two macrophage cell lines, while iNOS induction appeared to require humoral signaling by LPS/IFN-ɣ. Our findings indicated that direct energy transfer by trauma can activate macrophages directly without humoral mediators, which comprises a novel activation mechanism of macrophages.

The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Kun; Li, Shi-zheng, E-mail: ychozon@yahoo.com.cn; Zhang, Yun-li

2011-11-11

Highlights: Black-Right-Pointing-Pointer Conditional medium and dan-shen root were used for cardiomyogenic differentiation. Black-Right-Pointing-Pointer They all could induce hPDMSCs to differentiate into cardiomyocytes. Black-Right-Pointing-Pointer The induction effect of the latter was slightly higher compared to that of the former. Black-Right-Pointing-Pointer Dan-shen root could be a good inducer for cardiomyogenic differentiation. -- Abstract: The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10 mg/L dan-shen root for 20 days. Morphologic characteristics weremore » analyzed by confocal and electron microscopy. Expression of {alpha}-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain ({beta}-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with {alpha}-sarcomeric actin and TnI antibody. ANF and {beta}-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.« less

Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation

PubMed Central

Gerami-Nejad, Maryam; Kumamoto, Yosuke; Mohammed, Javed A.; Jarrett, Elizabeth; Drummond, Rebecca A.; Zurawski, Sandra M.; Zurawski, Gerard; Berman, Judith; Iwasaki, Akiko; Brown, Gordon D.; Kaplan, Daniel H.

2015-01-01

Summary Candida albicans is a dimorphic fungus responsible for chronic mucocutaneous and systemic infections. Mucocutaneous immunity to C. albicans requires T helper-17 (Th17) cell differentiation that is thought to depend on recognition of filamentous C. albicans. Systemic immunity is considered T cell independent. Using a murine skin infection model, we compared T helper cell responses to yeast and filamentous C. albicans, We found that only yeast induced Th17 cell responses through a mechanism that required Dectin-1 mediated expression of interleukin-6 (IL-6) by Langerhans cells. Filamentous forms induced Th1 without Th17 cell responses due to the absence of Dectin-1 ligation. Notably, Th17 cell responses provided protection against cutaneous infection while Th1 cell responses provided protection against systemic infection. Thus, C. albicans morphology drives distinct T helper cell responses that provide tissue specific protection. These findings provide insight into compartmentalization of Th responses, C. albicans pathogenesis and have critical implications for vaccine strategies. PMID:25680275

S179D prolactin diminishes the effects of UV light on epidermal gamma delta T cells

PubMed Central

Guzmán, Esther A.; Langowski, John L.; De Guzman, Ariel; Konrad Muller, H.; Walker, Ameae M.; Owen, Laurie B.

2008-01-01

Epidermal gamma delta T cells (γδ T) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the two major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV irradiation. Under control conditions, both PRLs reduced the number of γδ T, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in γδ T number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC: γδ T ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis. PMID:17945411

Betanodavirus Induces Oxidative Stress-Mediated Cell Death That Prevented by Anti-Oxidants and Zfcatalase in Fish Cells

PubMed Central

Chang, Chih-Wei; Su, Yu-Chin; Her, Guor-Mour; Ken, Chuian-Fu; Hong, Jiann-Ruey

2011-01-01

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12–24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24–48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis. PMID:21991373

Alterations in zebrafish development induced by simvastatin: Comprehensive morphological and physiological study, focusing on muscle

PubMed Central

Campos, Laise M; Rios, Eduardo A; Guapyassu, Livia; Midlej, Victor; Atella, Georgia C; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia

2016-01-01

The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin. PMID:27444151

Alterations in zebrafish development induced by simvastatin: Comprehensive morphological and physiological study, focusing on muscle.

PubMed

Campos, Laise M; Rios, Eduardo A; Guapyassu, Livia; Midlej, Victor; Atella, Georgia C; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia; Costa, Manoel L

2016-11-01

The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin. © 2016 by the Society for Experimental Biology and Medicine.

Epithelial–mesenchymal transition during oncogenic transformation induced by hexavalent chromium involves reactive oxygen species-dependent mechanism in lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Song-Ze, E-mail: dingsongze@hotmail.com; Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536; Yang, Yu-Xiu

2013-05-15

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Exposure to Cr(VI) induces DNA damage, cell morphological change and malignant transformation in human lung epithelial cells. Despite extensive studies, the molecular mechanisms remain elusive, it is also not known if Cr(VI)-induced transformation might accompany with invasive properties to facilitate metastasis. We aimed to study Cr(VI)-induced epithelial–mesenchymal transition (EMT) and invasion during oncogenic transformation in lung epithelial cells. The results showed that Cr(VI) at low doses represses E-cadherin mRNA and protein expression, enhances mesenchymal marker vimentin expression and transforms the epithelial cell into fibroblastoid morphology.more » Cr(VI) also increases cell invasion and promotes colony formation. Further studies indicated that Cr(VI) uses multiple mechanisms to repress E-cadherin expression, including activation of E-cadherin repressors such as Slug, ZEB1, KLF8 and enhancement the binding of HDAC1 in E-cadherin gene promoter, but DNA methylation is not responsible for the loss of E-cadherin. Catalase reduces Cr(VI)-induced E-cadherin and vimentin protein expression, attenuates cell invasion in matrigel and colony formation on soft agar. These results demonstrate that exposure to a common human carcinogen, Cr(VI), induces EMT and invasion during oncogenic transformation in lung epithelial cells and implicate in cancer metastasis and prevention. - Graphical abstract: Epithelial–mesenchymal transition during oncogenic transformation induced by hexavalent chromium involves reactive oxygen species-dependent mechanisms in lung epithelial cells. - Highlights: • We study if Cr(VI) might induce EMT and invasion in epithelial cells. • Cr(VI) induces EMT by altering E-cadherin and vimentin expression. • It also increases cell invasion and promotes oncogenic transformation. • Catalase reduces Cr(VI)-induced EMT, invasion and transformation.« less

Investigating free radical generation in HepG2 cells using immuno-spin trapping.

PubMed

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

2014-10-01

Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

Microarray analysis on gene regulation by estrogen, progesterone and tamoxifen in human endometrial stromal cells.

PubMed

Ren, Chun-E; Zhu, Xueqiong; Li, Jinping; Lyle, Christian; Dowdy, Sean; Podratz, Karl C; Byck, David; Chen, Hai-Bin; Jiang, Shi-Wen

2015-03-13

Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

PubMed

Legartová, S; Sbardella, G; Kozubek, S; Bártová, E

2015-01-01

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

Morphological, anatomical, and ultrastructural changes (visualized through scanning electron microscopy) induced in Triticum aestivum by Pb²⁺ treatment.

PubMed

Kaur, Gurpreet; Singh, Harminder Pal; Batish, Daizy Rani; Kohli, Ravinder Kumar

2014-11-01

Lead (Pb) causes severe damage to crops, ecosystems, and humans, and alters the physiology and biochemistry of various plant species. It is hypothesized that Pb-induced metabolic alterations could manifest as structural variations in the roots of plants. In light of this, the morphological, anatomical, and ultrastructural variations (through scanning electron microscopy, SEM) were studied in 4-day-old seedlings of Triticum aestivum grown under Pb stress (0, 8, 16, 40, and 80 mg Pb(2+) l(-1); mild to highly toxic). The toxic effect was more pronounced in radicle growth than on the plumule growth. The SEM of the root of T. aestivum depicted morphological alterations and surface ultrastructural changes. Compared to intact and uniform surface cells in the control roots, cells were irregular and desiccated in Pb(2+)-treated roots. In Pb(2+)-treated roots, the number of root hairs increased manifold, showing dense growth, and these were apparently longer. Apart from the deformity in surface morphology and anatomy of the roots in response to Pb(2+) toxicity, considerable anatomical alterations were also observed. Pb(2+)-treated root exhibited signs of injury in the form of cell distortion, particularly in the cortical cells. The endodermis and pericycle region showed loss of uniformity post Pb(2+) exposure (at 80 mg l(-1) Pb(2+)). The cells appeared to be squeezed with greater depositions observed all over the tissue. The study concludes that Pb(2+) treatment caused structural anomalies and induced anatomical and surface ultrastructural changes in T. aestivum.

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Yasuhiko, E-mail: 97318@ib.k.u-tokyo.ac.jp; Furuyama, Isao; Oda, Shoji

2011-04-01

Highlights: {yields} A novel major transcript, AIFL-I4, is found. {yields} Nuclear localization of AIFL-I4 induces mitochondrial morphology change and suppression of cell proliferation. {yields} AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site does not induce these phenotypes. {yields} [2Fe-2S] cluster binding site is essential for these phenotypes. -- Abstract: Apoptosis-inducing factor-like (AIFL) protein contains a Rieske domain and pyridine nucleotide-disulfide oxidoreductase (Pyr{sub r}edox) domain that shows 35% homology to that of apoptosis-inducing factor (AIF) protein. We identified a novel major transcript of the medaka (Oryzias latipes) AIFL gene that retained intron 4 (AIFL-I4) in embryos and tissues frommore » adult fish. The product of this transcript, AIFL-I4 protein, lacked the Pyr{sub r}edox domain because of a nonsense codon in intron 4. Both AIFL-I4 and full-length AIFL (fAIFL) transcripts were highly expressed in the brain and late embryos, and relative fAIFL and AIFL-I4 expression levels differed among tissues. Transient expression of AIFL-I4 and fAIFL tagged with GFP showed that AIFL-I4 localized in the nucleus, while fAIFL localized throughout the cytoplasm. We also found that overexpression of AIFL-I4 induced a change in mitochondrial morphology and suppression of cell proliferation. AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site of the Rieske domain did not induce these phenotypes. This report is the first to demonstrate nuclear localization of a Rieske-type protein translated from the AIFL gene. Our data suggested that the [2Fe-2S] cluster binding site was essential for the nuclear localization and involved in mitochondrial morphology and suppression of cell proliferation.« less

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Dexamethasone reduces mitomycin C-related inflammatory cytokine expression without inducing further cell death in corneal fibroblasts.

PubMed

Chang, Shu-Wen; Chou, San-Fang; Yu, Shuen-Yuen

2010-01-01

The purpose of this study was to investigate the effect of dexamethasone (DEX) on mitomycin C (MMC)-induced inflammatory cytokine expression in corneal fibroblasts. Primary human corneal fibroblasts were treated with MMC, dexamethasone, or in combination. Morphological changes and cell growth were documented using phase-contrast microscopy and PicoGreen assay, respectively. Cell apoptosis was evaluated by annexin V/propidium iodide staining, whereas viability was tested by the live/dead assay and analyzed by flow cytometry. The relative expression of interleukin-8 and monocyte chemoattractant protein-1 was investigated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Mitogen-activated protein kinase activation and mitogen-activated protein kinase phosphatase-1 expression were documented by Western blot analysis. We found that MMC induced corneal fibroblast elongation, apoptosis, and retarded cell growth, whereas DEX did not significantly alter cell morphology or viability. The combination of DEX and MMC did not induce additional apoptosis and cell death. DEX dose dependently down-regulated basal and MMC-induced interleukin-8 and monocyte chemoattractant protein-1 mRNA expression and protein secretion. DEX attenuated MMC-induced p38 and Jun N-terminal kinases activation and up-regulated expression. These suggested that DEX may inhibit MMC-induced interleukin-8 and monocyte chemoattractant protein-1 by up-regulating MKP-1 expression, which subsequently deactivated p38 and Jun N-terminal kinases activation. Combined MMC and DEX treatment may facilitate corneal wound healing.

Repeated folding stress-induced morphological changes in the dermal equivalent.

PubMed

Arai, Koji Y; Sugimoto, Mami; Ito, Kanako; Ogura, Yuki; Akutsu, Nobuko; Amano, Satoshi; Adachi, Eijiro; Nishiyama, Toshio

2014-11-01

Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent. We developed a novel device to apply repeated folding stress to the dermal equivalent. After applying the mechanical stress, morphological changes of the dermal equivalent and expression of several genes related to extracellular matrix turn over and cell contraction were examined. The repeated folding stress induced a noticeable decrease in the width of the dermal equivalent. The mechanical stress altered orientations of collagen fibrils. Hydroxyproline contents, dry weights and cell viability of the dermal equivalents were not affected by the mechanical stress. On the other hand, Rho-associated coiled-coil-containing kinase (ROCK) specific inhibitor Y27632 completely suppressed the decrease in the width of the dermal equivalent. The present results revealed that either degradation of collagen or changes in the number of cells were not responsible for the decrease in the width of the dermal equivalent and indicate that the repeated mechanical stress induces unidirectional contraction in the dermal equivalent through the RhoA-ROCK signaling pathway. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Retinol induces morphological alterations and proliferative focus formation through free radical-mediated activation of multiple signaling pathways.

PubMed

Gelain, Daniel Pens; Pasquali, Matheus Augusto de Bittencourt; Caregnato, Fernanda Freitas; Castro, Mauro Antonio Alves; Moreira, José Claudio Fonseca

2012-04-01

Toxicity of retinol (vitamin A) has been previously associated with apoptosis and/or cell malignant transformation. Thus, we investigated the pathways involved in the induction of proliferation, deformation and proliferative focus formation by retinol in cultured Sertoli cells of rats. Sertoli cells were isolated from immature rats and cultured. The cells were subjected to a 24-h treatment with different concentrations of retinol. Parameters of oxidative stress and cytotoxicity were analyzed. The effects of the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor Gö6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L) on morphological and proliferative/transformation-associated modifications were studied. Retinol (7 and 14 μmol/L) significantly increases the reactive species production in Sertoli cells. Inhibition of p38, JNK, ERK1/2, Akt, and PKA suppressed retinol-induced [(3)H]dT incorporation into the cells, while PKC inhibition had no effect. ERK1/2 and p38 inhibition also blocked retinol-induced proliferative focus formation in the cells, while Akt and JNK inhibition partially decreased focus formation. ERK1/2 and p38 inhibition hindered transformation-associated deformation in retinol-treated cells, while other treatments had no effect. Our results suggest that activation of multiple kinases is responsible for morphological and proliferative changes associated to malignancy development in Sertoli cells by retinol at the concentrations higher than physiological level.

Low intensity ultrasound induces apoptosis via MPT channel on mitochondrial membrane: Target for regulating cancer therapy or not?

NASA Astrophysics Data System (ADS)

Feng, Yi; Wan, Mingxi

2017-03-01

To discuss how the mitochondrion is involved in low intensity ultrasound induced apoptosis, HepG2 cells were irradiated by low intensity focused ultrasound (ISPTA = 3W/cm2, 1 min) and then cultured from 3-12 h post irradiation in the study. The morphological alteration was examined by light and fluorescent microscopy respectively. Cell viability and apoptosis were examined by trypan blue staining and flow cytometry with double staining of FITC-labelled Annexin-V/PI. Key proteins responded to irradiation were screened out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shotgun proteomic methods with Agilent 1100 HPLC-Chip-MS technology. Representative apoptotic morphological characteristics and increased percentage of apoptotic cells were achieved. Six important proteins (4 up-regulated and 2 down-regulated) were selected and analyzed. It revealed low intensity focused ultrasound could induce apoptosis in HepG2 cells and the US-induced apoptosis was mitochondria-dependent and caspases-dependent. Moreover, mitochondrial membrane permeability transition (MPT) is related to ultrasound induced apoptosis, but VDAC may be not the main MPT channel. Understanding it could help to assist the cancer therapy by regulating the MPT as the target.

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

PubMed

Nesnow, Stephen; Davis, Christine; Nelson, Garret B; Lambert, Guy; Padgett, William; Pimentel, Maria; Tennant, Alan H; Kligerman, Andrew D; Ross, Jeffrey A

2002-11-26

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

Overexpression of interleukin-6 and -8, cell growth inhibition and morphological changes in 2-hydroxyethyl methacrylate-treated human dental pulp mesenchymal stem cells.

PubMed

Trubiani, O; Cataldi, A; De Angelis, F; D'Arcangelo, C; Caputi, S

2012-01-01

To evaluate morphological features, cell growth and interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion in expanded ex vivo human dental pulp mesenchymal stem cells (DP-MSCs) after exposure to 2-hydroxyethyl methacrylate (HEMA).   Dental pulp mesenchymal stem cells were derived from the dental pulps of 10 young donors. After in vitro isolation, DP-MSCs were treated with 3 and 5 mmol L(-1) HEMA, and after 24, 48 and 72 h of incubation, their morphological features, cell growth, IL-6 and IL-8 secretion were analysed. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons.   Dental pulp mesenchymal stem cells revealed a decrease in cell growth with both treatments (P < 0.05), more evident at 5 mmol L(-1) . Microscopic analysis displayed extensive cytotoxic effects in treated cells, which lost their fibroblastoid features and became retracted, even roundish, with a large number of granules. An up-regulation of IL-6 and IL-8 in treated cells cytokines was evident (P < 0.05).   2-Hydroxyethyl methacrylate exhibited cytotoxicity, inhibited cell growth and induced morphological changes in cultured DP-MSCs. Moreover, in treated samples, an up-regulation of soluble mediators of inflammation such as IL-6 and IL-8 cytokines was found. The direct application of HEMA potentially induces an inflammation process that could be the starting point for toxic response and cell damage in DP-MSCs. © 2011 International Endodontic Journal.

Phototodynamic activity of zinc monocarboxyphenoxy phthalocyane (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells

NASA Astrophysics Data System (ADS)

Manoto, Sello L.; Oluwole, David O.; Malabi, Rudzani; Maphanga, Charles; Ombinda-Lemboumba, Saturnin; Nyokong, Tebello; Mthunzi-Kufa, Patience

2017-02-01

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality for the treatment of neoplastic and non-neoplastic diseases. In PDT of cancer, irradiation with light of a specific wavelength leads to activation of a photosensitizer which results in generation of reactive oxygen species (ROS) which induces cell death. Many phthalocyanine photosensitizers are hydrophobic and insoluble in water, which limits their therapeutic efficiency. Consequently, advanced delivery systems and strategies are needed to improve the effectiveness of these photosensitizers. Nanoparticles have shown promising results in increasing aqueous solubility, bioavailability, stability and delivery of photosensitizers to their target. This study investigated the photodynamic activity of zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells. The photodynamic activity of ZnMCPPc conjugated to AuAg nanoparticles were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated ZnMCPPc conjugated to AuAg nanoparticles showed changes in cell morphology and a dose dependent decrease in cellular viability, proliferation and an increase in cell membrane damage. The ZnMCPPc conjugated to AuAg nanoparticles used in this study was highly effective in inducing cell death of melanoma cancer cells.

High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui

The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovariesmore » in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27{sup Kip1} and p21{sup Cip1}, were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure. - Highlights: • HFD induced-obesity leads to abnormal ovarian morphology. • HFD induced-obesity triggers excessive apoptosis in the ovary. • HFD induced-obesity up-regulates cell cycle inhibitors p21{sup Cip1} and p27{sup Kip1} in the ovary. • HFD induced-obesity causes cell cycle arrest in the ovary.« less

CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology.

PubMed

Gutsch, Romina; Kandemir, Judith D; Pietsch, Daniel; Cappello, Christian; Meyer, Johann; Simanowski, Kathrin; Huber, René; Brand, Korbinian

2011-07-01

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβ(WT) macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβ(KO). The typical macrophage morphology was only observed in C/EBPβ(WT), whereas C/EBPβ(KO) stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβ(KO) macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβ(WT) cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.

Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

NASA Astrophysics Data System (ADS)

Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

2016-11-01

In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

PubMed Central

Irani, Niloufer G; Grotewold, Erich

2005-01-01

Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin. PMID:15907203

CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

PubMed

Haraguchi, Misako; Sato, Masahiro; Ozawa, Masayuki

2015-01-01

Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

In Vitro Therapeutic Potential of Tio2 Nanoparticles Against Human Cervical Carcinoma Cells.

PubMed

Pandurangan, Muthuraman; Enkhtaivan, Gansukh; Young, Jung A; Hoon, Hur Ji; Lee, Hannah; Lee, SooBin; Kim, Doo Hwan

2016-06-01

Cellular and physiological responses to the degradation products of titanium implants are key indicators to determine the quality of biocompatibility of implant devices. The present study investigated titanium dioxide (TiO2) nanoparticle-induced cytotoxicity, apoptotic morphological modification, and apoptotic-related gene expressions in the human cervical carcinoma cells. TiO2 nanoparticle-induced cytotoxicity on cancer cells was determined by the sulphorhodamine-B assay. Apoptotic morphological modification such as nuclear fragmentation, rounding, cytoplasm shrinkage, loss of adhesion, and reduced cell volume were observed by an inverted, fluorescence, and confocal laser scanning microscope (CLSM). The DNA fragmentation study showed the occurrence of necrosis and apoptosis in nanoparticle-treated cells. The qPCR study showed the increased p53 and bax mRNA expression in the nanoparticle-treated cells compared to control. In addition, caspase 3 activity was increased in nanoparticle-treated cells, which indicates the increased auto-catalysis. Taking all these data together, it may suggest that TiO2 nanoparticle could inhibit the growth of HeLa cells.

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

PubMed

Czeisler, Catherine; Short, Aaron; Nelson, Tyler; Gygli, Patrick; Ortiz, Cristina; Catacutan, Fay Patsy; Stocker, Ben; Cronin, James; Lannutti, John; Winter, Jessica; Otero, José Javier

2016-12-01

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protective effect of NSA on intestinal epithelial cells in a necroptosis model

PubMed Central

Dong, Wei; Zhang, Min; Zhu, Yaxi; Chen, Yuanhan; Zhao, Xingchen; Li, Ruizhao; Zhang, Li; Ye, Zhiming; Liang, Xingling

2017-01-01

Objective This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD). Methods 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF-α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected. Results In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF-α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF-α and Z-VAD-fmk. Conclusion NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD. PMID:29156831

Protective effect of NSA on intestinal epithelial cells in a necroptosis model.

PubMed

Dong, Wei; Zhang, Min; Zhu, Yaxi; Chen, Yuanhan; Zhao, Xingchen; Li, Ruizhao; Zhang, Li; Ye, Zhiming; Liang, Xingling

2017-10-17

This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD). 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF- α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected. In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF- α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF- α and Z-VAD-fmk. NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.

PubMed

Dwane, Susan; Durack, Edel; Kiely, Patrick A

2013-09-11

Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Tailorable Surface Morphology of 3D Scaffolds by Combining Additive Manufacturing with Thermally Induced Phase Separation.

PubMed

Di Luca, Andrea; de Wijn, Joost R; van Blitterswijk, Clemens A; Camarero-Espinosa, Sandra; Moroni, Lorenzo

2017-08-01

The functionalization of biomaterials substrates used for cell culture is gearing towards an increasing control over cell activity. Although a number of biomaterials have been successfully modified by different strategies to display tailored physical and chemical surface properties, it is still challenging to step from 2D substrates to 3D scaffolds with instructive surface properties for cell culture and tissue regeneration. In this study, additive manufacturing and thermally induced phase separation are combined to create 3D scaffolds with tunable surface morphology from polymer gels. Surface features vary depending on the gel concentration, the exchanging temperature, and the nonsolvent used. When preosteoblasts (MC-3T3 cells) are cultured on these scaffolds, a significant increase in alkaline phosphatase activity is measured for submicron surface topography, suggesting a potential role on early cell differentiation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vitamin K3 induces antiproliferative effect in cervical epithelial cells transformed by HPV 16 (SiHa cells) through the increase in reactive oxygen species production.

PubMed

de Carvalho Scharf Santana, Natália; Lima, Natália Alves; Desoti, Vânia Cristina; Bidóia, Danielle Lazarin; de Souza Bonfim Mendonça, Patrícia; Ratti, Bianca Altrão; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Consolaro, Marcia Edilaine Lopes; Ximenes, Valdecir Farias; de Oliveira Silva, Sueli

2016-10-01

Cervical cancer is characterized as an important public health problem. According to latest estimates, cancer of the cervix is the fourth most common cancer among women. Due to its high prevalence, the search for new and efficient drugs to treat this infection is continuous. The progression of HPV-associated cervical cancer involves the expression of two viral proteins, E6 and E7, which are rapidly degraded by the ubiquitin-proteasome system through the increase in reactive oxygen species generation. Vitamins are essential to human substances, participate in the regulation of metabolism, and facilitate the process of energy transfer. Some early studies have indicated that vitamin K3 exerts antitumor activity by inducing cell death by apoptosis through an increase in the generation of reactive oxygen species. Thus, we evaluated the antiproliferative effect and a likely mechanism of action of vitamin K3 against cervical epithelial cells transformed by HPV 16 (SiHa cells) assessing the production of total ROS, the mitochondrial membrane potential, the cell morphology, the cell volume, and the cell membrane integrity. Our results show that vitamin K3 induces an increase in ROS production in SiHa cells, triggering biochemical and morphological events, such as depolarization of mitochondrial membrane potential and decreasing cell volume. Our data showed that vitamin K3 generates an oxidative imbalance in SiHa cells, leading to mechanisms that induce cell death by apoptosis.

Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

PubMed

Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

2013-10-01

To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

Myasthenia gravis sera have no effect on cardiomyocytes in vitro.

PubMed

Helgeland, Geir; Luckman, Steven P; Romi, Fredrik R; Jonassen, Anne K; Gilhus, Nils Erik

2008-09-15

Myasthenia gravis (MG) is an autoimmune disorder primarily caused by circulating autoantibodies targeting the nicotinic acetylcholine receptor. Several studies have suggested a link between MG and heart disease. Girardi heart cells were treated with MG sera, measuring cytotoxic effects using flow cytometry, adenylate kinase (AK) release and evaluating morphology. MG sera did not induce morphological changes in the cells. AK release from cells treated with MG sera did not exceed controls and flow cytometric examination did not reveal any increase in dead or apoptotic cells. We conclude that MG sera have no cytotoxic effect in our heart cell culture system.

Morphological examination of the effects of defibrotide on experimentally induced bladder injury and its relation to interstitial cystitis.

PubMed

Aydin, H; Ercan, F; Cetinel, S; San, T

2001-08-01

This morphological study aims to investigate the effects of defibrotide, a deoxyribonucleic acid derivative drug with cytoprotective, immunosuppressive and vasorelaxant effects, on protamine sulfate induced bladder injury. Wistar albino female rats were catheterized and intravesically infused with phosphate buffered solution (control group) or, either protamine sulfate (bladder injury group) or protamine sulfate+defibrotide (bladder injury+defibrotide group) dissolved in phosphate buffered solution. The morphology of the urinary bladder was investigated using light and electron microscopy. The number of mast cells in the mucosa, mucosal alterations, intercellular junctions, surface topography and the glycosaminoglycan (GAG) layer as well as microvillus formation on the luminal surface were evaluated. In the bladder injury group, ulcerated areas, irregularity of the GAG layer, increased number of mast cells, vacuole formation, dilated perinuclear cistern, formation of pleomorphic and uniform microvilli and dilatations in the intercellular spaces in the urothelium were observed. In the bladder injury+defibrotide group a relatively normal urothelial topography, GAG layer and a few mast cells in the mucosa, some dilatations between the intercellular areas, less uniform microvilli, regular perinuclear cistern and tight junctions were observed. These results show that defibrotide can inhibit PS induced bladder damage.

Carbon Ion-Irradiated Hepatoma Cells Exhibit Coupling Interplay between Apoptotic Signaling and Morphological and Mechanical Remodeling

PubMed Central

Zhang, Baoping; Li, Long; Li, Zhiqiang; Liu, Yang; Zhang, Hong; Wang, Jizeng

2016-01-01

A apoptotic model was established based on the results of five hepatocellular carcinoma cell (HCC) lines irradiated with carbon ions to investigate the coupling interplay between apoptotic signaling and morphological and mechanical cellular remodeling. The expression levels of key apoptotic proteins and the changes in morphological characteristics and mechanical properties were systematically examined in the irradiated HCC lines. We observed that caspase-3 was activated and that the Bax/Bcl-2 ratio was significantly increased over time. Cellular morphology and mechanics analyses indicated monotonic decreases in spatial sizes, an increase in surface roughness, a considerable reduction in stiffness, and disassembly of the cytoskeletal architecture. A theoretical model of apoptosis revealed that mechanical changes in cells induce the characteristic cellular budding of apoptotic bodies. Statistical analysis indicated that the projected area, stiffness, and cytoskeletal density of the irradiated cells were positively correlated, whereas stiffness and caspase-3 expression were negatively correlated, suggesting a tight coupling interplay between the cellular structures, mechanical properties, and apoptotic protein levels. These results help to clarify a novel arbitration mechanism of cellular demise induced by carbon ions. This biomechanics strategy for evaluating apoptosis contributes to our understanding of cancer-killing mechanisms in the context of carbon ion radiotherapy. PMID:27731354

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

PubMed Central

2005-01-01

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process. PMID:15707390

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

PubMed

Soysa, Preethi; Jayarthne, Panchima; Ranathunga, Imali

2018-03-05

Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Reversal of Myoblast Aging by Tocotrienol Rich Fraction Posttreatment

PubMed Central

Wan Ngah, Wan Zurinah; Mouly, Vincent; Abdul Karim, Norwahidah

2013-01-01

Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response to the aging-related deterioration of the skeletal muscle mass, strength, and regenerative capacity, termed as sarcopenia. This study focused on the effect of tocotrienol rich fraction (TRF) on regenerative capacity of myoblasts in stress-induced premature senescence (SIPS). The myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pretreatment, and TRF posttreatment. Optimum dose of TRF, morphological observation, activity of senescence-associated β-galactosidase (SA-β-galactosidase), and cell proliferation were determined. 50 μg/mL TRF treatment exhibited the highest cell proliferation capacity. SIPS-induced myoblasts exhibit large flattened cells and prominent intermediate filaments (senescent-like morphology). The activity of SA-β-galactosidase was significantly increased, but the proliferation capacity was significantly reduced as compared to young control. The activity of SA-β-galactosidase was significantly reduced and cell proliferation was significantly increased in the posttreatment group whereas there was no significant difference in SA-β-galactosidase activity and proliferation capacity of pretreatment group as compared to SIPS-induced myoblasts. Based on the data, we hypothesized that TRF may reverse the myoblasts aging through replenishing the regenerative capacity of the cells. However, further investigation on the mechanism of TRF in reversing the myoblast aging is needed. PMID:24349615

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

PubMed Central

Fernández-Sánchez, Laura; Rondón, Netxibeth; Esquiva, Gema; Germain, Francisco; de la Villa, Pedro; Cuenca, Nicolás

2015-01-01

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss. PMID:26379056

Neurogenesis-Promoting Natural Product α-Asarone Modulates Morphological Dynamics of Activated Microglia

PubMed Central

Cai, Qing; Li, Yuanyuan; Mao, Jianxin; Pei, Gang

2016-01-01

α-Asarone is an active constituent of Acori Tatarinowii, one of the widely used traditional Chinese Medicine to treat cognitive defect, and recently is shown to promote neurogenesis. Here, we demonstrated that low level (3 μM) of α-asarone attenuated LPS-induced BV2 cell bipolar elongated morphological change, with no significant effect on the LPS-induced pro-inflammatory cytokine expressions. In addition, time-lapse analysis also revealed that α-asarone modulated LPS-induced BV2 morphological dynamics. Consistently a significant reduction in the LPS-induced Monocyte Chemoattractant Protein (MCP-1) mRNA and protein levels was also detected along with the morphological change. Mechanistic study showed that the attenuation effect to the LPS-resulted morphological modulation was also detected in the presence of MCP-1 antibodies or a CCR2 antagonist. This result has also been confirmed in primary cultured microglia. The in vivo investigation provided further evidence that α-asarone reduced the proportion of activated microglia, and reduced microglial tip number and maintained the velocity. Our study thus reveals α-asarone effectively modulates microglial morphological dynamics, and implies this effect of α-asarone may functionally relate to its influence on neurogenesis. PMID:28018174

Molecular mechanism of cardol, isolated from Trigona incisa stingless bee propolis, induced apoptosis in the SW620 human colorectal cancer cell line.

PubMed

Kustiawan, Paula Mariana; Lirdprapamongkol, Kriengsak; Palaga, Tanapat; Puthong, Songchan; Phuwapraisirisan, Preecha; Svasti, Jisnuson; Chanchao, Chanpen

2017-05-04

Cardol is a major bioactive constituent in the Trigona incisa propolis from Indonesia, with a strong in vitro antiproliferative activity against the SW620 colorectal adenocarcinoma cell line (IC 50 of 4.51 ± 0.76 μg/mL). Cardol induced G 0 /G 1 cell cycle arrest and apoptotic cell death. The present study was designed to reveal the mechanism of cardol's antiproliferative effect and induction of apoptosis. Changes in cell morphology were observed by light microscopy. To determine whether the mitochondrial apoptotic pathway was involved in cell death, caspase-3 and caspase-9 activities, western blot analysis, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assayed. Changes in the cell morphology and the significantly increased caspase-3 and caspase-9 activities, plus the cleavage of pro-caspase-3, pro-caspase-9 and PARP, supported that cardol caused apoptosis in SW620 cells within 2 h after treatment by cardol. In addition, cardol decreased the mitochondrial membrane potential while increasing the intracellular ROS levels in a time- and dose-dependent manner. Antioxidant treatment supported that the cardol-induced cell death was dependent on ROS production. Cardol induced cell death in SW620 cells was mediated by oxidative stress elevation and the mitochondrial apoptotic pathway, and these could be the potential molecular mechanism for the antiproliferative effect of cardol.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

PubMed

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Mammalian cell-transforming potential of traffic-linked ultrafine particulate matter PM0.056 in urban roadside atmosphere.

PubMed

Verma, Mukesh K; Poojan, Shiv; Sultana, Sarwat; Kumar, Sushil

2014-09-01

We examined the clastogenic and cell-transforming potential of ultrafine particulate matter fraction PM0.056 of urban ambient aerosol using mammalian cells. PM1.0, PM0.56 and PM0.056 fractions were sampled from roadside atmosphere of an urban area using the cascade impactor MOUDI-NR-110. The potential to induce cytotoxicity, DNA damage and micronuclei formation was examined at the test concentrations of 3, 6, 12.5, 25, 50 and 100 μg/ml using the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the plasmid relaxation assay and the C3H10T1/2 (10T1/2) cells. The cell-transforming potential was investigated in vitro using 10T1/2 cell transformation assay and the soft agar assay. PM1, PM0.56 and PM0.056 fractions were found to be toxic in dose-dependent manner. These induced cytotoxicity at five test concentrations, the ultrafine particle fraction PM0.056 showed greater cytotoxic potential. PM0.056 induced micronucleus formation in 10T1/2 cells. The effect was statistically significant. The DNA-damaging potential was measured in a plasmid relaxation assay. Both fine and ultrafine particle fraction PM0.56 and PM0.056 displayed greater effect as compared to larger PM1 fraction. DNA damage was found to be dependent on particulate matter intrinsic pro-oxidant chemicals. The ability of the ultrafine particle fraction PM0.056 to induce morphological cell transformation was demonstrated by significant and dose-dependent increases in type III focus formation by morphologically transformed cells in culture flasks and their clonal expansion in soft agar. It is concluded that the traffic-linked ultrafine particle fraction PM0.056 in the atmosphere by the roadside of an urban area is clastogenic and able to induce morphological transformation of mammalian cells. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Activation of Meiosis-Specific Genes is Associated with Depolyploidization of Human Tumor Cells Following Radiation-Induced Mitotic Catastrophe

PubMed Central

Ianzini, Fiorenza; Kosmacek, Elizabeth A.; Nelson, Elke S.; Napoli, Eleonora; Erenpreisa, Jekaterina; Kalejs, Martins; Mackey, Michael A.

2009-01-01

Cancer is frequently characterized histologically by the appearance of large cells that are either aneuploid or polyploid. Aneuploidy and polyploidy are hallmarks of radiation-induced mitotic catastrophe (MC), a common phenomenon occurring in tumor cells with impaired p53 function exposed to various cytotoxic and genotoxic agents. MC is characterized by altered expression of mitotic regulators, untimely and abnormal cell division, delayed DNA damage, and changes in morphology. We report here that cells undergoing radiation-induced MC are more plastic with regards to ploidy and that this plasticity allows them to reorganize their genetic material through reduction divisions to produce smaller cells morphologically indistinguishable from control cells. Experiments conducted with the Large Scale Digital Cell Analysis System (LSDCAS) are discussed that show that a small fraction of polyploid cancer cells formed via radiation-induced MC can survive and start a process of depolyploidization that yields various outcomes. While most multipolar divisions failed and cell fusion occurred; some of these divisions were successful and originated a variety of cell progeny characterized by different ploidy. Among these ploidy phenotypes, a progeny of small mononucleated cells, indistinguishable from the untreated control cells, is often seen. We report here evidence that meiosis-specific genes are expressed in the polyploid cells during depolyploidization. Tumor cells might take advantage of the temporary change from a pro-mitotic to a pro-meiotic division regimen to facilitate depolyploidization and restore the proliferative state of the tumor cell population. These events might be mechanisms by which tumor progression and resistance to treatment occur in vivo. PMID:19258501

Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms

PubMed Central

Emmons-Bell, Maya; Durant, Fallon; Hammelman, Jennifer; Bessonov, Nicholas; Volpert, Vitaly; Morokuma, Junji; Pinet, Kaylinnette; Adams, Dany S.; Pietak, Alexis; Lobo, Daniel; Levin, Michael

2015-01-01

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria. PMID:26610482

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage.

PubMed

Carranza-Rosales, Pilar; Said-Fernández, Salvador; Sepúlveda-Saavedra, Julio; Cruz-Vega, Delia E; Gandolfi, A Jay

2005-06-01

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl2. At concentrations of 1 and 10 microM of HgCl2 there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 microM HgCl2, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 microM HgCl2 ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 microM HgCl2 apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9h with 15 microM HgCl2. This effect was not observed at short times (1 and 3h) with this same concentration, neither with 1 and 10 microM HgCl2 in all the studied times. Taken together, these findings indicate that low concentrations of HgCl2 induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy.

PubMed

Chan, C W; Hussain, I; Waugh, D G; Lawrence, J; Man, H C

2014-09-01

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by hemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology. Copyright © 2014 Elsevier B.V. All rights reserved.

Leaf extract of Rhus verniciflua Stokes protects dopaminergic neuronal cells in a rotenone model of Parkinson's disease.

PubMed

Kim, Seung; Park, Se-Eun; Sapkota, Kumar; Kim, Myung-Kon; Kim, Sung-Jun

2011-10-01

The present study investigated the neuroprotective effects of Rhus verniciflua Stokes (RVS) leaf extract on rotenone-induced apoptosis in human dopaminergic cells, SH-SY5Y. Cells were pretreated with RVS extract for 1 h then treated with vehicle or rotenone for 24 h. Cell viability, cell cytotoxicity, cell morphology and nuclear morphology were examined by MTT assay, lactate dehydrogenase release assay, phase contrast microscopy and staining with Hoechast 33342, respectively. Reactive oxygen species were measured by 2'7'-dichlorofluorescein diacetate and fragmented DNA was observed by TUNEL assay. Mitochondrial membrane potential was determined by Rhodamine 123. Pro-apoptotic and anti-apoptotic proteins and tyrosine hydroxylase were analysed by Western blotting. Results showed that RVS suppressed rotenone-induced reactive oxygen species generation, cellular injury and apoptotic cell death. RVS also prevented rotenone-mediated changes in Bax/Bcl-2 levels, mitochondrial membrane potential dissipation and Caspase 3 activation. Moreover, RVS pretreatment increased the tyrosine hydroxylase levels in SH-SY5Y cells. These findings demonstrate that RVS protects SH-SY5Y cells against rotenone-induced injury and suggest that RVS may have potential therapeutic value for neurodegenerative disease associated with oxidative stress. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

PubMed Central

Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Imaoka, Yuka; Itagaki, Yoshitsuna; Suzuki, Yuki

2018-01-01

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. PMID:29723197

Glycans coated silver nanoparticles induces autophagy and necrosis in HeLa cells

NASA Astrophysics Data System (ADS)

Panzarini, Elisa; Mariano, Stefania; Dini, Luciana

2015-06-01

This study reports the induction of autophagy by two concentrations (2×103 or 2×104 NPs/cell) of 30 nm sized β-D-Glucose- and β-D-Glucose/Sucrose-coated silver NanoParticles (AgNPs-G and AgNPs-GS respectively) in HeLa cells treated for 6, 12, 24 and 48 hrs. Cell viability was assessed by Neutral Red (NR) test and morphological evaluation. In addition ROS generation (NBT test) and induction of apoptosis/necrosis (Annexin V/Propidium Iodide-Annexin V/PI staining) and autophagy (Monodansylcadaverine-MDC staining) were evaluated. Cytotoxicity, ROS generation and morphology changes depend on NPs type and amount, and incubation time. As a general result, AgNPs-G are more toxic than AgNPs-GS. Moreover, the lowest AgNPs-GS concentration is ineffective on cell viability and ROS generation. Only 10% and 25% of viable HeLa cells were found at the end of incubation time in the presence of higher amount of AgNPs - G and AgNPs-GS respectively and in parallel ROS generation is induced. To elucidate the type of cell death, Annexin V/PI and MDC staining was performed. Interestingly, irrespective of coating type and NPs amount the percentage of apoptotic cells (Annexin V+/PI-) is similar to viable HeLa cells. At contrary, we observed a NPs amount dependent autophagy and necrosis induction. In fact, the lower amount of NPs induces autophagy (MDC+/PI- cells) whereas the higher one induces necrosis (Annexin V+/PI+ cells). Our findings suggest that AgNPs-induced cytotoxicity depends on AgNPs amount and type and provide preliminary evidence of induction of autophagy in HeLa cells cultured in the presence of AgNPs.

Apoptosis of human gastric cancer SGC-7901 cells induced by podophyllotoxin.

PubMed

Ji, Chen-Feng; Ji, Yu-Bin

2014-05-01

Numerous studies have demonstrated that podophyllotoxin and its derivatives exhibit antitumor effects. The aim of the present study was to investigate SGC-7901 cell apoptosis and the underlying mechanism induced by podophyllotoxin. SGC-7901 cells were treated with varying concentrations of podophyllotoxin. MTT assays and flow cytometry were used to evaluate the effects of podophyllotoxin on the proliferation and apoptosis of SGC-7901 cells, while fluorescence inverted microscopy was used to observe the morphology of SGC-7901 cells that had been dyed with Hoechst 33258. In addition, laser scanning confocal microscopy was used to analyze the mitochondrial membrane potential (MMP) of SGC-7901 cells dyed with Rhodamine 123. Western blotting was performed to analyze the expression levels of cytochrome c (cyt- c ), caspase-9 and caspase-3 in the SGC-7901 cells. The results indicated that podophyllotoxin was capable of inhibiting growth and inducing the apoptosis of SGC-7901 cells in a dose-dependent manner, causing cell cycle arrest at the G 2 /M phase. After 48 h of treatment, the apoptotic morphology of SGC-7901 cells was clear, exhibiting cell protuberance, concentrated cytoplasms and apoptotic bodies. Following 24 h of treatment, the MMP of the SGC-7901 cells decreased. In addition, after 48 h, the expression of cyt- c was shown to be upregulated, while the expression levels of pro-caspase-9 and pro-caspase-3 in the SGC-7901 cells were shown to be downregulated. In conclusion, apoptosis can be induced in SGC-7901 cells by podophyllotoxin, potentially via a mitochondrial pathway, indicating that podophyllotoxin may be a potent agent for cancer treatment.

[Study on thaspine in inducing apoptosis of A549 cell].

PubMed

Zhang, Yan-min; He, Lang-chong

2007-04-01

To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. Thaspine has the effects of anti-tumor and inducing apoptosis.

Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kabiri, Azadeh, E-mail: z_kabiri@resident.mui.ac.ir; Esfandiari, Ebrahim, E-mail: esfandiari@med.mui.ac.ir; Hashemibeni, Batool, E-mail: hashemibeni@med.mui.ac.ir

2012-07-27

Highlights: Black-Right-Pointing-Pointer We investigated effects of FGF-2 on hADSCs. Black-Right-Pointing-Pointer We examine changes in the level of gene expressions of SOX-9, aggrecan and collagen type II and type X. Black-Right-Pointing-Pointer FGF-2 induces chondrogenesis in hADSCs, which Bullet Increasing information will decrease quality if hospital costs are very different. Black-Right-Pointing-Pointer The result of this study may be beneficial in cartilage tissue engineering. -- Abstract: Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs)more » are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.« less

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells.

PubMed

Pi, Jiang; Jin, Hua; Liu, Ruiying; Song, Bing; Wu, Qing; Liu, Li; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Cai, Jiye

2013-02-01

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA-Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA-Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA-Se NPs in MCF-7 cells. The results indicated that the 70-nm FA-Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA-Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca(2)+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA-Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA-Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA-Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.

Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae

PubMed Central

Lorenz, Michael C.; Cutler, N. Shane; Heitman, Joseph

2000-01-01

Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol–insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation. PMID:10637301

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

PubMed

Buyse, M A; Lefebvre, R A; Fraeyman, N H

1989-01-01

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

Effect of Overproduction of Mitochondrial Uncoupling Protein 2 on Cos7 Cells: Induction of Senescent-like Morphology and Oncotic Cell Death.

PubMed

Nishio, Koji; Ma, Qian

2016-01-01

The maintenance of mitochondrial membrane potential is essential for cell growth and survival. Mitochondrial uncoupling protein 2 plays the most important roles in uncoupling oxidative phosphorylation and decreasing mitochondrial O2- production by regulating the mitochondrial membrane potential. We propose that mouse UCP2 has two glycine-rich motifs, motif 1: EGIRGLWKG (170-178) and a known Walker A-like motif 2: EGPRAFYKG (264-272). These motifs seem to be important for the function of UCP2. We investigated the biological effects of overproduced-UCP2 and its physiological consequence in Cos7 cells. We introduced several amino acid changes in the motif 1. The expression vectors of the green fluorescent protein (GFP)-fused UCP2 and mutant UCP2 were constructed and expressed in Cos7 cells. The UCP2-GFP-expressed cells significantly down-regulated the mitochondrial membrane potentials and induced the enlarged cell shapes. Next we generated the stably UCP2-GFP-expressed Cos7 cells by selection with the antibiotic Genecitin (G418). Within the first few weeks following G418-selection, the stably UCP2-GFP-expressed cells could not divide well and gradually manifested the irregular and enlarged senescent-like cell morphology. The UCP2/K177E- or UCP2/G174L-expressed cells did not induce the enlarged cell shapes. Hence, UCP2/K177E and UCP2/G174L produced the functional incompetence of the glycine-rich motif 1. The senescent-like cells significantly decreased the mitochondrial membrane potentials and finally died nearly one month. Overproduction of UCP2 irreversibly reduces the mitochondrial membrane potentials and induces the senescent-like morphology and finally oncotic cell death in Cos7 cells. These changes seem to occur from the irreversible metabolic changes following total loss of cellular ATP.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

PubMed Central

Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

2017-01-01

ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

High-dose ascorbic acid induces carcinostatic effects through hydrogen peroxide and superoxide anion radical generation-induced cell death and growth arrest in human tongue carcinoma cells.

PubMed

Ohwada, Ryouhei; Ozeki, Yu; Saitoh, Yasukazu

2017-01-01

High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3 mM of AsA for 60 min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H 2 O 2 ) scavenger catalase and a superoxide anion radical (O 2 - ) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H 2 O 2 and O 2 - generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer cells.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheu, C.W.; Moreland, F.M.; Dunkel, V.C.

1987-01-01

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO/sub 2/ incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14more » had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.« less

Three-dimensional morphological imaging of human induced pluripotent stem cells by using low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Kakuno, Yumi; Goto, Kentaro; Fukami, Tadashi; Sugiyama, Norikazu; Iwai, Hidenao; Mizuguchi, Yoshinori; Yamashita, Yutaka

2014-03-01

There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4{sup +} T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependentmore » phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. - Highlights: • Jurkat T cells expressing the HIV-1 envelope fuse with THP-1 monocytes. • Heterokaryons display a dominant myeloid phenotype and monocyte function. • Heterokaryons exhibit activation features in the absence of activation agents. • Activation is not due to cell-cell interaction but requires cell-cell fusion. • The activated monocyte-like phenotype is mediated by TLR2/TLR4 signaling.« less

Induced sensitivity of Bacillus subtilis colony morphology to mechanical media compression

PubMed Central

Polka, Jessica K.

2014-01-01

Bacteria from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly) or merely physical. Here we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained) state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells. These results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus can be modulated by altering a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof. PMID:25289183

Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells.

PubMed

Rivera, Francisco J; Sierralta, Walter D; Minguell, Jose J; Aigner, Ludwig

2006-10-02

Bone marrow-derived mesenchymal stem cells (MSCs) are not restricted in their differentiation fate to cells of the mesenchymal lineage. They acquire a neural phenotype in vitro and in vivo after transplantation in the central nervous system. Here we investigated whether soluble factors derived from different brain regions are sufficient to induce a neuronal phenotype in MSCs. We incubated bone marrow-derived MSCs in conditioned medium (CM) derived from adult hippocampus (HCM), cortex (CoCM) or cerebellum (CeCM) and analyzed the cellular morphology and the expression of neuronal and glial markers. In contrast to muscle derived conditioned medium, which served as control, conditioned medium derived from the different brain regions induced a neuronal morphology and the expression of the neuronal markers GAP-43 and neurofilaments in MSCs. Hippocampus derived conditioned medium had the strongest activity. It was independent of NGF or BDNF; and it was restricted to the neuronal differentiation fate, since no induction of the astroglial marker GFAP was observed. The work indicates that soluble factors present in the brain are sufficient to induce a neuronal phenotype in MSCs.

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration

PubMed Central

2013-01-01

Background Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. Results The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. Conclusions We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events. PMID:24025096

Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

NASA Astrophysics Data System (ADS)

Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

2008-02-01

Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

A comparative study of corneal incisions induced by diamond and steel knives and two ultraviolet radiations from an excimer laser.

PubMed Central

Marshall, J; Trokel, S; Rothery, S; Krueger, R R

1986-01-01

This paper reviews the potential role of excimer lasers in corneal surgery. The morphology of incisions induced by two wavelengths of excimer laser radiation, 193 nm and 248 nm, are compared with the morphology of incisions produced by diamond and steel knives. Analysis suggests that ablation induced by excimer laser results from highly localised photochemical reactions and that 193 nm is the optimal wavelength for surgery. The only significant complication of laser surgery is loss of endothelial cells when incisions are within 40 micron of Descemet's membrane. Images PMID:3013283

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging.

PubMed

Olivier, Elodie; Dutot, Mélody; Regazzetti, Anne; Dargère, Delphine; Auzeil, Nicolas; Laprévote, Olivier; Rat, Patrice

2017-05-01

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

PubMed

Rogers, Danny A; Schor, Nina F

2013-03-10

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

NASA Astrophysics Data System (ADS)

Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

2015-04-01

Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

Cortical astrocytes exposed to tributyltin undergo morphological changes in vitro.

PubMed

Mizuhashi, S; Ikegaya, Y; Nishiyama, N; Matsuki, N

2000-11-01

We investigated the effect of tributyltin (TBT), an endocrine-disrupting chemical, on the morphology and viability of cultured rat cortical astrocytes. Cultured astrocytes exhibited smooth and planiform morphology under normal conditions. Following exposure to TBT, however, they showed rapid morphological changes that are characterized by asteriated cell bodies and process formation in a time- and concentration-dependent manner. Higher concentrations of TBT produced progressive cell death of the astrocytes. In serum-free medium, TBT at a concentration as low as 200 nM induced the stellation. Pharmacological studies revealed that the morphological changes were alleviated by application of diverse free radical scavengers or antioxidants such as catalase, superoxide dismutase, Trolox, ascorbic acid and N-acetyl-L-cysteine, suggesting that TBT-induced stellation is caused by oxidative stress involving free radicals, particularly reactive oxygen species. Furthermore, we found that the astrocyte stellation was abolished by treatment with inhibitors of phospholipase C, mitogen-activated protein kinase kinase or tyrosine phosphatase. The data suggest that TBT causes the stellation through intracellular signaling cascades rather than its non-specific toxicity. These findings provide an important insight for reconciling the problems in assumed aversive actions of this environmental pollutant for mammals.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

PubMed

Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

2015-07-31

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology

PubMed Central

2013-01-01

Background The APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation. Methods The localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity. Results APC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton. Conclusions These findings indicate that membrane protrusions with APC/β-catenin-containing puncta control the migratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later stages of tumor progression might impact tumor cell dissemination or colonization. PMID:23302090

Microglia is activated by astrocytes in trimethyltin intoxication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roehl, Claudia; Sievers, Jobst

2005-04-01

Microglia participates in most acute and chronic neuropathologies and its activation appears to involve interactions with neurons and other glial cells. Trimethyltin (TMT)-induced brain damage is a well-characterized model of neurodegeneration, in which microglial activation occurs before neuronal degeneration. The aim of this in vitro study was to investigate the role of astroglia in TMT-induced microgliosis by using nitric oxide (NO), inducible NO synthase (iNOS), and morphological changes as parameters for microglial activation. Our investigation discusses (a) whether microglial cells can be activated directly by TMT; (b) if astroglial cells are capable of triggering or modulating microglial activation; (c) howmore » the morphology and survival of microglia and astrocytes are affected by TMT treatment; and (d) whether microglial-astroglial interactions depend on direct cell contact or on soluble factors. Our results show that microglia are more vulnerable to TMT than astrocytes are and cannot be activated directly by TMT with regard to the examined parameters. In bilayer coculture with viable astroglial cells, microglia produce NO in significant amounts at subcytotoxic concentrations of TMT (20 {mu}mol/l). At these TMT concentrations, microglial cells in coculture convert into small round cells without cell processes, whereas flat, fibroblast-like astrocytes convert into thin process bearing stellate cells with a dense and compact cell body. We conclude that astrocytes trigger microglial activation after treatment with TMT, although the mechanisms of this interaction remain unknown.« less

Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fiber formation without inducing epithelial-to-mesenchymal transition.

PubMed

Jensen, Helene H; Holst, Mikkel R; Login, Frédéric H; Morgen, Jeanette J; Nejsum, Lene N

2018-06-01

Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.

Morphometric analysis of cisplatin-induced neurite outgrowth in N1E-115 neuroblastoma cells.

PubMed

Konings, P N; Philipsen, R L; van den Broek, J H; Ruigt, G S

1994-08-29

Cisplatin, a widely used cytostatic drug for the control of a variety of neoplastic tumors, unexpectedly induced neurite outgrowth in N1E-115 neuroblastoma cells and this phenomenon was studied further in detail with morphometric analysis. As expected, cisplatin dose-dependently reduced cell number. At the same time, however, cisplatin affected the morphology of the neuroblastoma cells that changed from small rounded cell bodies into large flat cell bodies with neurites. The neurite length/cell as a function of cisplatin concentration showed a bell-shaped curve. The maximal effect (1200% of control) on neurite length/cell was observed at 1 microgram/ml cisplatin. In conclusion, cisplatin induced cellular differentiation in N1E-115 neuroblastoma cells at and just above threshold doses for cytostatic activity.

Dopamine is a key regulator in the signalling pathway underlying predator-induced defences in Daphnia

PubMed Central

Weiss, Linda C.; Leese, Florian; Laforsch, Christian; Tollrian, Ralph

2015-01-01

The waterflea Daphnia is a model to investigate the genetic basis of phenotypic plasticity resulting from one differentially expressed genome. Daphnia develops adaptive phenotypes (e.g. morphological defences) thwarting predators, based on chemical predator cue perception. To understand the genomic basis of phenotypic plasticity, the description of the precedent cellular and neuronal mechanisms is fundamental. However, key regulators remain unknown. All neuronal and endocrine stimulants were able to modulate but not induce defences, indicating a pathway of interlinked steps. A candidate able to link neuronal with endocrine responses is the multi-functional amine dopamine. We here tested its involvement in trait formation in Daphnia pulex and Daphnia longicephala using an induction assay composed of predator cues combined with dopaminergic and cholinergic stimulants. The mere application of both stimulants was sufficient to induce morphological defences. We determined dopamine localization in cells found in close association with the defensive trait. These cells serve as centres controlling divergent morphologies. As a mitogen and sclerotization agent, we anticipate that dopamine is involved in proliferation and structural formation of morphological defences. Furthermore, dopamine pathways appear to be interconnected with endocrine pathways, and control juvenile hormone and ecdysone levels. In conclusion, dopamine is suggested as a key regulator of phenotypic plasticity. PMID:26423840

Investigation of dynamic morphological changes of cancer cells during photoimmuno therapy (PIT) by low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Ogawa, Mikako; Yamauchi, Toyohiko; Iwai, Hidenao; Magata, Yasuhiro; Choyke, Peter L.; Kobayashi, Hisataka

2014-03-01

We have reported a new molecular-targeted cancer phototherapy, photoimmunotherapy (PIT), which killed implanted tumors in mice without side-effects. To understand the mechanism of cell killing with PIT, three-dimentional dynamic low-coherence quantitative phase microscopy (3D LC-QPM), a device developed by Hamamatsu Photonics K.K, was used to detect morphologic changes in cancer cells during PIT. 3T3/HER2 cells were incubated with anti-HER2 trastuzumab-IR700 (10 μg/mL, 0.1 μM as IR700) for 24 hours, then, three-dimensionally imaged with the LC-QPM during the exposure of two different optically filtered lights for excitation of IR700 (500-780 nm) and imaging (780-950 nm). For comparison with traditional PDT, the same experiments were performed with Photofrin (10 and 1 μM). Serial changes in the cell membrane were readily visualized on 3D LC-QPM. 3T3/HER2 cells began to swell rapidly after exposure to 500-780 nm light excitation. The cell volume reached a maximum within 1 min after continuous exposure, and then the cells appeared to burst. This finding suggests that PIT damages the cell membrane by photo-reaction inducing an influx of water into the cell causing swelling and bursting of the cells. Interestingly, even after only 5 seconds of light exposure, the cells demonstrated swelling and bursting albeit more slowly, implying that sufficient cumulative damage occurs on the cell membrane to induce lethal damage to cells even at minimal light exposure. Similar but non-selective membrane damage was shown in PDT-treated cells Photofrin. Thus, PIT induces sufficient damage to the cell membrane within 5 seconds to induce rapid necrotic cell death which can be observed directly with 3D LC-QPM. Further investigation is needed to evaluate the biochemical mechanisms underlying PIT-induced cellular membrane damage.

Cytoprotective effects of essential oil of Pinus halepensis L. against aspirin-induced toxicity in IEC-6 cells.

PubMed

Bouzenna, Hafsia; Hfaiedh, Najla; Bouaziz, Mouhamed; Giroux-Metges, Marie-Agnès; Elfeki, Abdelfattah; Talarmin, Hélène

2017-12-01

Essential oils from Pinus species have been reported to have various therapeutic properties. This study was undertaken to identify the chemical composition and cytoprotective effects of the essential oil of Pinus halepensis L. against aspirin-induced damage in cells in vitro. The cytoprotection of the oil against toxicity of aspirin on the small intestine epithelial cells IEC-6 was tested. The obtained results have shown that 35 different compounds were identified. Aspirin induced a decrease in cell viability, and exhibited significant damage to their morphology and an increase in superoxide dismutase (SOD) and catalase (CAT) activities. However, the co-treatment of aspirin with the essential oil of Pinus induced a significant increase in cell viability and a decrease in SOD and CAT activities. Overall, these finding suggest that the essential oil of Pinus halepensis L. has potent cytoprotective effect against aspirin-induced toxicity in IEC-6 cells.

Cell–material interactions on biphasic polyurethane matrix

PubMed Central

Dicesare, Patrick; Fox, Wade M.; Hill, Michael J.; Krishnan, G. Rajesh; Yang, Shuying; Sarkar, Debanjan

2013-01-01

Cell–matrix interaction is a key regulator for controlling stem cell fate in regenerative tissue engineering. These interactions are induced and controlled by the nanoscale features of extracellular matrix and are mimicked on synthetic matrices to control cell structure and functions. Recent studies have shown that nanostructured matrices can modulate stem cell behavior and exert specific role in tissue regeneration. In this study, we have demonstrated that nanostructured phase morphology of synthetic matrix can control adhesion, proliferation, organization and migration of human mesenchymal stem cells (MSCs). Nanostructured biodegradable polyurethanes (PU) with segmental composition exhibit biphasic morphology at nanoscale dimensions and can control cellular features of MSCs. Biodegradable PU with polyester soft segment and hard segment composed of aliphatic diisocyanates and dipeptide chain extender were designed to examine the effect polyurethane phase morphology. By altering the polyurethane composition, morphological architecture of PU was modulated and its effect was examined on MSC. Results show that MSCs can sense the nanoscale morphology of biphasic polyurethane matrix to exhibit distinct cellular features and, thus, signifies the relevance of matrix phase morphology. The role of nanostructured phases of a synthetic matrix in controlling cell–matrix interaction provides important insights for regulation of cell behavior on synthetic matrix and, therefore, is an important tool for engineering tissue regeneration. PMID:23255285

Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration.

PubMed

Blum, Walter; Pecze, László; Felley-Bosco, Emanuela; Schwaller, Beat

2015-12-22

The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared. Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system. CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space. We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.

Kynurenic Acid Prevents Cytoskeletal Disorganization Induced by Quinolinic Acid in Mixed Cultures of Rat Striatum.

PubMed

Pierozan, Paula; Biasibetti-Brendler, Helena; Schmitz, Felipe; Ferreira, Fernanda; Pessoa-Pureur, Regina; Wyse, Angela T S

2018-06-01

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan known to modulate a number of mechanisms involved in neural dysfunction. Although its activity in the brain has been widely studied, the effect of KYNA counteracting the actions of quinolinic acid (QUIN) remains unknown. The present study aims at describing the ability of 100 μM KYNA preventing cytoskeletal disruption provoked by QUIN in astrocyte/neuron/microglia mixed culture. KYNA totally preserved cytoskeletal organization, cell morphology, and redox imbalance in mixed cultures exposed to QUIN. However, KYNA partially prevented morphological alteration in isolated primary astrocytes and failed to protect the morphological alterations of neurons caused by QUIN exposure. Moreover, KYNA prevented QUIN-induced microglial activation and upregulation of ionized calcium-binding adapter molecule 1 (Iba-1) and partially preserved tumor necrosis factor-α (TNF-α) level in mixed cultures. TNF-α level was also partially preserved in astrocytes. In addition to the mechanisms dependent on redox imbalance and microglial activation, KYNA prevented downregulation of connexin-43 and the loss of functionality of gap junctions (GJs), preserving cell-cell contact, cytoskeletal organization, and cell morphology in QUIN-treated cells. Furthermore, the toxicity of QUIN targeting the cytoskeleton of mixed cultures was not prevented by the N-methyl-D-aspartate (NMDA) antagonist MK-801. We suggest that KYNA protects the integrity of the cytoskeleton of mixed cultures by complex mechanisms including modulating microglial activation preventing oxidative imbalance and misregulated GJs leading to disrupted cytoskeleton in QUIN-treated cells. This study contributed to elucidate the molecular basis of KYNA protection against QUIN toxicity.

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

PubMed

Shao, Genbao; Lai, Wensheng; Wan, Xiaolei; Xue, Jing; Wei, Ye; Jin, Jie; Zhang, Liuping; Lin, Qiong; Shao, Qixiang; Zou, Shengqiang

2017-05-01

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclin D1. Significantly, blockage of epidermal growth factor receptor (EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling.

PubMed

Khalo, Irina V; Konokhova, Anastasiya I; Orlova, Darya Y; Trusov, Konstantin V; Yurkin, Maxim A; Bartova, Eva; Kozubek, Stanislav; Maltsev, Valeri P; Chernyshev, Andrei V

2018-05-30

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Monitoring cell morphology during necrosis and apoptosis by quantitative phase imaging

NASA Astrophysics Data System (ADS)

Mugnano, Martina; Calabuig, Alejandro; Grilli, Simonetta; Miccio, Lisa; Ferraro, Pietro

2015-05-01

Cellular morphology changes and volume alterations play significant roles in many biological processes and they are mirrors of cell functions. In this paper, we propose the Digital Holographic microscope (DH) as a non-invasive imaging technique for a rapid and accurate extraction of morphological information related to cell death. In particular, we investigate the morphological variations that occur during necrosis and apoptosis. The study of necrosis is extremely important because it is often associated with unwarranted loss of cells in human pathologies such as ischemia, trauma, and some forms of neurodegeneration; therefore, a better elucidation in terms of cell morphological changes could pave the way for new treatments. Also, apoptosis is extremely important because it's involved in cancer, both in its formation and in medical treatments. Because the inability to initiate apoptosis enhances tumour formation, current cancer treatments target this pathway. Within this framework, we have developed a transmission off-axis DH apparatus integrated with a micro incubator for investigation of living cells in a temperature and CO2 controlled environment. We employ DH to analyse the necrosis cell death induced by laser light (wavelength 473 nm, light power 4 mW). We have chosen as cellular model NIH 3T3 mouse embryonic fibroblasts because their adhesive features such as morphological changes, and the time needed to adhere and spread have been well characterized in the literature. We have monitored cell volume changes and morphological alterations in real time in order to study the necrosis process accurately and quantitatively. Cell volume changes were evaluated from the measured phase changes of light transmitted through cells. Our digital holographic experiments showed that after exposure of cells to laser light for 90-120 min., they swell and then take on a balloon-like shape until the plasma membrane ruptures and finally the cell volume decreases. Furthermore, we present a preliminary study on the variation of morphological parameters in case of cell apoptosis induced by exposure to 10 μM cadmium chloride. We employ the same cell line, monitoring the process for 18 hours. In the vast group of environmental pollutants, the toxic heavy metal cadmium is considered a likely candidate as a causative agent of several types of cancers. Widely distributed and used in industry, and with a broad range of target organs and a long half-life (10-30 years) in the human body, this element has been long known for its multiple adverse effects on human health, through occupational or environmental exposure. In apoptosis, we measure cell volume decrease and cell shrinking. Both data of apoptosis and necrosis were analysed by means of a Sigmoidal Statistical Distribution function, which allows several quantitative data to be established, such as swelling and cell death time, flux of intracellular material from inside to outside the cell, initial and final volume versus time. In addition, we can quantitatively study the cytoplasmatic granularity that occurs during necrosis. As a future application, DH could be employed as a non-invasive and label-free method to distinguish between apoptosis and necrosis in terms of morphological parameters.

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

Redifferentiation of human hepatoma cells (SMMC-7721) induced by two new highly oxygenated bisabolane-type sesquiterpenes.

PubMed

Miao, Ruidong; Wei, Juan; Zhang, Qi; Sajja, Venkateswara; Yang, Jinbo; Wang, Qin

2008-12-01

Bisabolane-type sesquiterpenes are a class of biologically active compounds that has antitumour,antifungal, antibacterial,antioxidant and antivenom properties.We investigated the effect of two new highly oxygenated bisabolane-type sesquiterpenes (HOBS)isolated from Cremanthodium discoideum (C.discoideum) on tumour cells. Our results showed that HOBS induced morphological differentiation and reduced microvilli formation on the cell surface in SMMC-7721 cells.Flow cytometry analysis demonstrated that HOBS could induce cell-cycle arrest in the G1 phase. Moreover,HOBS was able to increase tyrosine-alpha ketoglutarate transaminase activity,decrease alpha- foetoprotein level and gamma-glutamyl transferase activity. In addition,we found that HOBS inhibited the anchorage- independent growth of SMMC-7721 cells in a dose-dependent manner.Taken together,all the above observations indicate that HOBS might be able to normalize malignant SMMC-7721 cells by inhibiting cell proliferation and inducing redifferentiation.

Effects of hydrogen peroxide on vestibular hair cells in the guinea pig: importance of cell membrane impairment preceding cell death.

PubMed

Tanigawa, Tohru; Tanaka, Hirokazu; Hayashi, Ken; Nakayama, Meiho; Iwasaki, Satoshi; Banno, Shinya; Takumida, Masaya; Brodie, Hirally; Inafuku, Shigeru

2008-11-01

Our findings indicate that oxidative stress induces morphological changes in vestibular hair cells and subsequently leads to cell death after 2.5 h. The aim of this study was to confirm the direct effects of oxidative stress on vestibular hair cells. Vestibular hair cells isolated from guinea pigs were loaded with 1 or 10 mM H2O2, and morphological changes were observed. In addition, in a viability/cytotoxicity assay system, the numbers of dead cells in isolated cristae ampullares were counted 1, 3, and 5 h after loading with H2O2 or artificial perilymph (control). Reactive oxygen, in the form of H2O2, directly affects the cell membrane of isolated vestibular hair cells and causes swelling of the cell body, bleb formation, and shortening of the neck region. Morphological changes occur within 30 min after loading with H2O2, but a significant increase in the number of dead cells is noted only after 3 h.

Apoptosis evaluation in epithelial cells exposed to different chemicals: relevance of floating cells.

PubMed

Turco, L; De Angelis, I; Stammati, A; Zucco, F

2000-01-01

The recent increase in understanding of cell death has promoted new approaches in toxicological studies, mainly those dealing with in vitro systems where the evaluation of cell death has been the most widely adopted end-point in measuring the effects of chemical toxicants. The aim of this study was to investigate the possibility of improving the traditional cytotoxicity test protocols in order to produce more specific information on the type of cell death induced by exposure to toxicants. In particular, we characterized the mode of cell death in an established epithelial cell line, HEp-2 cells, which is frequently used in cytotoxicity testing owing to its easy handling and standardization of culture conditions. Reference chemicals for apoptosis and necrosis were selected as controls, together with other molecules that have been shown, in preliminary studies, to induce various morphological and structural modifications in relation to cell death. The results obtained show that: (a) the floating fraction of treated cells gives the clearest picture of the necrotic/apoptotic distribution; (b) morphological analysis is crucial for characterization of apoptosis; (c) more than one cytotoxic end-point is necessary to clearly identify the type of cell death.

Tailoring vessel morphology in vivo

NASA Astrophysics Data System (ADS)

Gould, Daniel Joseph

Tissue engineering is a rapidly growing field which seeks to provide alternatives to organ transplantation in order to address the increasing need for transplantable tissues. One huge hurdle in this effort is the provision of thick tissues; this hurdle exists because currently there is no way to provide prevascularized or rapidly vascularizable scaffolds. To design thick, vascularized tissues, scaffolds are needed that can induce vessels which are similar to the microvasculature found in normal tissues. Angiogenic biomaterials are being developed to provide useful scaffolds to address this problem. In this thesis angiogenic and cell signaling and adhesion factors were incorporated into a biomimetic poly(ethylene glycol) (PEG) hydrogel system. The composition of these hydrogels was precisely tuned to induce the formation of differing vessel morphology. To sensitively measure induced microvascular morphology and to compare it to native microvessels in several tissues, this thesis developed an image-based tool for quantification of scale invariant and classical measures of vessel morphology. The tool displayed great utility in the comparison of native vessels and remodeling vessels in normal tissues. To utilize this tool to tune the vessel response in vivo, Flk1::myr-mCherry fluorescently labeled mice were implanted with Platelet Derived Growth Factor-BB (PDGF-BB) and basic Fibroblast Growth Factor (FGF-2) containing PEG-based hydrogels in a modified mouse corneal angiogenesis assay. Resulting vessels were imaged with confocal microscopy, analyzed with the image based tool created in this thesis to compare morphological differences between treatment groups, and used to create a linear relationship between space filling parameters and dose of growth factor release. Morphological parameters of native mouse tissue vessels were then compared to the linear fit to calculate the dose of growth factors needed to induce vessels similar in morphology to native vessels. Resulting induced vessels did match in morphology to the target vessels. Several other covalently bound signals were then analyzed in the assay and resulting morphology of vessels was compared in several studies which further highlighted the utility of the micropocket assay in conjunction with the image based tool for vessel morphological quantification. Finally, an alternative method to provide rapid vasculature to the constructs, which relied on pre-seeded hydrogels encapsulated endothelial cells was also developed and shown to allow anastamosis between induced host vessels and the implanted construct within 48 hours. These results indicate great promise in the rational design of synthetic, bioactive hydrogels, which can be used as a platform to study microvascular induction for regenerative medicine and angiogenesis research. Future applications of this research may help to develop therapeutic strategies to ameliorate human disease by replacing organs or correcting vessel morphology in the case of ischemic diseases and cancer.

Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells.

PubMed

Lukhele, Sindiswa T; Motadi, Lesetja R

2016-09-01

Cervical cancer remains a global health related issue among females of Sub-Saharan Africa, with over half a million new cases reported each year. Different therapeutic regimens have been suggested in various regions of Africa, however, over a quarter of a million women die of cervical cancer, annually. This makes it the most lethal cancer amongst black women and calls for urgent therapeutic strategies. In this study we compare the anti-proliferative effects of crude extract of Cannabis sativa and its main compound cannabidiol on different cervical cancer cell lines. To achieve our aim, phytochemical screening, MTT assay, cell growth analysis, flow cytometry, morphology analysis, Western blot, caspase 3/7 assay, and ATP measurement assay were conducted. Results obtained indicate that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations. They further revealed that apoptosis was induced by cannabidiol as shown by increased subG0/G1 and apoptosis through annexin V. Apoptosis was confirmed by overexpression of p53, caspase 3 and bax. Apoptosis induction was further confirmed by morphological changes, an increase in Caspase 3/7 and a decrease in the ATP levels. In conclusion, these data suggest that cannabidiol rather than Cannabis sativa crude extracts prevent cell growth and induce cell death in cervical cancer cell lines.

Cytotoxic effect of artocarpin on T47D cells.

PubMed

Arung, Enos Tangke; Wicaksono, Britanto Dani; Handoko, Yohana Ayupriyanti; Kusuma, Irawan Wijaya; Shimizu, Kuniyoshi; Yulia, Dina; Sandra, Ferry

2010-10-01

In our screening projects for anticancer agents from natural resources, artocarpin [6-(3-methyl-1-butenyl)-5,2',4'-trihydroxy-3-isoprenyl-7-methoxyflavone] isolated from wood of jack fruit (Artocarpus heterophyllus) showed potent cytotoxic activity on human T47D breast cancer cells. The mode of action of artocarpin was evaluated by its effect on cell viability, nuclear morphology, cell cycle progression, expression of protein markers for apoptosis, and mitochondrial membrane potential (Delta psi m). These results showed that artocarpin caused a reduction of cell viability in a concentration-dependent manner and an alteration of cell and nuclear morphology. Moreover, the percentage of the sub-G1 phase formation was elevated dose-dependently. Artocarpin induced activation of caspase 8 and 10 as indicated by stronger signal intensity of cleaved-caspase 8 and weaker signal intensity of caspase 10 markers detected after artocarpin treatment. In addition, we also noticed the activation of caspase 3 by artocarpin. There were negligible changes in mitochondrial membrane potential (Delta psi m) due to artocarpin treatment. All together, these data indicated that artocarpin induced apoptosis in T47D cells possibly via an extrinsic pathway.

New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

2016-03-01

Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

[Essential oil from Artemisia lavandulaefolia induces apoptosis and necrosis of HeLa cells].

PubMed

Zhang, Lu-min; Lv, Xue-wei; Shao, Lin-xiang; Ma, Yan-fang; Cheng, Wen-zhao; Gao, Hai-tao

2013-12-01

To investigate the effects of Artemisia lavandulaefolia essential oil on apoptosis and necrosis of HeLa cells. Cell viability was assayed using MTT method. The morphological and structure alterations in HeLa cells were observed by microscopy. Furthermore, cell apoptosis was measured by DNA Ladder and flow cytometry. DNA damage was measured by comet assay, and the protein expression was examined by Western blot analysis. MTT assay displayed essential oil from Artemisia lavandulaefolia could inhibit the proliferation of HeLa cells in a dose-dependent manner. After treated with essential oil of Artemisia lavadulaefolia for 24 h, HeLa cells in 100 and 200 microg/mL experiment groups exhibited the typical morphology changes of undergoing apoptosis, such as cell shrinkage and nucleus chromatin condensed. However, the cells in the 400 microg/mL group showed the necrotic morphology changes including cytomembrane rupture and cytoplasm spillover. In addition, DNA Ladder could be demonstrated by DNA electrophoresis in each experiment group. Apoptosis peak was also evident in flow cytometry in each experiment group. After treating the HeLa cells with essential oil of Artemisia lavadulaefolia for 6 h, comet tail was detected by comet assay. Moreover, western blotting analysis showed that caspase-3 was activated and the cleavage of PARP was inactivated. Essential oil from Artemisia lavadulaefolia can inhibit the proliferation of HeLa cells in vitro. Low concentration of essential oil from Artemisia lavadulaefolia can induce apoptosis, whereas high concentration of the compounds result in necrosis of HeLa cells. And,the mechanism may be related to the caspase-3-mediated-PARP apoptotic signal pathway.

Monitoring the change of mitochondrial morphology and its metabolism of the breast cancer cells with the treatment of Hsp70 inhibitor during heat shock by fluorescence imaging

NASA Astrophysics Data System (ADS)

Yu, Biying; Yang, Hongqin; Zhang, Xiaoman; Li, Hui

2016-10-01

Heat shock (HS) is one of the best-studied exogenous cellular stresses, and all cellular compartments and metabolic processes are involved in HS response. The heat shock proteins (Hsps) expression enhanced during HS mainly localized in subcellular compartments, such as cytosol, endoplasmic reticulum and mitochandria. The major inducible heat shock protein 70 (Hsp70) modulate cellular homeostasis and promote cellular survival by blocking a caspase independent cell death through its association with apoptosis inducing factor. Mitochondria as the critical elements of HS response that participate in key metabolic reactions, and the changes in mitochonrial morphology may impact on mitochondrial metabolism. In this paper, the changes of mitorchondrial morphology in breast cancer cell have been monitored in real time after heat shock (43 °) by the fluorescence imaging, and the influence of Hsp70 inhibitor on mitochandrial structures have also been investigated. Then the information of mitochondrial metabolism which can be characterized by the level of the mitochondrial membrane potential has also been obtained wihout/with the treatment of Hsp70 inhibitor. Our data indicated that the mitochandrial morphology were related with the mitochandrial membrane potential, and the mitochandrial membrane potential was influenced significantly with the treatment of Hsp70 inhibitor during HS.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

PubMed

Baumgold, J; Spector, I

1987-04-01

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

IMPACT OF SMOKING HABITS ON THE STATE OF CHROMATIN AND MORPHOLOGY OF BUCCAL EPITHELIAL CELLS AMONG MEDICAL STUDENTS.

PubMed

Volkova, O; Ryabokon, E; Magda, I; Shckorbatov, Y

2017-01-01

The cells of buccal epithelium were investigated in groups of smoking and non-smoking students. Cell samples were collected by scraping with blunt sterile spatula, stained with orcein and photographed. The smoking of cigarettes and hookah induces significant decrease in nuclear and cell perimeter and cell area in cells of buccal epithelium. Smoking of hookah induces, besides, the heterochromatization in cell nuclei and the decrease of nuclear area. The data obtained indicate stress reaction in cells (heterochromatinization) and apoptosis-related changes in cells (decrease of nuclear and cell perimeter and cell area). These data show unfavorable effects of smoking cigarettes and even more harmful effect of hookah smoking.

Adaptive stress response to menadione-induced oxidative stress in Saccharomyces cerevisiae KNU5377.

PubMed

Kim, Il-Sup; Sohn, Ho-Yong; Jin, Ingnyol

2011-10-01

The molecular mechanisms involved in the ability of yeast cells to adapt and respond to oxidative stress are of great interest to the pharmaceutical, medical, food, and fermentation industries. In this study, we investigated the time-dependent, cellular redox homeostasis ability to adapt to menadione-induced oxidative stress, using biochemical and proteomic approaches in Saccharomyces cerevisiae KNU5377. Time-dependent cell viability was inversely proportional to endogenous amounts of ROS measured by a fluorescence assay with 2',7'-dichlorofluorescin diacetate (DCFHDA), and was hypersensitive when cells were exposed to the compound for 60 min. Morphological changes, protein oxidation and lipid peroxidation were also observed. To overcome the unfavorable conditions due to the presence of menadione, yeast cells activated a variety of cell rescue proteins including antioxidant enzymes, molecular chaperones, energy-generating metabolic enzymes, and antioxidant molecules such as trehalose. Thus, these results show that menadione causes ROS generation and high accumulation of cellular ROS levels, which affects cell viability and cell morphology and there is a correlation between resistance to menadione and the high induction of cell rescue proteins after cells enter into this physiological state, which provides a clue about the complex and dynamic stress response in yeast cells.

Assessment of sulforaphane-induced protective mechanisms against cadmium toxicity in human mesenchymal stem cells.

PubMed

Alkharashi, Nouf Abdulkareem Omer; Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

2018-04-01

Cd is a hazardous substance and carcinogen that is present in the environment; it is known to cause toxic effects in living organisms. Sulforaphane is a naturally available phytochemical with antioxidant, anti-inflammatory, and anticarcinogenic properties. However, the effects of sulforaphane on Cd toxicity in human mesenchymal stem cells (hMSCs) are unknown. In the present study, we investigated the molecular mechanisms of the effects of sulforaphane on Cd toxicity in hMSCs by using MTT assays, acridine orange/ethidium bromide staining, Hoechst staining, LysoRed staining, assessment of mitochondrial membrane potential, and gene expression analysis. Cd decreased hMSC viability in a dose-dependent manner with an IC 50 value of 56.5 μM. However, sulforaphane did not induce any significant reduction in cell viability. Nuclear morphological analysis revealed that Cd induced necrotic cell death. Additionally, Cd caused mitochondrial membrane potential loss in hMSCs. The treatment of Cd-exposed cells with sulforaphane (Cd-sulforaphane co-treatment) resulted in a significant recovery of the cell viability and nuclear morphological changes compared with that of cells treated with Cd only. The gene expression pattern of cells co-treated with Cd-sulforaphane was markedly different from that of Cd-treated cells, owing to the reduction in Cd toxicity. Our results clearly indicated that sulforaphane reduced Cd-induced toxic effects in hMSCs. Overall, the results of our study suggested that sulforaphane-rich vegetables and fruits can help to improve human health through amelioration of the molecular effects of Cd poisoning.

Effect of fiber diameter and orientation on fibroblast morphology and proliferation on electrospun poly(D,L-lactic-co-glycolic acid) meshes.

PubMed

Bashur, Chris A; Dahlgren, Linda A; Goldstein, Aaron S

2006-11-01

Engineered ligament tissues are promising materials for the repair of tears and ruptures, but require the development of biomaterial scaffolds that not only support physiologically relevant loads, but also possess architectures capable of orienting cell adhesion and extracellular matrix deposition. Based on evidence that micron-scale topographic features induce cell orientation through a contact guidance phenomenon, we postulate that oriented micron-scale fiber meshes-formed by the electrospinning process-can regulate cell morphology. To test this, fused fiber meshes of poly(d,l-lactic-co-glycolic acid) (PLGA) were electrospun onto rigid supports under conditions that produced mean fiber diameters of 0.14-3.6 microm, and angular standard deviations of 31-60 degrees . Analysis of the morphology of adherent NIH 3T3 fibroblasts indicated that projected cell area and aspect ratio increased systematically with both increasing fiber diameter and degree of fiber orientation. Importantly, cell morphology on 3.6 microm fibers was similar to that on spincoated PLGA films. Finally, cell densities on electrospun meshes were not significantly different from spincoated PLGA, indicating that cell proliferation is not sensitive to fiber diameter or orientation.

Real-time QCM-D monitoring of cancer cell death early events in a dynamic context.

PubMed

Nowacki, Laetitia; Follet, Julie; Vayssade, Muriel; Vigneron, Pascale; Rotellini, Laura; Cambay, Florian; Egles, Christophe; Rossi, Claire

2015-02-15

Since a few years, the acoustic sensing of whole cell is the focus of increasing interest for monitoring the cytoskeletal cellular response to morphological modulators. We aimed at illustrating the potentialities of the quartz crystal microbalance with dissipation (QCM-D) technique for the real-time detection of the earliest morphological changes that occur at the cell-substrate interface during programmed cell death. Human breast cancer cells (MCF-7) grown on serum protein-coated gold sensors were placed in dynamic conditions under a continuous medium flow. The mass and viscoelasticity changes of the cells were tracked by monitoring the frequency and dissipation shifts during the first 4h of cell exposure to staurosporine, a well-known apoptosis inducer. We have identified a QCM-D signature characteristic of morphological modifications and cell detachment from the sensing surface that are related to the pro-apoptotic treatment. In particular, for low staurosporine doses below 1 µM, we showed that recording the dissipation shift allows to detect an early cell response which is undetectable after the same duration by the classical analytical techniques in cell biology. Furthermore, this sensing method allows quantifying the efficiency of the drug effect in less than 4h without requiring labeling and without interfering in the system, thus preventing any loss of information. In the actual context of targeted cancer therapy development, we believe that these results bring new insights in favor of the use of the non invasive QCM-D technique for quickly probing the cancer cell sensitivity to death inducer drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Pulsed DC Electric Field–Induced Differentiation of Cortical Neural Precursor Cells

PubMed Central

Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K.; Cheng, Ji-Yen

2016-01-01

We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

Customization of Advia 120 thresholds for canine erythrocyte volume and hemoglobin concentration, and effects on morphology flagging results.

PubMed

Grimes, Carolyn N; Fry, Michael M

2014-12-01

This study sought to develop customized morphology flagging thresholds for canine erythrocyte volume and hemoglobin concentration [Hgb] on the ADVIA 120 hematology analyzer; compare automated morphology flagging with results of microscopic blood smear evaluation; and examine effects of customized thresholds on morphology flagging results. Customized thresholds were determined using data from 52 clinically healthy dogs. Blood smear evaluation and automated morphology flagging results were correlated with mean cell volume (MCV) and cellular hemoglobin concentration mean (CHCM) in 26 dogs. Customized thresholds were applied retroactively to complete blood (cell) count (CBC) data from 5 groups of dogs, including a reference sample group, clinical cases, and animals with experimentally induced iron deficiency anemia. Automated morphology flagging correlated more highly with MCV or CHCM than did blood smear evaluation; correlation with MCV was highest using customized thresholds. Customized morphology flagging thresholds resulted in more sensitive detection of microcytosis, macrocytosis, and hypochromasia than default thresholds.

Neonatal exposure to monosodium glutamate induces morphological alterations in suprachiasmatic nucleus of adult rat.

PubMed

Rojas-Castañeda, Julio César; Vigueras-Villaseñor, Rosa María; Chávez-Saldaña, Margarita; Rojas, Patricia; Gutiérrez-Pérez, Oscar; Rojas, Carolina; Arteaga-Silva, Marcela

2016-02-01

Neonatal exposure to monosodium glutamate (MSG) induces circadian disorders in several physiological and behavioural processes regulated by the suprachiasmatic nucleus (SCN). The objective of this study was to evaluate the effects of neonatal exposure to MSG on locomotor activity, and on morphology, cellular density and expression of proteins, as evaluated by optical density (OD), of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and glial fibrillary acidic protein (GFAP)-immunoreactive cells in the SCN. Male Wistar rats were used: the MSG group was subcutaneously treated from 3 to 10 days of age with 3.5 mg/g/day. Locomotor activity was evaluated at 90 days of age using 'open-field' test, and the brains were processed for immunohistochemical studies. MSG exposure induced a significant decrease in locomotor activity. VP- and VIP-immunoreactive neuronal densities showed a significant decrease, while the somatic OD showed an increase. Major axes and somatic area were significantly increased in VIP neurons. The cellular and optical densities of GFAP-immunoreactive sections of SCN were significantly increased. These results demonstrated that newborn exposure to MSG induced morphological alterations in SCN cells, an alteration that could be the basis for behavioural disorders observed in the animals. © 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.

A drug-induced accelerated senescence (DIAS) is a possibility to study aging in time lapse.

PubMed

Alili, Lirija; Diekmann, Johanna; Giesen, Melanie; Holtkötter, Olaf; Brenneisen, Peter

2014-06-01

Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. Accordingly, a stress-induced senescence-like phenotype of human dermal fibroblasts can be induced in vitro by the exposure of human diploid fibroblasts to subcytotoxic concentrations of hydrogen peroxide. However, several biomarkers of replicative senescence e.g. cell cycle arrest and enlarged morphology are abrogated 14 days after treatment, indicating that reactive oxygen species (ROS) rather acts as a trigger for short-term senescence (1-3 days) than being responsible for the maintenance of the senescence-like phenotype. Further, DNA-damaging factors are discussed resulting in a permanent senescent cell type. To induce long-term premature senescence and to understand the molecular alterations occurring during the aging process, we analyzed mitomycin C (MMC) as an alkylating DNA-damaging agent and ROS producer. Human dermal fibroblasts (HDF), used as model for skin aging, were exposed to non-cytotoxic concentrations of MMC and analyzed for potential markers of cellular aging, for example enlarged morphology, activity of senescence-associated-ß-galactosidase, cell cycle arrest, increased ROS production and MMP1-activity, which are well-documented for HDF in replicative senescence. Our data show that mitomycin C treatment results in a drug-induced accelerated senescence (DIAS) with long-term expression of senescence markers, demonstrating that a combination of different susceptibility factors, here ROS and DNA alkylation, are necessary to induce a permanent senescent cell type.

Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

PubMed

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Mason, Ronald P

2015-01-01

Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA. Copyright © 2014 Elsevier Inc. All rights reserved.

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimamura, Mai; Kyotani, Akane; Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585

2014-08-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdownmore » of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.« less

Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments

PubMed Central

Inoue, H; Tani, K

2014-01-01

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases. PMID:23832118

Mitochondrial control by DRP1 in brain tumor initiating cells.

PubMed

Xie, Qi; Wu, Qiulian; Horbinski, Craig M; Flavahan, William A; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N; Kashatus, David F; Bao, Shideng; Rich, Jeremy N

2015-04-01

Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca(2+)-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

The Alternaria alternata Mycotoxin Alternariol Suppresses Lipopolysaccharide-Induced Inflammation

PubMed Central

Grover, Shivani; Lawrence, Christopher B.

2017-01-01

The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage cell line (RAW264.7) were investigated. During these studies, it was discovered that AOH and to a lesser extent AME potently suppressed lipopolysaccharide (LPS)-induced innate immune responses in a dose-dependent manner. Treatment of BEAS-2B cells with AOH resulted in morphological changes including a detached pattern of growth as well as elongated arms. AOH/AME-related immune suppression and morphological changes were linked to the ability of these mycotoxins to cause cell cycle arrest at the G2/M phase. This model was also used to investigate the AOH/AME mechanism of immune suppression in relation to aryl hydrocarbon receptor (AhR). AhR was not found to be important for the immunosuppressive properties of AOH/AME, but appeared important for the low levels of cell death observed in BEAS-2B cells. PMID:28726766

Xylitol induces cell death in lung cancer A549 cells by autophagy.

PubMed

Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

2015-05-01

Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

Single-cell-precision microplasma-induced cancer cell apoptosis.

PubMed

Tan, Xiao; Zhao, Shasha; Lei, Qian; Lu, Xinpei; He, Guangyuan; Ostrikov, Kostya

2014-01-01

The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure.

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

PubMed

Wagoner, Michael D; Bohrer, Laura R; Aldrich, Benjamin T; Greiner, Mark A; Mullins, Robert F; Worthington, Kristan S; Tucker, Budd A; Wiley, Luke A

2018-05-08

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4 , SOX2 , KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3 , SOX9 , TFAP2A , SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1 , COL8A1 , COL8A2 , AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na + /K + ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics. © 2018. Published by The Company of Biologists Ltd.

Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells.

PubMed

Tang, Zi-Hua; Chen, Jia-Rong; Zheng, Jing; Shi, Hao-Song; Ding, Jie; Qian, Xiao-Dan; Zhang, Cui; Chen, Jian-Ling; Wang, Cui-Cui; Li, Liang; Chen, Jun-Zhen; Yin, Shan-Kai; Huang, Tao-Sheng; Chen, Ping; Guan, Min-Xin; Wang, Jin-Fu

2016-05-01

The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A(WT/WT); C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs. ©AlphaMed Press.

Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells

PubMed Central

Tang, Zi-Hua; Chen, Jia-Rong; Zheng, Jing; Shi, Hao-Song; Ding, Jie; Qian, Xiao-Dan; Zhang, Cui; Chen, Jian-Ling; Wang, Cui-Cui; Li, Liang; Chen, Jun-Zhen; Yin, Shan-Kai; Huang, Tao-Sheng; Chen, Ping; Guan, Min-Xin

2016-01-01

The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7AWT/WT; C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. Significance Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs. PMID:27013738

Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs.

PubMed

Tripathi, Anima; PremKumar, Karuppanan V; Pandey, Ashutosh N; Khatun, Sabana; Mishra, Surabhi Kirti; Shrivastav, Tulsidas G; Chaube, Shail K

2011-09-30

The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs. Copyright © 2011 Elsevier B.V. All rights reserved.

Composite vascular scaffold combining electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves structure.

PubMed

Liu, Yuanyuan; Jiang, Chen; Li, Shuai; Hu, Qingxi

2016-08-01

While the field of tissue engineered vascular grafts has greatly advanced, many inadequacies still exist. Successfully developed scaffolds require mechanical and structural properties that match native vessels and optimal microenvironments that foster cell integration, adhesion and growth. We have developed a small diameter, three-layered composite vascular scaffold which consists of electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves by combining the electrospinning and dip-coating methods. Scaffold morphology and mechanics were assessed, quantified and compared to native vessels. Scaffolds were seeded with Human Umbilical Vein Endothelial Cells (HUVECs), cultured in vitro for 3 days and were evaluated for cell viability and morphology. The results showed that composite scaffolds had adjustable mechanical strength and favorable biocompatibility, which is important in the future clinical application of Tissue-engineered vascular grafts (TEVGs). Copyright © 2016 Elsevier Ltd. All rights reserved.

Nutrient-induced intestinal adaption and its effect in obesity.

PubMed

Dailey, Megan J

2014-09-01

Obese and lean individuals respond differently to nutrients with changes in digestion, absorption and hormone release. This may be a result of differences in intestinal epithelial morphology and function driven by the hyperphagia or the type of diet associated with obesity. It is well known that the maintenance and growth of the intestine is driven by the amount of luminal nutrients, with high nutrient content resulting in increases in cell number, villi length and crypt depth. In addition, the type of nutrient appears to contribute to alterations in the morphology and function of the epithelial cells. This intestinal adaptation may be what is driving the differences in nutrient processing in lean versus obese individuals. This review describes how nutrients may be able to induce changes in intestinal epithelial cell proliferation, differentiation and function and the link between intestinal adaptation and obesity. Copyright © 2014 Elsevier Inc. All rights reserved.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

PubMed

Kitasaka, Hiroya; Kawai, Tomoko; Hoque, S A Masudul; Umehara, Takashi; Fujita, Youko; Shimada, Masayuki

2018-01-01

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

Fast Neutron Induced Autophagy Leads To Necrosis In Glioblastoma Multiforme Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yasui, Linda; Gladden, Samantha; Andorf, Christine

Fast neutrons are highly effective at killing glioblastoma multiforme (GBM), U87 and U251 cells. The mode of cell death was investigated using transmission electron microscopy (TEM) to identify the fraction of irradiated U87 or U251 cells having morphological features of autophagy and/or necrosis. U87 or U251 cells were irradiated with 2 Gy fast neturons or 10 Gy {gamma} rays. A majority of U87 and U251 cells exhibit features of cell death with autophagy after irradiation with either 10 Gy {gamma} rays or 2 Gy fast neutrons. Very few {gamma} irradiated cells had features of necrosis (U87 or U251 cell samplesmore » processed for TEM 1 day after 10 Gy {gamma} irradiation). In contrast, a significant increase was observed in necrotic U87 and U251 cells irradiated with fast neutrons. These results show a greater percentage of cells exhibit morphological evidence of necrosis induced by a lower dose of fast neutron irradiation compared to {gamma} irradiation. Also, the evidence of necrosis in fast neutron irradiated U87 and U251 cells occurs in a background of autophagy. Since autophagy is observed before necrosis, autophagy may play a role in signaling programmed necrosis in fast neutron irradiated U87 and U251 cells.« less

Protein carboxyl methylation increases in parallel with differentiation of neuroblastoma cells.

PubMed

Kloog, Y; Axelrod, J; Spector, I

1983-02-01

Cells of mouse neuroblastoma clone N1E-115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane-bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylene-bisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6-7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane-bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest, and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property of neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation, and a major group that may be related to ionic permeability mechanisms of the excitable membrane.

Cyclic mechanical stretch contributes to network development of osteocyte-like cells with morphological change and autophagy promotion but without preferential cell alignment in rat.

PubMed

Inaba, Nao; Kuroshima, Shinichiro; Uto, Yusuke; Sasaki, Muneteru; Sawase, Takashi

2017-09-01

Osteocytes play important roles in controlling bone quality as well as preferential alignment of biological apatite c -axis/collagen fibers. However, the relationship between osteocytes and mechanical stress remains unclear due to the difficulty of three-dimensional (3D) culture of osteocytes in vitro . The aim of this study was to investigate the effect of cyclic mechanical stretch on 3D-cultured osteocyte-like cells. Osteocyte-like cells were established using rat calvarial osteoblasts cultured in a 3D culture system. Cyclic mechanical stretch (8% amplitude at a rate of 2 cycles min -1 ) was applied for 24, 48 and 96 consecutive hours. Morphology, cell number and preferential cell alignment were evaluated. Apoptosis- and autophagy-related gene expression levels were measured using quantitative PCR. 3D-cultured osteoblasts became osteocyte-like cells that expressed osteocyte-specific genes such as Dmp1 , Cx43 , Sost , Fgf23 and RANKL , with morphological changes similar to osteocytes. Cell number was significantly decreased in a time-dependent manner under non-loaded conditions, whereas cyclic mechanical stretch significantly prevented decreased cell numbers with increased expression of anti-apoptosis-related genes. Moreover, cyclic mechanical stretch significantly decreased cell size and ellipticity with increased expression of autophagy-related genes, LC3b and atg7 . Interestingly, preferential cell alignment did not occur, irrespective of mechanical stretch. These findings suggest that an anti-apoptotic effect contributes to network development of osteocyte-like cells under loaded condition. Spherical change of osteocyte-like cells induced by mechanical stretch may be associated with autophagy upregulation. Preferential alignment of osteocytes induced by mechanical load in vivo may be partially predetermined before osteoblasts differentiate into osteocytes and embed into bone matrix.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

PubMed Central

Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

2013-01-01

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. PMID:23941992

N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

PubMed

Hossain, Md Motarab; Banik, Naren L; Ray, Swapan K

2013-10-15

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. © 2013 Elsevier B.V. All rights reserved.

Biological Effects of Protracted Exposure to Ionizing Radiation: Review, Analysis, and Model Development

DTIC Science & Technology

1991-11-01

dynamics, physiological changes, morphologi- cal changes, cell/tissue damage and recovery mechanisms, and existing radiobiological injury and recovery...humans and the ferret. The gut injury model (GIM) is a three-compartment hierarchial- type tissue model to simulate radiation-induced changes in the...Prodromal Symptoms Diarrhea Gastrointestinal Symptoms Dose Rate Cell Survival Intestinal Injury Fatigability Cell Damage Cell Repair Cell Proliferation

VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

PubMed

Tenn, Catherine C; Wang, Yushan

2007-05-01

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

Analysis of epothilone B-induced cell death in normal ovarian cells.

PubMed

Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells.

PubMed Central

Bryant, A E; Stevens, D L

1996-01-01

Clostridium perfringens phospholipase C (PLC) and perfringolysin O (PFO) differentially induced human umbilical vein endothelial cell expression and synthesis of endothelial cell-leukocyte adherence molecule-1 (ELAM-1), intracellular leukocyte adherence molecule-1 (ICAM-1), and interleukin-8 (IL-8). PLC strongly induced expression of ELAM-1, ICAM-1, and IL-8, while PFO stimulated early ICAM-1 expression but did not promote ELAM-1 expression or IL-8 synthesis. PLC caused human umbilical vein endothelial cells to assume a fibroblastoid morphology, whereas PFO, in high concentrations or after prolonged low-dose toxin exposure, caused cell death. The toxin-induced expression of proadhesive and activational proteins and direct cytopathic effects may contribute to the leukostasis, vascular compromise, and capillary leak characteristics of C. perfringens gas gangrene. PMID:8557365

Abnormal morphology of the penis in male rats exposed neonatally to diethylstilbestrol is associated with altered profile of estrogen receptor-alpha protein, but not of androgen receptor protein: a developmental and immunocytochemical study.

PubMed

Goyal, H O; Braden, T D; Williams, C S; Dalvi, P; Mansour, M M; Mansour, M; Williams, J W; Bartol, F F; Wiley, A A; Birch, L; Prins, G S

2004-05-01

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.

Detrimental impact of silica nanoparticles on the nanomechanical properties of Escherichia coli, studied by AFM.

PubMed

Mathelié-Guinlet, Marion; Grauby-Heywang, Christine; Martin, Axel; Février, Hugo; Moroté, Fabien; Vilquin, Alexandre; Béven, Laure; Delville, Marie-Hélène; Cohen-Bouhacina, Touria

2018-05-29

Despite great innovative and technological promises, nanoparticles (NPs) can ultimately exert an antibacterial activity by affecting the cell envelope integrity. This envelope, by conferring the cell its rigidity and protection, is intimately related to the mechanical behavior of the bacterial surface. Depending on their size, surface chemistry, shape, NPs can induce damages to the cell morphology and structure among others, and are therefore expected to alter the overall mechanical properties of bacteria. Although Atomic Force Microscopy (AFM) stands as a powerful tool to study biological systems, with high resolution and in near physiological environment, it has rarely been applied to investigate at the same time both morphological and mechanical degradations of bacteria upon NPs treatment. Consequently, this study aims at quantifying the impact of the silica NPs (SiO 2 -NPs) on the mechanical properties of E. coli cells after their exposure, and relating it to their toxic activity under a critical diameter. Cell elasticity was calculated by fitting the force curves with the Hertz model, and was correlated with the morphological study. SiO 2 -NPs of 100 nm diameter did not trigger any significant change in the Young modulus of E. coli, in agreement with the bacterial intact morphology and membrane structure. On the opposite, the 4 nm diameter SiO 2 -NPs did induce a significant decrease in E. coli Young modulus, mainly associated with the disorganization of lipopolysaccharides in the outer membrane and the permeation of the underlying peptidoglycan layer. The subsequent toxic behavior of these NPs is finally confirmed by the presence of membrane residues, due to cell lysis, exhibiting typical adhesion features. Copyright © 2018 Elsevier Inc. All rights reserved.

Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

PubMed

Wang, Xiaobing; Liu, Quanhong; Wang, Zhezhi; Wang, Pan; Hao, Qiao; Li, Chendi

2009-01-01

The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis. Copyright 2009 S. Karger AG, Basel.

Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma

PubMed Central

Landreville, Solange; Agapova, Olga A.; Matatall, Katie A.; Kneass, Zachary T.; Onken, Michael D.; Lee, Ryan S.; Bowcock, Anne M.; Harbour, J. William

2011-01-01

Purpose Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma and metastasis (UM). The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. Experimental Design In silico screens were performed to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid, trichostatin A, LBH-589 and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, BrdU incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. Results HDAC inhibitors induced morphologic differentiation, cell cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. Valproic acid inhibited the growth of UM tumors in vivo. Conclusions These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM. PMID:22038994

Response of the macaque nasal epithelium to ambient levels of ozone. A morphologic and morphometric study of the transitional and respiratory epithelium.

PubMed Central

Harkema, J. R.; Plopper, C. G.; Hyde, D. M.; St George, J. A.; Wilson, D. W.; Dungworth, D. L.

1987-01-01

Although ozone (O3)-induced bronchiolitis has been morphologically characterized, effects of O3 on the upper respiratory tract have not been thoroughly investigated. The purpose of this study was to determine whether exposures to ambient levels of O3 induce lesions in the nasal mucosa. Bonnet monkeys were exposed to 0.00, 0.15, or 0.30 ppm O3 for 6 or 90 days, 8 hours/day. After exposure, nasal mucosa was processed for light and electron microscopy. Quantitative changes were evident in the nasal transitional and respiratory epithelium. At 6 or 90 days of exposure to 0.15 or 0.30 ppm O3 lesions consisted of ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia. Inflammatory cell influx was only present at 6 days of exposure. Ultrastructural changes in goblet cells were evident at 90 days. Ambient levels of O3 can induce significant nasal epithelial lesions, which may compromise upper respiratory defense mechanisms. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:3605312

Intranasal cotinine improves memory, and reduces depressive-like behavior, and GFAP+ cells loss induced by restraint stress in mice.

PubMed

Perez-Urrutia, Nelson; Mendoza, Cristhian; Alvarez-Ricartes, Nathalie; Oliveros-Matus, Patricia; Echeverria, Florencia; Grizzell, J Alex; Barreto, George E; Iarkov, Alexandre; Echeverria, Valentina

2017-09-01

Posttraumatic stress disorder (PTSD), chronic psychological stress, and major depressive disorder have been found to be associated with a significant decrease in glial fibrillary acidic protein (GFAP) immunoreactivity in the hippocampus of rodents. Cotinine is an alkaloid that prevents memory impairment, depressive-like behavior and synaptic loss when co-administered during restraint stress, a model of PTSD and stress-induced depression, in mice. Here, we investigated the effects of post-treatment with intranasal cotinine on depressive- and anxiety-like behaviors, visual recognition memory as well as the number and morphology of GFAP+ immunoreactive cells, in the hippocampus and frontal cortex of mice subjected to prolonged restraint stress. The results revealed that in addition to the mood and cognitive impairments, restraint stress induced a significant decrease in the number and arborization of GFAP+ cells in the brain of mice. Intranasal cotinine prevented these stress-derived symptoms and the morphological abnormalities GFAP+ cells in both of these brain regions which are critical to resilience to stress. The significance of these findings for the therapy of PTSD and depression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins

PubMed Central

Stephens, Andrew D.; Liu, Patrick Z.; Banigan, Edward J.; Almassalha, Luay M.; Backman, Vadim; Adam, Stephen A.; Goldman, Robert D.; Marko, John F.

2018-01-01

Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed “blebs” are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson–Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity. PMID:29142071

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Low-intensity red and infrared laser effects at high fluences on Escherichia coli cultures

PubMed Central

Barboza, L.L.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.; Fonseca, A.S.

2015-01-01

Semiconductor laser devices are readily available and practical radiation sources providing wavelength tenability and high monochromaticity. Low-intensity red and near-infrared lasers are considered safe for use in clinical applications. However, adverse effects can occur via free radical generation, and the biological effects of these lasers from unusually high fluences or high doses have not yet been evaluated. Here, we evaluated the survival, filamentation induction and morphology of Escherichia coli cells deficient in repair of oxidative DNA lesions when exposed to low-intensity red and infrared lasers at unusually high fluences. Cultures of wild-type (AB1157), endonuclease III-deficient (JW1625-1), and endonuclease IV-deficient (JW2146-1) E. coli, in exponential and stationary growth phases, were exposed to red and infrared lasers (0, 250, 500, and 1000 J/cm2) to evaluate their survival rates, filamentation phenotype induction and cell morphologies. The results showed that low-intensity red and infrared lasers at high fluences are lethal, induce a filamentation phenotype, and alter the morphology of the E. coli cells. Low-intensity red and infrared lasers have potential to induce adverse effects on cells, whether used at unusually high fluences, or at high doses. Hence, there is a need to reinforce the importance of accurate dosimetry in therapeutic protocols. PMID:26445339

Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

PubMed

Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

2016-11-07

An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism

PubMed Central

Nagy, J; Kobolák, J; Berzsenyi, S; Ábrahám, Z; Avci, H X; Bock, I; Bekes, Z; Hodoscsek, B; Chandrasekaran, A; Téglási, A; Dezső, P; Koványi, B; Vörös, E T; Fodor, L; Szél, T; Németh, K; Balázs, A; Dinnyés, A; Lendvai, B; Lévay, G; Román, V

2017-01-01

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD. PMID:28742076

Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

PubMed Central

Bozic, Iva; Savic, Danijela; Jovanovic, Marija; Bjelobaba, Ivana; Laketa, Danijela; Nedeljkovic, Nadezda; Stojiljkovic, Mirjana; Pekovic, Sanja; Lavrnja, Irena

2015-01-01

Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation. PMID:26413464

Mutagenic and morphologic impacts of 1.8GHz radiofrequency radiation on human peripheral blood lymphocytes (hPBLs) and possible protective role of pre-treatment with Ginkgo biloba (EGb 761).

PubMed

Esmekaya, Meric Arda; Aytekin, Ebru; Ozgur, Elcin; Güler, Göknur; Ergun, Mehmet Ali; Omeroğlu, Suna; Seyhan, Nesrin

2011-12-01

The mutagenic and morphologic effects of 1.8GHz Global System for Mobile Communications (GSM) modulated RF (radiofrequency) radiation alone and in combination with Ginkgo biloba (EGb 761) pre-treatment in human peripheral blood lymphocytes (hPBLs) were investigated in this study using Sister Chromatid Exchange (SCE) and electron microscopy. Cell viability was assessed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay. The lymphocyte cultures were exposed to GSM modulated RF radiation at 1.8GHz for 6, 8, 24 and 48h with and without EGb 761. We observed morphological changes in pulse-modulated RF radiated lymphocytes. Longer exposure periods led to destruction of organelle and nucleus structures. Chromatin change and the loss of mitochondrial crista occurred in cells exposed to RF for 8h and 24h and were more pronounced in cells exposed for 48h. Cytoplasmic lysis and destruction of membrane integrity of cells and nuclei were also seen in 48h RF exposed cells. There was a significant increase (p<0.05) in SCE frequency in RF exposed lymphocytes compared to sham controls. EGb 761 pre-treatment significantly decreased SCE from RF radiation. RF radiation also inhibited cell viability in a time dependent manner. The inhibitory effects of RF radiation on the growth of lymphoctes were marked in longer exposure periods. EGb 761 pre-treatment significantly increased cell viability in RF+EGb 761 treated groups at 8 and 24h when compared to RF exposed groups alone. The results of our study showed that RF radiation affects cell morphology, increases SCE and inhibits cell proliferation. However, EGb 761 has a protective role against RF induced mutagenity. We concluded that RF radiation induces chromosomal damage in hPBLs but this damage may be reduced by EGb 761 pre-treatment. Copyright © 2011 Elsevier B.V. All rights reserved.

Pharmacoethnicity in Paclitaxel-Induced Sensory Peripheral Neuropathy

PubMed Central

Komatsu, Masaaki; Wheeler, Heather E.; Chung, Suyoun; Low, Siew-Kee; Wing, Claudia; Delaney, Shannon M.; Gorsic, Lidija K.; Takahashi, Atsushi; Kubo, Michiaki; Kroetz, Deanna L.; Zhang, Wei; Nakamura, Yusuke; Dolan, M. Eileen

2015-01-01

Purpose Paclitaxel is used worldwide in the treatment of breast, lung, ovarian and other cancers. Sensory peripheral neuropathy is an associated adverse effect that cannot be predicted, prevented or mitigated. To better understand the contribution of germline genetic variation to paclitaxel-induced peripheral neuropathy, we undertook an integrative approach that combines genome-wide association study (GWAS) data generated from HapMap lymphoblastoid cell lines (LCLs) and Asian patients. Methods GWAS was performed with paclitaxel-induced cytotoxicity generated in 363 LCLs and with paclitaxel-induced neuropathy from 145 Asian patients. A gene-based approach was used to identify overlapping genes and compare to a European clinical cohort of paclitaxel-induced neuropathy. Neurons derived from human induced pluripotent stem cells were used for functional validation of candidate genes. Results SNPs near AIPL1 were significantly associated with paclitaxel-induced cytotoxicity in Asian LCLs (P < 10−6). Decreased expression of AIPL1 resulted in decreased sensitivity of neurons to paclitaxel by inducing neurite morphological changes as measured by increased relative total outgrowth, number of processes and mean process length. Using a gene-based analysis, there were 32 genes that overlapped between Asian LCL cytotoxicity and Asian patient neuropathy (P < 0.05) including BCR. Upon BCR knockdown, there was an increase in neuronal sensitivity to paclitaxel as measured by neurite morphological characteristics. Conclusion We identified genetic variants associated with Asian paclitaxel-induced cytotoxicity and functionally validated the AIPL1 and BCR in a neuronal cell model. Furthermore, the integrative pharmacogenomics approach of LCL/patient GWAS may help prioritize target genes associated with chemotherapeutic-induced peripheral neuropathy. PMID:26015512

Docosapentaenoic acid (DPA) is a critical determinant of cubic membrane formation in amoeba Chaos mitochondria.

PubMed

Deng, Yuru; Almsherqi, Zakaria A; Shui, Guanghou; Wenk, Markus R; Kohlwein, Sepp D

2009-09-01

Very long-chain polyunsaturated fatty acids (VLC-PUFAs), such as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), have recently made it to the realm of "magical molecules" based on their multiple presumably beneficial effects in biological systems, making these PUFAs particularly interesting in biomedicine. Their specific biological functions, however, remain enigmatic. Here we provide evidence derived from studies in the amoeba Chaos that indicates a structural role for omega-6 DPA in cell membrane organization, which may help to explain the multiple diverse effects of VLC-PUFA in healthy and diseased states. Amoeba Chaos mitochondria undergo a remarkable and reversible morphological transition into cubic morphology on starvation. This morphological transition is reflected in major changes in fatty acid and lipid composition, as determined by gas liquid chromatography and mass spectrometry, in particular by a drastic increase in C22:5 modified phosphatidylcholine plasmalogen, phosphatidylethanolamine plasmalogen, and phosphatidylinositol species. Liposomes produced in vitro from lipids of starved amoeba cells show a high propensity to form hexagonal tubular and cubic morphologies. Addition of omega-6 DPA, but not of omega-3 DPA, to the cell culture also induced mitochondrial membrane transformation into cubic morphology in fed cells, demonstrating for the first time an important structural role of omega-6 DPA-containing lipids in cell membrane organization.

A supercritical-CO2 extract of Ganoderma lucidum spores inhibits cholangiocarcinoma cell migration by reversing the epithelial-mesenchymal transition.

PubMed

Li, Lian; Guo, Hui-Jun; Zhu, Ling-Yan; Zheng, Limin; Liu, Xin

2016-05-15

Ganoderma lucidum (G. lucidum) is an oriental medical mushroom that has been widely used in Asian countries for centuries to prevent and treat different diseases, including cancer. The objective of this study was to investigate the effect of A supercritical-CO2 extract of G. lucidum spores on the transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of cholangiocarcinoma cells. This was an in vitro study with human cholangiocarcinoma TFK-1 cells treated with varying concentrations of G. lucidum. A supercritical-CO2 extract of G. lucidum spores (GLE) was obtained from completely sporoderm-broken germinating G. lucidum spores by supercritical fluid carbon dioxide (SCF-CO2) extraction. GLE pre-incubated with human cholangiocarcinoma TFK-1 cells prior to TGF-β1 treatment (2ng/ml) for 48h. Changes in EMT markers were analyzed by western blotting and immunofluorescence. The formation of F-actin stress fibers was assessed via immunostaining with phalloidin and examined using confocal microscopy. Additionally, the effect of the GLE on TGF-β1-induced migration was investigated by a Boyden chamber assay. TGF-β1-induced reduction in E-cadherin expression was associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin and Fibronectin were evident in predominantly elongated fibroblast-like cells. The GLE suppressed the TGF-β1-induced morphological changes and the changes in cadherin expression, and also inhibited the formation of F-actin stress fibers, which are a hallmark of EMT. The GLE also inhibited TGF-β1-induced migration of TFK-1 cells. Our findings provide new evidence that GLE suppress cholangiocarcinoma migration in vitro through inhibition of TGF-β1-induced EMT. The GLE may be clinically applied in the prevention and/or treatment of cancer metastasis. Copyright © 2016. Published by Elsevier GmbH.

Odorous Compounds from Poultry Manure Induce DNA Damage, Nuclear Changes, and Decrease Cell Membrane Integrity in Chicken Liver Hepatocellular Carcinoma Cells

PubMed Central

Matusiak, Katarzyna; Gałęcki, Remigiusz; Borowski, Sebastian; Gutarowska, Beata

2017-01-01

Animal breeding and management of organic wastes pose a serious problem to the health of livestock and workers, as well as the nearby residents. The aim of the present study was to determine the mechanisms of toxicity of selected common odorous compounds from poultry manure, including ammonia, dimethylamine (DMA), trimethylamine (TMA), butyric acid, phenol, and indole. We measured their genotoxic and cytotoxic activity in the model chicken cell line (LMH), in vitro, by comet assay and lactate dehydrogenase assay, respectively. We also made microscopic observations of any morphological changes in these cells by DAPI staining. Four compounds, namely ammonia, DMA, TMA, and butyric acid increased DNA damage in a dose-dependent manner (p < 0.05), reaching genotoxicity as high as 73.2 ± 1.9%. Phenol and indole induced extensive DNA damage independent of the concentration used. Ammonia, DMA, and TMA caused a dose-dependent release of lactate dehydrogenase (p < 0.05). The IC50 values were 0.02%, 0.05%, and 0.1% for DMA, ammonia and TMA, respectively. These compounds also induced nuclear morphological changes, such as chromatin condensation, shrinkage, nuclear fragmentation (apoptotic bodies), and chromatin lysis. Our study exhibited the damaging effects of odorous compounds in chick LMH cell line. PMID:28820500

Anti-hepatoma activity of a novel compound glaucocalyxin H in vivo and in vitro.

PubMed

Hai, Guangfan; Zhang, Chong; Jia, Yanlong; Bai, Suping; Han, Jinfen; Guo, Lanqing; Cui, Taizhen; Niu, Bingxuan; Huang, Feng; Song, Yu

2015-06-01

Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG2 cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG2 cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG2 cells to apoptosis in a dose-dependent manner. Bcl2 and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl2 and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl2 protein was downregulated in HepG2 cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl2 and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma.

Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

PubMed

Afriyie, D K; Asare, G A; Bugyei, K; Lin, J; Peng, J; Hong, Z

2015-01-15

Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G₀/G₁ sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

PubMed

Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

2016-03-01

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

Molecular mechanisms of transformation of C3H/10T1/2 C1 8 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds.

PubMed Central

Landolph, J R

1994-01-01

Carcinogenic arsenic, nickel, and chromium compounds induced morphological and neoplastic transformation but no mutation to ouabain resistance in 10T1/2 mouse embryo cells; lead chromate also did not induce mutation to ouabain or 6-thioguanine resistance in Chinese hamster ovary cells. The mechanism of metal-induced morphological transformation was likely not due to the specific base substitution mutations measured in ouabain resistance mutation assays, and for lead chromate, likely not due to this type of base substitution mutation or to frameshift mutations. Preliminary data indicate increases in steady-state levels of c-myc RNA in arsenic-, nickel-, and chromium-transformed cell lines. We also showed that carcinogenic nickel, chromium, and arsenic compounds and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) induced stable anchorage independence (Al) in diploid human fibroblasts (DHF) but no focus formation or immortality. Nickel subsulfide and lead chromate induced Al but not mutation to 6-thioguanine resistance. The mechanism of induction of Al by metal salts in DHF was likely not by the type of base substitution or frameshift mutations measured in these assays. MNNG induced Al, mutation to 6-thioguanine resistance, and mutation to ouabain resistance, and might induce Al by base substitution or frameshift mutations. Dexamethasone, aspirin, and salicylic acid inhibited nickel subsulfide, MNNG, and 12-O-tetrade-canoylphorbol-13-acetate (TPA)-induced Al in DHF, suggesting that arachidonic acid metabolism and oxygen radical generation play a role in induction of Al. We propose that nickel compounds stimulate arachidonic acid metabolism, consequent oxygen radical generation, and oxygen radical attack upon DNA.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. PMID:7843085

Ketamine-induced apoptosis in cultured rat cortical neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takadera, Tsuneo; Ishida, Akira; Ohyashiki, Takao

2006-01-15

Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cellmore » death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.« less

Surface-Acoustic-Wave (SAW)-Driven Device for Dynamic Cell Cultures.

PubMed

Greco, Gina; Agostini, Matteo; Tonazzini, Ilaria; Sallemi, Damiano; Barone, Stefano; Cecchini, Marco

2018-06-19

In the last few decades, new types of cell cultures have been introduced to provide better cell survival and development, with micro- and nanoenvironmental physicochemical conditions aimed at mimicking those present in vivo. However, despite the efforts made, the systems available to date are often difficult to replicate and use. Here, an easy-to-use surface-acoustic-wave (SAW)-based platform is presented for realizing dynamic cell cultures that is compatible with standard optical microscopes, incubators, and cell-culture dishes. The SAW chip is coupled to a standard Petri dish via a polydimethylsiloxane (PDMS) disc and consists of a lithium niobate (LN) substrate on which gold interdigital transducers (IDTs) are patterned to generate the SAWs and induce acoustic streaming in the dish. SAW excitation is verified and characterized by laser Doppler vibrometry, and the fluid dynamics is studied by microparticle image velocimetry (μPIV). Heating is measured by an infrared (IR) thermal camera. We finally tested this device with the U-937 monocyte cell line for viability and proliferation and cell-morphological analysis. The data demonstrate that it is possible to induce significant fluid recirculation within the Petri dish while maintaining negligible heating. Remarkably, cell proliferation in this condition was enhanced by 36 ± 12% with respect to those of standard static cultures. Finally, we show that cell death does not increase and that cell morphology is not altered in the presence of SAWs. This device is the first demonstration that SAW-induced streaming can mechanically improve cell proliferation and further supports the great versatility and biocompatibility of the SAW technology for cell manipulation.

Green synthesis of platinum nanoparticles that induce cell death and G2/M-phase cell cycle arrest in human cervical cancer cells.

PubMed

Alshatwi, Ali A; Athinarayanan, Jegan; Vaiyapuri Subbarayan, Periasamy

2015-01-01

Platinum-based chemotherapeutic drugs, including cisplatin, carboplatin, and oxaliplatin, have been used to manage cancer in spite of dose-dependent side effects, including nephrotoxicity, neurotoxicity and ototoxicity. These disadvantages have prompted the development of new strategies for cancer therapy that utilize functionalized nanoparticles as nanomedicines. In the present investigation, we have synthesized platinum nanoparticles using tea polyphenol (TPP) as both a reducing and surface modifying agent. The crystalline nature and morphology of the prepared TPP-functionalized platinum nanoparticles (TPP@Pt) were analyzed using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD results revealed that the TPP@Pt had a crystalline nature with a face-centered cubic structure. TEM imaging suggested that the TTP@Pt are flower shaped with a well-dispersed 30-60 nm-sized TPP@Pt formation. Cervical cancer cells (SiHa) were then treated with different concentrations of TPP@Pt. The effects of TPP@Pt on cell viability, nuclear morphology and cell cycle distribution were investigated. A cell viability assay revealed that the proliferation of SiHa cells was inhibited by TPP@Pt. Propidium iodide nuclear staining indicated that TPP@Pt induced nuclear fragmentation and chromatin condensation. Treatment with TPP@Pt significantly increased the percentage of cells in the G2/M phase, which indicates induced cell cycle arrest in the G2/M phase and an increased number of cells in the subG0 cell death phase. These findings highlight a potential use of TPP@Pt in cervical cancer treatment.

The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology.

PubMed

Murk, Kai; Blanco Suarez, Elena M; Cockbill, Louisa M R; Banks, Paul; Hanley, Jonathan G

2013-09-01

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

Comparative study of the effect of chloro-, dichloro-, bromo-, and dibromoacetic acid on necrotic, apoptotic and morphological changes in human peripheral blood mononuclear cells (in vitro study).

PubMed

Michałowicz, Jaromir; Wróblewski, Wojciech; Mokra, Katarzyna; Maćczak, Aneta; Kwiatkowska, Marta

2015-10-01

In this study, the effect of monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA) on human peripheral blood mononuclear cells (PBMCs) was assessed. HAAs studied induced at millimolar concentrations necrotic alterations in PBMCs with the strongest effect noted for MBAA and DBAA. Chloro- and bromoacetic acids also provoked changes in PBMCs morphology because they caused a strong decrease in cell size (particularly DCAA and DBAA) and increase in cell granulation (mainly MBAA and DBAA). All HAAs studied, and DCAA and DBAA in particular (at lower concentrations than those, which caused necrosis) induced apoptotic changes, which was confirmed by analysis of alterations in cell membrane permeability and caspase 8, 9 and 3 activation. Moreover, HAAs examined (mainly dihalogenated acids) strongly increased transmembrane mitochondrial potential and enhanced ROS (mainly hydroxyl radical) formation, which was possibly associated with apoptotic changes provoked by those substances. The results showed that DBAA exhibited the strongest effects on PBMCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Time resolved impedance spectroscopy analysis of lithium phosphorous oxynitride - LiPON layers under mechanical stress

NASA Astrophysics Data System (ADS)

Glenneberg, Jens; Bardenhagen, Ingo; Langer, Frederieke; Busse, Matthias; Kun, Robert

2017-08-01

In this paper we present investigations on the morphological and electrochemical changes of lithium phosphorous oxynitride (LiPON) under mechanically bent conditions. Therefore, two types of electrochemical cells with LiPON thin films were prepared by physical vapor deposition. First, symmetrical cells with two blocking electrodes (Cu/LiPON/Cu) were fabricated. Second, to simulate a more application-related scenario cells with one blocking and one non-blocking electrode (Cu/LiPON/Li/Cu) were analyzed. In order to investigate mechanical distortion induced transport property changes in LiPON layers the cells were deposited on a flexible polyimide substrate. Morphology of the as-prepared samples and deviations from the initial state after applying external stress by bending the cells over different radii were investigated by Focused Ion Beam- Scanning Electron Microscopy (FIB-SEM) cross-section and surface images. Mechanical stress induced changes in the impedance were evaluated by time-resolved electrochemical impedance spectroscopy (EIS). Due to the formation of a stable, ion-conducting solid electrolyte interphase (SEI), cells with lithium show decreased impedance values. Furthermore, applying mechanical stress to the cells results in a further reduction of the electrolyte resistance. These results are supported by finite element analysis (FEA) simulations.

Structural phenotyping of stem cell-derived cardiomyocytes.

PubMed

Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

2015-03-10

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

LRSAM1 Depletion Affects Neuroblastoma SH-SY5Y Cell Growth and Morphology: The LRSAM1 c.2047-1G>A Loss-of-Function Variant Fails to Rescue The Phenotype.

PubMed

Minaidou, Anna; Nicolaou, Paschalis; Christodoulou, Kyproula

2018-10-01

Deleterious variants in LRSAM1, a RING finger ubiquitin ligase which is also known as TSG101-associated ligase (TAL), have recently been associated with Charcot-Marie-Tooth disease type 2P (CMT2P). The mechanism by which mutant LRSAM1 contributes to the development of neuropathy is currently unclear. The aim of this study was to induce LRSAM1 deficiency in a neuronal cell model, observe its effect on cell growth and morphology and attempt to rescue the phenotype with ancestral and mutant LRSAM1 transfections. In this experimental study, we investigated the effect of LRSAM1 downregulation on neuroblastoma SH-SY5Y cells by siRNA technology where cells were transfected with siRNA against LRSAM1. The effects on the expression levels of TSG101, the only currently known LRSAM1 interacting molecule, were also examined. An equal dosage of ancestral or mutant LRSAM1 construct was transfected in LRSAM1-downregulated cells to investigate its effect on the phenotype of the cells and whether cell proliferation and morphology could be rescued. A significant reduction in TSG101 levels was observed with the downregulation of LRSAM1. In addition, LRSAM1 knockdown significantly decreased the growth rate of SH-SY5Y cells which is caused by a decrease in cell proliferation. An effect on cell morphology was also observed. Furthermore, we overexpressed the ancestral and the c.2047-1G>A mutant LRSAM1 in knocked down cells. Ancestral LRSAM1 recovered cell proliferation and partly the morphology, however, the c.2047-1G>A mutant did not recover cell proliferation and further aggravated the observed changes in cell morphology. Our findings suggest that depletion of LRSAM1 affects neuroblastoma cells growth and morphology and that overexpression of the c.2047-1G>A mutant form, unlike the ancestral LRSAM1, fails to rescue the phenotype. Copyright© by Royan Institute. All rights reserved.

Cell wall carbohydrates content of pathogenic Candida albicans strain morphological forms.

PubMed

Staniszewska, Monika; Bondaryk, Małgorzata; Rabczenko, Daniel; Smoleńska-Sym, Gabriela; Kurzatkowski, Wiesław

2013-01-01

The study evaluated the cell wall carbohydrates fraction in blastoconidia grown in YEPD medium at 30 degrees C and in the conglomerate of true hyphae grown in human serum at 37 degrees C. The clinical isolate obtained from a child with widespread C. albicans infection was used in the study. The cells were broken with glass beads, centrifuged to harvest the cell wall followed by subjection to TFA hydrolysis and in the result of that released monosaccharides were detected by HPAEC-PAD. Both, serum and temperature conditions (37 degrees C) affected germination process influencing the cell wall carbohydrates content when incubation in serum was prolonged from 1 to 18 h. The mannan content of blastoconidia was almost twofold higher compared to filamentous forms (149.25 +/- 299.24 vs 77.26 +/- 122.07). The glucan content was threefold lower in blastoconidia compared to hyphae (251.86 +/- 243.44 vs 755.81 +/- 1299.30). The chitin level was fourfold lower in blastoconidia compared to filaments (23.86 +/- 54.09 vs 106.29 +/- 170.12). The reason for the differences in the carbohydrates content may be related to type of morphology induced in different environmental conditions. Among tested carbohydrates, glucan appeared to be present in appreciably larger amounts in both tested morphological fractions. The ultrastructure of the blastoconidial cell wall revealed striking differences compared to the hyphae indicating the carbohydrates content alterations for wall assembly during hyphal growth at alkaline pH and temp. 37 degrees C. The study provided evidence for the relationship between morphogenesis, cell-cell adhesion induced by serum and changes in the level of carbohydrates content.

Toward Improved Lifetimes of Organic Solar Cells under Thermal Stress: Substrate-Dependent Morphological Stability of PCDTBT:PCBM Films and Devices.

PubMed

Li, Zhe; Ho Chiu, Kar; Shahid Ashraf, Raja; Fearn, Sarah; Dattani, Rajeev; Cheng Wong, Him; Tan, Ching-Hong; Wu, Jiaying; Cabral, João T; Durrant, James R

2015-10-15

Morphological stability is a key requirement for outdoor operation of organic solar cells. We demonstrate that morphological stability and lifetime of polymer/fullerene based solar cells under thermal stress depend strongly on the substrate interface on which the active layer is deposited. In particular, we find that the stability of benchmark PCDTBT/PCBM solar cells under modest thermal stress is substantially increased in inverted solar cells employing a ZnO substrate compared to conventional devices employing a PSS substrate. This improved stability is observed to correlate with PCBM nucleation at the 50 nm scale, which is shown to be strongly influenced by different substrate interfaces. Employing this approach, we demonstrate remarkable thermal stability for inverted PCDTBT:PC70BM devices on ZnO substrates, with negligible (<2%) loss of power conversion efficiency over 160 h under 85 °C thermal stress and minimal thermally induced "burn-in" effect. We thus conclude that inverted organic solar cells, in addition to showing improved environmental stability against ambient humidity exposure as widely reported previously, can also demonstrate enhanced morphological stability. As such we show that the choice of suitable substrate interfaces may be a key factor in achieving prolonged lifetimes for organic solar cells under thermal stress conditions.

Phase contrast microscopy of living cells within the whole lens: spatial correlations and morphological dynamics

PubMed Central

Kong, Zhiying; Zhu, Xiangjia; Zhang, Shenghai; Wu, Jihong

2012-01-01

Purpose Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a real-time, convenient approach for monitoring label-free imaging of dynamic processes of living cells within the whole lens is urgently needed. Methods Female Wistar rat lenses were kept in organ culture. Insulin-like growth factor-I was added to the culture medium to induce cell mitosis. A novel method of ultraviolet (UV) irradiation was used to induce cell apoptosis and fiber damage. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy. Apoptosis was assessed using a commercial kit with Hoechst 33342/YO-PRO®-1/propidium iodide (PI). Results The intrinsic transparency and low-light scattering property of the rat lens permitted direct imaging of the lens epithelial cells (LECs) and the superficial fiber cells. We visualized the processes of mitosis and apoptosis of the LECs, and we obtained dynamic images of posterior fiber cells following UVA irradiation. Conclusions This method opens a new window for observing lens cells in their physiologic location, and it can be readily applied in studies on lens physiology and pathology. PMID:22879736

Effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells.

PubMed

Sun, Li; Wang, Xu

2003-09-01

To investigate the effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells. The gastric cancer SGC-7901 adenocarcinoma cells were treated with allicin and the cell cycle, inhibitory rate, apoptosis, telomerase activity and morphologic changes were studied by MTT assay, flow cytometry (FCM), TRAP-PCR-ELISA assay, light microscope, electron microscope respectively. Results were compared with that of AZT (3'-Azido-3'-deoxythymidine). SGC-7901 cells were suppressed after exposure to allicin of 0.016 mg/ml, 0.05 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P<0.05). Allicin could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G(2)/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P<0.05). Allicin could inhibit telomerase activity in a time-dependent and dose-dependent pattern. After exposure to allicin at 0.016 mg/ml for 24 hours, SGC-7901 cells showed typical morphologic change. Allicin can inhibit telomerase activity and induce apoptosis of gastric cancer SGC-7901 cells. Allicin may be more effective than AZT.

Diallyl disulfide attenuated carbon ion irradiation-induced apoptosis in mouse testis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway

PubMed Central

Di, Cui-xia; Han, Lu; Zhang, Hong; Xu, Shuai; Mao, Ai-hong; Sun, Chao; Liu, Yang; Si, Jing; Li, Hong-yan; Zhou, Xin; Liu, Bing; Miao, Guo-ying

2015-01-01

Diallyl disulfide (DADS), a major organosulfur compound derived from garlic, has various biological properties, including anti-cancer effects. However, the protective mechanism of DADS against radiation-induced mouse testis cell apoptosis has not been elucidated. In this study, the magnitude of radiation effects evoked by carbon ion irradiation was marked by morphology changes, significant rise in apoptotic cells, activation expression of p53, up regulation the ratio of pro-apoptotic Tap73/anti-apoptotic ΔNp73, as well as alterations of crucial mediator of the mitochondrial pathway. Interestingly, pretreatment with DADS attenuated carbon ion irradiation-induced morphology damages and apoptotic cells. Additionally, DADS elevated radiation-induced p53 and p21 expression, suggesting that p53 might be involved in the inhibition of cell cycle progression through up regulation of p21. Furthermore, administration with DADS prevented radiation-induced Tap73/ΔNp73 expression and consequently down regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression, indicating that the balance between Tap73 and ΔNp73 had potential to activate p53 responsive genes. Thus, our results showed that radio protection effect of DADS on mouse testis is mediated by blocking apoptosis through changing the ratio of Tap73/ΔNp73 via mitochondrial pathway, suggesting that DADS could be used as a potential radio protection agent for the testis against heavy-ion radiation. PMID:26526304

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

PubMed Central

Lionakis, Michail S.; Nickerson, Kenneth W.

2016-01-01

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

The natural insect peptide Neb-colloostatin induces ovarian atresia and apoptosis in the mealworm Tenebrio molitor.

PubMed

Czarniewska, Elżbieta; Rosiński, Grzegorz; Gabała, Elżbieta; Kuczer, Mariola

2014-01-30

The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown. We tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. Neb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.

The natural insect peptide Neb-colloostatin induces ovarian atresia and apoptosis in the mealworm Tenebrio molitor

PubMed Central

2014-01-01

Background The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown. Results We tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. Conclusion Neb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes. PMID:24479487

Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells.

PubMed

Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

2016-10-25

Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Antisense sequences of the nbl gene induce apoptosis in the human promyelocytic leukemia cell line HL-60.

PubMed

Naora, H; Nishida, T; Shindo, Y; Adachi, M; Naora, H

1998-04-01

Apoptosis is induced by the transcriptional inhibitor actinomycin D (Act D) in various cell types, particularly many leukemic cell lines such as HL-60. A common feature of these cell lines is their high constitutive expression level of the nbl gene, which was originally isolated by virtue of its abundance in a Namalwa Burkitt lymphoma cDNA library. In contrast, cell lines which constitutively express nbl at low levels appear not to undergo typical apoptotic death in response to Act D. Apoptotic induction by Act D in cells which normally express nbl at high levels was found in this study to be closely associated with a decline in nbl mRNA levels, raising the possibility that apoptosis could be induced by lowering nbl expression levels in such cells. Transient expression of nbl antisense sequences in HL-60 cells decreased cell viability, and induced typical apoptotic morphology such as cell shrinkage, chromatin condensation and nuclear fragmentation. Incubation with nbl antisense oligomers also induced similar features in HL-60 cells and in another high nb-expressing cell line, Jurkat, but had little effect in HepG2 cells which constitutively express nbl at low levels. These findings suggest that lowering constitutively high levels of nbl expression can induce apoptosis.

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol.

PubMed

Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula O; Linne, Marja-Leena

2016-04-01

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K(+) depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells' population growth by inducing maturation and differentiation.

Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

NASA Astrophysics Data System (ADS)

Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

2011-07-01

Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% ( p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO-EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.

BH3-mimetic ABT-737 induces strong mitochondrial membrane depolarization in platelets but only weakly stimulates apoptotic morphological changes, platelet shrinkage and microparticle formation.

PubMed

Gyulkhandanyan, Armen V; Mutlu, Asuman; Allen, David J; Freedman, John; Leytin, Valery

2014-01-01

Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation. To elucidate this relationship in human platelets. Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis. We found that at optimal treatment conditions (90min, 37°C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P<0.0001). These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Renal-protective and ameliorating impacts of omega-3 fatty acids against aspartame damaged MDCK cells.

PubMed

Pandurangan, Muthuraman; Enkhtaivan, Gansukh; Veerappan, Muthuviveganandavel; Mistry, Bhupendra; Patel, Rahul; Moon, So Hyun; Nagajyothi, Patnamsetty Chidanandha; Kim, Doo Hwan

2017-11-01

Aspartame is widely used artificial sweeteners as food additives. Several researchers have pointed that the controversial report on the use of aspartame over more than decades. Omega-3 fatty acids are essential and unsaturated fatty acids, and it plays a remarkable role in vision, intelligence, neural development, and metabolism of neurotransmitters. Therefore, the present study was aimed to investigate the effect of omega-3 fatty acids on aspartame treated renal cells. Experimental groups were divided into three such as sham control, aspartame treated, and aspartame with omega-3 fatty acids. Cell viability was determined by sulforhodamine-b assay and flow cytometric analysis. The experimental results showed that the aspartame induced altered cell viability were reduced following treatment of aspartame with omega-3 fatty acids. Altered cell morphology was recovered by omega-3 fatty acids. DNA damage appeared in the highest concentration of aspartame used in this study. DNA damage characteristics such as comet tail and tiny head sections did not appear in the omega-3 fatty acids treated cells. Several microvilli and vesicular structures were found in aspartame treated cells. Altered morphology such as rounding, microvilli, and formation of dome-like structures did not appear in the omega-3 fatty acids with aspartame treated cells. Caspase-3 mRNA and protein expression were increased in aspartame treated cells, and these levels were reduced following omega-3 fatty acids treatment. Taking all these data together, it is suggested that the omega-3 fatty acids may be a therapeutic agent to reduce the aspartame induced biochemical and morphological alterations in normal renal cells. © 2017 BioFactors, 43(6):847-857, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Dendritic cells loaded with HeLa-derived exosomes simulate an antitumor immune response.

PubMed

Ren, Guoping; Wang, Yanhong; Yuan, Shexia; Wang, Baolian

2018-05-01

The aim of the present study was to investigate the effect of loading dendritic cells (DCs) with HeLa-derived exosomes on cytotoxic T-lymphocyte (CTL) responses, and the cytotoxic effects of CTL responses on the HeLa cell line. Ultrafiltration centrifugation combined with sucrose density gradient ultracentrifugation was applied to isolate exosomes (HeLa-exo) from the supernatant of HeLa cells. Morphological features of HeLa-exo were identified by transmission electron microscopy (TEM), and the expression of cluster of differentiation (CD)63 was detected by western blotting. Next, monocytes were isolated from peripheral blood and cultured with the removal of adherent cells to induce DC proliferation. DCs were then phenotypically characterized by flow cytometry. Finally, MTT assays were performed to analyze the effects of DCs loaded with HeLa-exo on T cell proliferation and cytotoxicity assays to evaluate the effect of CTL responses on HeLa cells. TEM revealed that HeLa-exo exhibit typical cup-shaped morphology with a diameter range of 30-100 nm. It was also identified that the CD63 surface antigen is expressed on HeLa-exo. Furthermore, monocyte-derived DCs were able to express CD1a, suggesting that DC induction was a success. DCs exhibited hair-like protrusions and other typical dendritic cell morphology. Furthermore, DCs loaded with HeLa-exo could enhance CTL proliferation and the cytotoxic activity of CTLs compared with DCs without HeLa-exo (P<0.05). In conclusion, DCs loaded with HeLa-exo may promote T cell proliferation and induce CTL responses to inhibit the growth of cervical cancer cells in vitro .

Genetic Analysis of Growth-Regulator-Induced Parthenocarpy in Arabidopsis1

PubMed Central

Vivian-Smith, Adam; Koltunow, Anna M.

1999-01-01

In Arabidopsis, seedless silique development or parthenocarpy can be induced by the application of various plant growth regulators (PGRs) to unfertilized pistils. Ecotype-specific responses were observed in the Arabidopsis ecotypes Columbia and Landsberg relative to the type of PGR and level applied. The parthenocarpic response was greatest in ecotype Landsberg, and comparisons of fruit growth and morphology were studied primarily in this ecotype. Gibberellic acid application (10 μmol pistil−1) caused development similar to that in pollinated pistils, while benzyladenine (1 μmol pistil−1) and naphthylacetic acid (10 μmol pistil−1) treatment produced shorter siliques. Naphthylacetic acid primarily modified mesocarp cell expansion. Arabidopsis mutants were employed to examine potential dependencies on gibberellin biosynthesis (ga1-3, ga4-1, and ga5-1) and perception (spy-4 and gai) during parthenocarpic silique development. Emasculated spy-4 pistils were neither obviously parthenocarpic nor deficient in PGR perception. By contrast, emasculated gai mutants did not produce parthenocarpic siliques following gibberellic acid application, but silique development occurred following pollination or application of auxin and cytokinin. Pollinated gai siliques had decreased cell numbers and morphologically resembled auxin-induced parthenocarpic siliques. This shows that a number of independent and possibly redundant pathways can direct hormone-induced parthenocarpy, and that endogenous gibberellins play a role in regulating cell expansion and promoting cell division in carpels. PMID:10517835

Adenovirus E1A and E1B-19K Proteins Protect Human Hepatoma Cells from Transforming Growth Factor β1-induced Apoptosis

PubMed Central

Tarakanova, Vera L.; Wold, William S. M.

2009-01-01

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor β1 (TGFβ). We report that infection with species C human adenovirus conferred resistance to TGFβ-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFβ-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFβ pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFβ-induced apoptosis. PMID:19854227

2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.

PubMed

Steele, Andrew J; Prentice, Archibald G; Hoffbrand, A Victor; Yogashangary, Birunthini C; Hart, Stephen M; Lowdell, Mark W; Samuel, Edward R; North, Janet M; Nacheva, Elisabeth P; Chanalaris, Anastasios; Kottaridis, Panagiotis; Cwynarski, Kate; Wickremasinghe, R Gitendra

2009-08-06

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

PubMed

Song, K-J; Jang, Y S; Lee, Y A; Kim, K A; Lee, S K; Shin, M H

2011-07-01

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells. © 2011 Blackwell Publishing Ltd.

The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.

PubMed

Ahmed, Ahmed Ashour; Mills, Anthony D; Ibrahim, Ashraf E K; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E; Iyer, N Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D; Earl, Helena M; Laskey, Ronald A; Caldas, Carlos; Brenton, James D

2007-12-01

The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Morphological changes in woody stem of Prunus jamasakura under simulated microgravity

NASA Technical Reports Server (NTRS)

Yoneyama, Emi; Ishimoto-Negishi, Yoko; Sano, Yuzou; Funada, Ryo; Yamada, Mitsuhiro; Nakamura, Teruko

2004-01-01

When the four-week-old woody stem of Prunus jamasakura was grown under simulated microgravity condition on a three-dimensional clinostat, it bent at growth, and width of its secondary xylem decreased due to the reduction of fiber cell numbers and a smaller microfibril angle in the secondary cell wall, as reported in our previous paper. Gravity induces the development of the secondary xylem that supports the stem upward against the action of gravity. In this study, morphological changes of the tissues and cells were microscopically observed. Disorder was found in the concentric structure of tissues that organize the stem. The radial arrangement of the cells was also disturbed in the secondary xylem, and in the secondary phloem secondary cell walls of the bast fiber cells were undeveloped. These findings suggest that differentiation and development of the secondary xylem and the bast fiber cells are strongly controlled by terrestrial gravity. These tissue and cells functions to support the stem under the action of gravity. Furthermore, clinorotation induced disorder in the straight joint of vessel elements and the lattice-like structure of radial parenchyma cells, which is responsible for water transportation and storage, respectively. Gravity is an essential factor for keeping the division and differentiation normal in woody stem.

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

PubMed

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-07-15

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

24(S)-Hydroxycholesterol Induces Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis*

PubMed Central

Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

2011-01-01

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis. PMID:21613228

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

SUB-ACUTE TREATMENT WITH METHYLMERCURY DURING DIFFERENTIATION OF PHEOCHROMOCYTOMA (PC12) CELLS DOES NOT ALTER BINDING OF ION CHANNEL LIGANDS OR CELL MORPHOLOGY.

EPA Science Inventory

We demonstrated recently that 6 days of exposure to nanomolar concentrations (3-10 nM) of methylmercury (MeHg) during nerve growth factor (NGF) induced PC12 cell differentiation reduced the amplitude and density of voltage-gated sodium and calcium currents. In the present study,...

Programming self-organizing multicellular structures with synthetic cell-cell signaling.

PubMed

Toda, Satoshi; Blauch, Lucas R; Tang, Sindy K Y; Morsut, Leonardo; Lim, Wendell A

2018-05-31

A common theme in the self-organization of multicellular tissues is the use of cell-cell signaling networks to induce morphological changes. We used the modular synNotch juxtacrine signaling platform to engineer artificial genetic programs in which specific cell-cell contacts induced changes in cadherin cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems: robust self-organization into multi-domain structures, well-choreographed sequential assembly, cell type divergence, symmetry breaking, and the capacity for regeneration upon injury. The ability of these networks to drive complex structure formation illustrates the power of interlinking cell signaling with cell sorting: signal-induced spatial reorganization alters the local signals received by each cell, resulting in iterative cycles of cell fate branching. These results provide insights into the evolution of multi-cellularity and demonstrate the potential to engineer customized self-organizing tissues or materials. Copyright © 2018, American Association for the Advancement of Science.

Proteomic and morphological changes produced by subinhibitory concentration of isoniazid in Mycobacterium tuberculosis.

PubMed

Campanerut-Sá, Paula Az; Ghiraldi-Lopes, Luciana D; Meneguello, Jean E; Fiorini, Adriana; Evaristo, Geisa Pc; Siqueira, Vera Ld; Scodro, Regiane Bl; Patussi, Eliana V; Donatti, Lucélia; Souza, Emanuel M; Cardoso, Rosilene F

2016-09-01

To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples' cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.

Valproic acid exhibits different cell growth arrest effect in three HPV-positive/negative cervical cancer cells and possibly via inducing Notch1 cleavage and E6 downregulation.

PubMed

Feng, Shuyu; Yang, Yue; Lv, Jingyi; Sun, Lichun; Liu, Mingqiu

2016-07-01

We investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of VPA-induced growth inhibition on three cervical cancer cell lines with different molecular and genetic background. We found that VPA induced proliferation suppression, cell apoptosis and cell cycle arrest in all tested cell lines, with an increase of Notch1 active form ICN1 as a tumor suppressor and its target gene HES1. Noteworthy, blocking of Notch signaling with DAPT resulted in growth inhibition in ICN1-overexpressing CaSki and HT-3 cells. Thus, endogenous Notch signaling may be necessary for survival of ICN1-overexpressing cervical cancer cell lines. Furthermore, G1 phase arrest was induced in HeLa and CaSki cells by VPA while G2 phase arrest was induced in HT-3 cells, suggesting different mechanism in this cycle arrest. We also found VPA suppressed oncogene E6 in a Notch-independent manner, and induced significant apoptosis in E6-overexpressing HPV positive CaSki cells. Cell morphological change was also observed in HeLa and HT-3 cell lines after VPA treatment with an upregulation of EMT transcription factor Snail1. Notch signaling inhibitor DAPT partly reversed VPA-induced Snail1 upregulation in HeLa cells. This discovery supports that VPA may induce EMT at least partly via Notch activation.

Pancreatic effect of andrographolide isolated from Andrographis paniculata (Burm. f.) Nees.

PubMed

Nugroho, Agung Endro; Rais, Ichwan Ridwan; Setiawan, Iwan; Pratiwi, Pramita Yuli; Hadibarata, Tony; Tegar, Maulana; Pramono, Suwidjiyo

2014-01-01

Andrographis paniculata (Burm. f.) Nees is a plant that originates from India and grows widely to Southeast which used for several purposes mainly as treatment of diabetes mellitus so the aim of this study was evaluate andrographolide for its pancreatic effect in neonatal streptozotocin (STZ)-induced diabetic rats, a model of type 2 diabetic rats. Diabetic condition was induced with an intraperitoneal injection of 90 mg kg(-1) streptozotocin in two-day-old rats. After three months, the neonatal STZ-induced diabetic rats were treated with andrographolide or andrographolide-enriched extract of A. paniculata (AEEAP) for 8 consecutive days. Pancreatic effect was evaluated by estimating mainly the preprandial and postprandial blood glucose levels and other parameters such as morphology of pancreatic islet, beta cells density and morphology and immunohistochemically pancreatic insulin. Andrographolide significantly (p < 0.05) decreased the levels of blood glucose and improved diabetic rat islet and beta cells. However, AEEAP exhibited moderate hypoglycaemic effects on the blood glucose levels. Moderate changes in beta cells were observed after AEEAP treatment. They could restore decreasing of pancreatic insulin contents. Based on these results andrographolide and AEEAP exhibited pancreatic actions in neonatal STZ-induced diabetic rats. The activity of andrographolide was more effective than this of AEEAP.

A dataset of images and morphological profiles of 30 000 small-molecule treatments using the Cell Painting assay

PubMed Central

Bray, Mark-Anthony; Gustafsdottir, Sigrun M; Rohban, Mohammad H; Singh, Shantanu; Ljosa, Vebjorn; Sokolnicki, Katherine L; Bittker, Joshua A; Bodycombe, Nicole E; Dančík, Vlado; Hasaka, Thomas P; Hon, Cindy S; Kemp, Melissa M; Li, Kejie; Walpita, Deepika; Wawer, Mathias J; Golub, Todd R; Schreiber, Stuart L; Clemons, Paul A; Shamji, Alykhan F

2017-01-01

Abstract Background Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at “The Cell Image Library” (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics. PMID:28327978

High hydrostatic pressure-induced cell death in human chondrocytes and chondrosarcoma cells.

PubMed

Naal, Florian-Dominique; Mengele, Karin; Schauwecker, Johannes; Gollwitzer, Hans; Gerdesmeyer, Ludger; Reuning, Ute; Mittelmeier, Wolfram; Gradinger, Reiner; Schmitt, Manfred; Diehl, Peter

2005-01-01

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in different ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used, although both methods have substantial disadvantages, e.g. loss of biomechanical and/or biological integrity of the bone and destabilization of the articular surface. In this regard, high hydrostatic pressure (HHP) treatment of bone is a new, advancing technology, now being used in preclinical testing to inactivate tumor cells. To find out if this technique is also suited for extracorporal inactivation of chondrocytes and chondral tumor cells, the effect of HHP on cell viability and morphology of human chondrocytes / chondrosarcoma cells was investigated in the present study. SW1353 chondrosarcoma cells and chondrocytes were subjected to HHP in the range of 50 to 350 MPa (10 min, 37 degrees C) and, subsequently, cell viability and cell morphology assessed. After exposure at 350 MPa, all HHP-treated chondral cells showed explicit morphological changes, evident by membrane ruffling and bleb formation; chondrosarcoma cells treated this way were irreversibly damaged and not alive. We anticipate that, in orthopedic surgery, HHP eventually can serve as a novel, promising technical approach for cell inactivation (including tumor cells) and allow subsequent reimplantation of the osteoarticular autograft.

Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl‑2 family proteins.

PubMed

Ji, Chen-Chen; Tang, Hai-Feng; Hu, Yi-Yang; Zhang, Yun; Zheng, Min-Hua; Qin, Hong-Yan; Li, San-Zhong; Wang, Xiao-Yang; Fei, Zhou; Cheng, Guang

2016-07-01

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL‑60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose‑ and time‑dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT‑22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6‑induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase‑3, ‑8 and ‑9, and decreases in the levels of B‑cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.

Control of Cellular Structural Networks Through Unstructured Protein Domains

DTIC Science & Technology

2016-07-01

stem cells (hPSCs), including embryonic and induced pluripotent stem cells . We had a third paper accepted to Scientific Reports in which we showed...2012 Stem Cells Young Investigator Award. We then had a followup paper accepted to Integrative Biology extending these ideas to human pluripotent ...morphology, mechanics, and neurogenesis in neural stem cells ; (3) To develop and use multiscale computational 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND

Dextran and Polymer Polyethylene Glycol (PEG) Coating Reduce Both 5 and 30 nm Iron Oxide Nanoparticle Cytotoxicity in 2D and 3D Cell Culture

PubMed Central

Yu, Miao; Huang, Shaohui; Yu, Kevin Jun; Clyne, Alisa Morss

2012-01-01

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles. PMID:22754315

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PubMed

Hoffman, Ewelina; Patel, Aateka; Ball, Doug; Klapwijk, Jan; Millar, Val; Kumar, Abhinav; Martin, Abigail; Mahendran, Rhamiya; Dailey, Lea Ann; Forbes, Ben; Hutter, Victoria

2017-12-01

Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Histopathology and pathogenesis of caerulein-, duct ligation-, and arginine-induced acute pancreatitis in Sprague-Dawley rats and C57BL6 mice.

PubMed

Zhang, Jun; Rouse, Rodney L

2014-09-01

Three classical rodent models of acute pancreatitis were created in an effort to identify potential pre-clinical models of drug-induced pancreatitis (DIP) and candidate non-invasive biomarkers for improved detection of DIP. Study objectives included designing a lexicon to minimize bias by capturing normal variation and spontaneous and injury-induced changes while maintaining the ability to statistically differentiate degrees of change, defining morphologic anchors for novel pancreatic injury biomarkers, and improved understanding of mechanisms responsible for pancreatitis. Models were created in male Sprague-Dawley rats and C57BL6 mice through: 1) administration of the cholecystokinin analog, caerulein; 2) administration of arginine; 3) surgical ligation of the pancreatic duct. Nine morphologically detectable processes were used in the lexicon; acinar cell hypertrophy; acinar cell autophagy; acinar cell apoptosis; acinar cell necrosis; vascular injury; interstitial edema, inflammation and hemorrhage; fat necrosis; ductal changes; acinar cell atrophy. Criteria were defined for scoring levels (0 = absent, 1 = mild, 2 = moderate, 3 = severe) for each lexicon component. Consistent with previous studies, histopathology scores were significant greater in rats compared to mice at baseline and after treatment. The histopathology scores in caerulein and ligation-treated rats and mice were significantly greater than those of arginine-treated rats and mice. The present study supports a multifaceted pathogenesis for acute pancreatitis in which intra-acinar trypsinogen activation, damage to acinar cells, fat cells, and vascular cells as well as activation/degranulation of mast cells and activated macrophages all contribute to the initiation and/or progression of acute inflammation of the exocrine pancreas.

Induction of insulin-producing cells from human pancreatic progenitor cells.

PubMed

Noguchi, H; Naziruddin, B; Shimoda, M; Fujita, Y; Chujo, D; Takita, M; Peng, H; Sugimoto, K; Itoh, T; Tamura, Y; Olsen, G S; Kobayashi, N; Onaca, N; Hayashi, S; Levy, M F; Matsumoto, S

2010-01-01

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells. Copyright 2010 Elsevier Inc. All rights reserved.

Hydrogen-rich saline attenuates spinal cord hemisection-induced testicular injury in rats.

PubMed

Ge, Li; Wei, Li-Hua; Du, Chang-Qing; Song, Guo-Hua; Xue, Ya-Zhuo; Shi, Hao-Shen; Yang, Ming; Yin, Xin-Xin; Li, Run-Ting; Wang, Xue-Er; Wang, Zhen; Song, Wen-Gang

2017-06-27

To study how hydrogen-rich saline (HS) promotes the recovery of testicular biological function in a hemi-sectioned spinal cord injury (hSCI) rat model, a right hemisection was performed at the T11-T12 of the spinal cord in Wistar rats. Animals were divided into four groups: normal group; vehicle group: sham-operated rats administered saline; hSCI group: subjected to hSCI and administered saline; HRST group: subjected to hSCI and administered HS. Hind limb neurological function, testis index, testicular morphology, mean seminiferous tubular diameter (MSTD) and seminiferous epithelial thickness (MSET), the expression of heme oxygenase-1 (HO-1), mitofusin-2 (MFN-2), and high-mobility group box 1 (HMGB-1), cell ultrastructure, and apoptosis of spermatogenic cells were studied. The results indicated that hSCI significantly decreased the hind limb neurological function, testis index, MSTD, and MSET, and induced severe testicular morphological injury. The MFN-2 level was decreased, and HO-1 and HMGB-1 were overexpressed in testicular tissues. In addition, hSCI accelerated the apoptosis of spermatogenic cells and the ultrastructural damage of cells in the hypophysis and testis. After HS administration, all these parameters were considerably improved, and the characteristics of hSCI testes were similar to those of normal control testes. Taken together, HS administration can promote the recovery of testicular biological function by anti-oxidative, anti-inflammatory, and anti-apoptotic action. More importantly, HS can inhibit the hSCI-induced ultrastructural changes in gonadotrophs, ameliorate the abnormal regulation of the hypothalamic-pituitary-testis axis, and thereby promote the recovery of testicular injury. HS administration also inhibited the hSCI-induced ultrastructural changes in testicular spermatogenic cells, Sertoli cells and interstitial cells.

Hydrogen-rich saline attenuates spinal cord hemisection-induced testicular injury in rats

PubMed Central

Ge, Li; Wei, Li-Hua; Du, Chang-Qing; Song, Guo-Hua; Xue, Ya-Zhuo; Shi, Hao-Shen; Yang, Ming; Yin, Xin-Xin; Li, Run-Ting; Wang, Xue-er; Wang, Zhen; Song, Wen-Gang

2017-01-01

To study how hydrogen-rich saline (HS) promotes the recovery of testicular biological function in a hemi-sectioned spinal cord injury (hSCI) rat model, a right hemisection was performed at the T11–T12 of the spinal cord in Wistar rats. Animals were divided into four groups: normal group; vehicle group: sham-operated rats administered saline; hSCI group: subjected to hSCI and administered saline; HRST group: subjected to hSCI and administered HS. Hind limb neurological function, testis index, testicular morphology, mean seminiferous tubular diameter (MSTD) and seminiferous epithelial thickness (MSET), the expression of heme oxygenase-1 (HO-1), mitofusin-2 (MFN-2), and high-mobility group box 1 (HMGB-1), cell ultrastructure, and apoptosis of spermatogenic cells were studied. The results indicated that hSCI significantly decreased the hind limb neurological function, testis index, MSTD, and MSET, and induced severe testicular morphological injury. The MFN-2 level was decreased, and HO-1 and HMGB-1 were overexpressed in testicular tissues. In addition, hSCI accelerated the apoptosis of spermatogenic cells and the ultrastructural damage of cells in the hypophysis and testis. After HS administration, all these parameters were considerably improved, and the characteristics of hSCI testes were similar to those of normal control testes. Taken together, HS administration can promote the recovery of testicular biological function by anti-oxidative, anti-inflammatory, and anti-apoptotic action. More importantly, HS can inhibit the hSCI-induced ultrastructural changes in gonadotrophs, ameliorate the abnormal regulation of the hypothalamic-pituitary-testis axis, and thereby promote the recovery of testicular injury. HS administration also inhibited the hSCI-induced ultrastructural changes in testicular spermatogenic cells, Sertoli cells and interstitial cells. PMID:28404953

Automated Quantification and Integrative Analysis of 2D and 3D Mitochondrial Shape and Network Properties

PubMed Central

Nikolaisen, Julie; Nilsson, Linn I. H.; Pettersen, Ina K. N.; Willems, Peter H. G. M.; Lorens, James B.; Koopman, Werner J. H.; Tronstad, Karl J.

2014-01-01

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells. PMID:24988307

Phenotypic and Physiological Characterization of the Epibiotic Interaction Between TM7x and Its Basibiont Actinomyces.

PubMed

Bor, Batbileg; Poweleit, Nicole; Bois, Justin S; Cen, Lujia; Bedree, Joseph K; Zhou, Z Hong; Gunsalus, Robert P; Lux, Renate; McLean, Jeffrey S; He, Xuesong; Shi, Wenyuan

2016-01-01

Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted quantitative real-time PCR (qRT-PCR) analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed-ends, and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth.

Quantification of a thermal damage threshold for astrocytes using infrared laser generated heat gradients.

PubMed

Liljemalm, Rickard; Nyberg, Tobias

2014-04-01

The response of cells and tissues to elevated temperatures is highly important in several research areas, especially in the area of infrared neural stimulation. So far, only the heat response of neurons has been considered. In this study, primary rat astrocytes were exposed to infrared laser pulses of various pulse lengths and the resulting cell morphology changes and cell migration was studied using light microscopy. By using a finite element model of the experimental setup the temperature distribution was simulated and the temperatures and times to induce morphological changes and migration were extracted. These threshold temperatures were used in the commonly used first-order reaction model according to Arrhenius to extract the kinetic parameters, i.e., the activation energy, E a, and the frequency factor, A c, for the system. A damage signal ratio threshold was defined and calculated to be 6% for the astrocytes to change morphology and start migrating.

Multiple effects of TRAIL in human carcinoma cells: Induction of apoptosis, senescence, proliferation, and cytokine production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levina, Vera; Marrangoni, Adele M.; DeMarco, Richard

TRAIL is a death ligand that induces apoptosis in malignant but not normal cells. Recently the ability of TRAIL to induce proliferation in apoptosis-resistant normal and malignant cells was reported. In this study, we analyzed TRAIL effects in apoptosis sensitive MCF7, OVCAR3 and H460 human tumor cell lines. TRAIL at low concentrations preferentially induced cell proliferation. At 100 ng/ml, apoptotic death was readily observed, however surviving cells acquired higher proliferative capacity. TRAIL-stimulated production of several cytokines, IL-8, RANTES, MCP-1 and bFGF, and activation of caspases 1 and 8 was essential for this effect. Antibodies to IL-8, RANTES, and bFGF blockedmore » TRAIL-induced cell proliferation and further stimulated apoptosis. For the first time, we report that high TRAIL concentrations induced cell senescence as determined by the altered morphology and expression of several senescence markers: SA-{beta}-gal, p21{sup Waf1/Cip1}, p16{sup INK4a}, and HMGA. Caspase 9 inhibition protected TRAIL-treated cells from senescence, whereas inhibition of caspases 1 and 8 increased the yield of SLP cells. In conclusion, in cultured human carcinoma cells, TRAIL therapy results in three functional outcomes, apoptosis, proliferation and senescence. TRAIL-induced proapoptotic and prosurvival responses correlate with the strength of signaling. TRAIL-induced cytokine production is responsible for its proliferative and prosurvival effects.« less

Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins

PubMed Central

1985-01-01

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells. PMID:2993312

Licochalcone C induces apoptosis via B-cell lymphoma 2 family proteins in T24 cells.

PubMed

Wang, Penglong; Yuan, Xuan; Wang, Yan; Zhao, Hong; Sun, Xiling; Zheng, Qiusheng

2015-11-01

The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase‑3 activity assay. Alterations in the gene expression levels of Bcl‑2 family members were measured by semi‑quantitative reverse transcription‑polymerase chain reaction assays. The protein levels of pro‑caspase‑3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration‑dependent manner. Licochalcone C treatment reduced the levels of the anti‑apoptotic mRNAs (Bcl‑2, Bcl‑w and Bcl‑XL) and increased expression of the pro‑apoptotic mRNAs (Bax and Bim). The Bcl‑2 family inhibitor (ABT‑737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer.

[Decursin reduces reactive oxygen species and inhibits cisplatin-induced apoptosis in rat renal tubular epithelial cells].

PubMed

Li, Cuiqiong; Li, Jianchun; Fan, Junming; Meng, Lifeng; Cao, Ling

2017-10-01

Objective To study the mechanism underlying the inhibitory effect of decursin on the apoptosis of rat renal tubular epithelial cells NRK-52E induced by cisplatin. Methods First, CCK-8 assay was used to detect the effects of 0, 10, 20, 40, 80, 100, 150, 200 μmol/L decursin and 0, 5, 10, 20, 30, 40, 50 μg/mL cispatin treatment for 24 hours on cell proliferation in NRK-52E cells via determining the half inhibitory concentration (IC 50 ). Then, NRK-52E cells were stimulated with 20 μg/mL cisplatin combined with 10, 50, 100 μmol/L decursin, and cell activity was detected by CCK-8 assay. The cells were divided into normal control group, 20 μg/mL cisplatin stimulation group, and 10, 50, 100 μmol/L decursin treated groups. Cell morphological changes was observed under inverted microscope, morphological changes of nucleus was detected by DAPI staining, cell apoptosis was detected by flow cytometry, the level of intracellular ROS was detected by DCFH-DA staining, and the apoptosis marker proteins cleaved-caspase-3 and cleaved-PARP were examined by Western blot analysis. Results Compared with the normal control group, cisplatin significantly inhibited the activity of the cells, and IC 50 was about 20 μg/mL; compared with the model group, in the decursin pretreatment groups, the level of intracellular ROS decreased remarkably, the expressions of cleaved-casspase-3 and cleaved-PARP proteins were reduced, and cell apoptosis was depressed. Conclusion Decursin can decrease the intracellular ROS level and inhibit the apoptosis of NRK-52E cells induced by cisplatin.

Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-β-catenin signaling to slow tumor progression.

PubMed

Froeling, Fieke E M; Feig, Christine; Chelala, Claude; Dobson, Richard; Mein, Charles E; Tuveson, David A; Clevers, Hans; Hart, Ian R; Kocher, Hemant M

2011-10-01

Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. PSCs and cancer cell lines (AsPc1 and Capan1) were exposed to doses and isoforms of retinoic acid (RA) in 2-dimensional and 3-dimensional culture conditions (physiomimetic organotypic culture). The effects of all-trans retinoic acid (ATRA) were studied in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, a model of human pancreatic ductal adenocarcinoma. After incubation with ATRA, PSCs were quiescent and had altered expression of genes that regulate proliferation, morphology, and motility; genes that encode cytoskeletal proteins and cytokines; and genes that control other functions, irrespective of culture conditions or dosage. In the organotypic model, and in mice, ATRA induced quiescence of PSCs and thereby reduced cancer cell proliferation and translocation of β-catenin to the nucleus, increased cancer cell apoptosis, and altered tumor morphology. ATRA reduced the motility of PSCs, so these cells created a "wall" at the junction between the tumor and the matrix that prevented cancer cell invasion. Restoring secreted frizzled-related protein 4 (sFRP4) secretion to quiescent PSCs reduced Wnt-β-catenin signaling in cancer cells and their invasive ability. Human primary and metastatic pancreatic tumor tissues stained strongly for cancer cell nuclear β-catenin but had low levels of sFRP4 (in cancer cells and PSCs). RA induces quiescence and reduces motility of PSCs, leading to reduced proliferation and increased apoptosis of surrounding pancreatic cancer cells. RA isoforms might be developed as therapeutic reagents for pancreatic cancer. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Mathematical modelling of the antibiotic-induced morphological transition of Pseudomonas aeruginosa

PubMed Central

Keen, Emma; Smith, David J.

2018-01-01

Here we formulate a mechanistic mathematical model to describe the growth dynamics of P. aeruginosa in the presence of the β-lactam antibiotic meropenem. The model is mechanistic in the sense that carrying capacity is taken into account through the dynamics of nutrient availability rather than via logistic growth. In accordance with our experimental results we incorporate a sub-population of cells, differing in morphology from the normal bacillary shape of P. aeruginosa bacteria, which we assume have immunity from direct antibiotic action. By fitting this model to experimental data we obtain parameter values that give insight into the growth of a bacterial population that includes different cell morphologies. The analysis of two parameters sets, that produce different long term behaviour, allows us to manipulate the system theoretically in order to explore the advantages of a shape transition that may potentially be a mechanism that allows P. aeruginosa to withstand antibiotic effects. Our results suggest that inhibition of this shape transition may be detrimental to bacterial growth and thus suggest that the transition may be a defensive mechanism implemented by bacterial machinery. In addition to this we provide strong theoretical evidence for the potential therapeutic strategy of using antimicrobial peptides (AMPs) in combination with meropenem. This proposed combination therapy exploits the shape transition as AMPs induce cell lysis by forming pores in the cytoplasmic membrane, which becomes exposed in the spherical cells. PMID:29481562

Chalcone Synthase (CHS) Gene Suppression in Flax Leads to Changes in Wall Synthesis and Sensing Genes, Cell Wall Chemistry and Stem Morphology Parameters

PubMed Central

Zuk, Magdalena; Działo, Magdalena; Richter, Dorota; Dymińska, Lucyna; Matuła, Jan; Kotecki, Andrzej; Hanuza, Jerzy; Szopa, Jan

2016-01-01

The chalcone synthase (CHS) gene controls the first step in the flavonoid biosynthesis. In flax, CHS down-regulation resulted in tannin accumulation and reduction in lignin synthesis, but plant growth was not affected. This suggests that lignin content and thus cell wall characteristics might be modulated through CHS activity. This study investigated the possibility that CHS affects cell wall sensing as well as polymer content and arrangement. CHS-suppressed and thus lignin-reduced plants showed significant changes in expression of genes involved in both synthesis of components and cell wall sensing. This was accompanied by increased levels of cellulose and hemicellulose. CHS-reduced flax also showed significant changes in morphology and arrangement of the cell wall. The stem tissue layers were enlarged averagely twofold compared to the control, and the number of fiber cells more than doubled. The stem morphology changes were accompanied by reduction of the crystallinity index of the cell wall. CHS silencing induces a signal transduction cascade that leads to modification of plant metabolism in a wide range and thus cell wall structure. PMID:27446124

Developmentally regulated GTP-binding protein 2 depletion leads to mitochondrial dysfunction through downregulation of dynamin-related protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vo, Mai-Tram; Ko, Myoung Seok; Lee, Unn Hwa

Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2more » depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1. - Highlights: • DRG2 depletion increased mitochondrial swelling. • DRG2 depletion inhibited the expression of Drp1. • Overexpression of DRG2 or Drp1 rescued mitochondrial shape in DRG2 depleted cells. • DRG2 depletion induced mitochondrial dysfunction.« less

Mitochondrial Control by DRP1 in Brain Tumor Initiating Cells

PubMed Central

Xie, Qi; Wu, Qiulian; Horbinski, Craig M.; Flavahan, William A.; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M.; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N.; Kashatus, David F.; Bao, Shideng; Rich, Jeremy N.

2015-01-01

Brain tumor initiating cells (BTICs) coopt the neuronal high affinity GLUT3 glucose transporter to withstand metabolic stress. Here, we investigated another mechanism critical to brain metabolism, mitochondrial morphology. BTICs displayed mitochondrial fragmentation relative to non-BTICs, suggesting that BTICs have increased mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), was activated in BTICs and inhibited in non-BTICs. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and AMPK targeting rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+–calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTICs, suggesting tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlates with poor prognosis in glioblastoma, suggesting mitochondrial dynamics may represent a therapeutic target for BTICs. PMID:25730670

Trehalose protects against cadmium-induced cytotoxicity in primary rat proximal tubular cells via inhibiting apoptosis and restoring autophagic flux

PubMed Central

Wang, Xin-Yu; Yang, Heng; Wang, Min-Ge; Yang, Du-Bao; Wang, Zhen-Yong; Wang, Lin

2017-01-01

Autophagy has an important renoprotective function and we recently found that autophagy inhibition is involved in cadmium (Cd)-induced nephrotoxicity. Here, we aimed to investigate the protective effect of trehalose (Tre), a novel autophagy activator, against Cd-induced cytotoxicity in primary rat proximal tubular (rPT) cells. First, data showed that Tre treatment significantly decreased Cd-induced apoptotic cell death of rPT cells via inhibiting caspase-dependent apoptotic pathway, evidenced by morphological analysis, flow cytometric and immunoblot assays. Also, administration with Tre protected rPT cells against Cd-induced lipid peroxidation. Inhibition of autophagic flux in Cd-exposed rPT cells was markedly restored by Tre administration, demonstrated by immunoblot analysis of autophagy marker proteins and GFP and RFP tandemly tagged LC3 method. Resultantly, Cd-induced autophagosome accumulation was obviously alleviated by Tre treatment. Meanwhile, blockage of autophagosome–lysosome fusion by Cd exposure was noticeably restored by Tre, which promoted the autophagic degradation in Cd-exposed rPT cells. Moreover, Tre treatment markedly recovered Cd-induced lysosomal alkalinization and impairment of lysosomal degradation capacity in rPT cells, demonstrating that Tre has the ability to restore Cd-impaired lysosomal function. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced cytotoxicity in rPT cells by inhibiting apoptosis and restoring autophagic flux. PMID:29022917

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Substrate Curvature Restricts Spreading and Induces Differentiation of Human Mesenchymal Stem Cells.

PubMed

Lee, Sang Joo; Yang, Shengyuan

2017-09-01

While cells attach, spread, migrate, proliferate, and differentiate in three-dimensional (3D) micromechanical environments, the mechanical factors of these environments influence the shapes, sizes, and adhesion forces of the cells. Here, the authors culture human mesenchymal stem cells (hMSCs) on a unique class of curvature-defined substrates, micro glass ball embedded polyacrylamide gels, prepared with an improved protocol, and investigate the spreading responses of the hMSCs on the glass balls to study the effects of substrate curvature on the spreading of hMSCs. The authors find that, among the used diameters of glass balls, the minimum diameter of a glass ball on which an hMSC can attach and spread is 500 μm. In contrast to the well-spread morphologies with randomly-multiple lamellipodia for the hMSCs growing on the flat glass plates, the morphologies of the hMSCs growing on the glass balls are almost uniformly spindle-shaped with two lamellipodia. The sensitivities of the attachment and spreading morphology of an hMSC to substrate curvature are very different from those of a fibroblast. The RT-PCR analysis reveals that the substrate curvature alone can induce adipogenesis of the hMSCs. These findings imply that substrate curvature has profound effects on stem cell behaviors, and detailed and in-depth studies on these effects and their underlying biophysical mechanisms are necessary. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of the Cytotoxic Potential of Anisomelic Acid Isolated from Anisomeles malabarica

PubMed Central

Preethy, Christo Paul; Alshatwi, Ali Abdullah; Gunasekaran, Muthukumaran; Akbarsha, Mohammad Abdulkadher

2013-01-01

Anisomelic acid (AA), one of the major compounds in Anisomeles malabarica, was tested for its cytotoxicity and apoptosis-inducing potential in breast and cervical cancer cells. The MTT assay for cell viability indicated that AA is cytotoxic to all of the four cell lines tested in a dose- and duration-dependent manner. Acridine Orange & Ethidium Bromide (AO & EB) and Hoechst 33258 staining of AA-treated cells revealed typical apoptotic morphology such as condensed chromatin and formation of apoptotic bodies. The comet assay revealed DNA strand break(s), indicating that AA induces DNA damage which culminates in apoptosis. Thus, the study revealed the anti-proliferative and apoptosis-inducing properties of AA in both breast and cervical cancer cells. Therefore, anisomelic acid offers potential for application in breast and cervical cancer therapy. PMID:23833721

Low-intensity vibrations normalize adipogenesis-induced morphological and molecular changes of adult mesenchymal stem cells.

PubMed

Baskan, Oznur; Mese, Gulistan; Ozcivici, Engin

2017-02-01

Bone marrow mesenchymal stem cells that are committed to adipogenesis were exposed daily to high-frequency low-intensity mechanical vibrations to understand molecular, morphological and ultrastructural adaptations to mechanical signals during adipogenesis. D1-ORL-UVA mouse bone marrow mesenchymal stem cells were cultured with either growth or adipogenic medium for 1 week. Low-intensity vibration signals (15 min/day, 90 Hz, 0.1 g) were applied to one group of adipogenic cells, while the other adipogenic group served as a sham control. Cellular viability, lipid accumulation, ultrastructure and morphology were determined with MTT, Oil-Red-O staining, phalloidin staining and atomic force microscopy. Semiquantitative reverse transcription polymerase chain reaction showed expression profile of the genes responsible for adipogenesis and ultrastructure of cells. Low-intensity vibration signals increased viability of the cells in adipogenic culture that was reduced significantly compared to quiescent controls. Low-intensity vibration signals also normalized the effects of adipogenic condition on cell morphology, including area, perimeter, circularization and actin cytoskeleton. Furthermore, low-intensity vibration signals reduced the expression of some adipogenic markers significantly. Mesenchymal stem cells are sensitive and responsive to mechanical loads, but debilitating conditions such as aging or obesity may steer mesenchymal stem cells toward adipogenesis. Here, daily application of low-intensity vibration signals partially neutralized the effects of adipogenic induction on mesenchymal stem cells, suggesting that these signals may provide an alternative and/or complementary option to reduce fat deposition.

The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

PubMed

Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

2015-10-30

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

PubMed Central

Pontier-Bres, Rodolphe; Rampal, Patrick; Peyron, Jean-François; Munro, Patrick; Lemichez, Emmanuel; Czerucka, Dorota

2015-01-01

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax. PMID:26529015

The Pathogenesis of Human Cervical Epithelium Cells Induced by Interacting with Trichomonas vaginalis

PubMed Central

Lin, Wei-Chen; Chang, Wei-Ting; Chang, Tsuey-Yu; Shin, Jyh-Wei

2015-01-01

Background Trichomonas vaginalis is a protozoan parasite that occurs in the urogenital-vaginal tract and is the primary causative agent of trichomoniasis, a common sexually transmitted disease in humans. The aggregation of this protozoan tends to destroy epithelial cells and induce pathogenesis. Principal Findings This study cultured T. vaginalis and human cervical epithelial cells (Z172) under the same conditions in the experiments. Following co-culturing for ten hours, the protozoans became attached to Z172, such that the cells presented a round shape and underwent shrinkage. Time-lapse recording and flow cytometry on interacted Z172 revealed that 70% had been disrupted, 18% presented a necrosis-like morphology and 8% showed signs of apoptosis. Gene expression profiling revealed in the seven inflammatory Z172 genes as well as in T. vaginalis genes that code for adhesion proteins 65 and 65-1. Significance These results suggest that cytopathogenic effects progress while Z172 is in contact with T. vaginalis, and the resulting morphological changes can be categorized as disruption. PMID:25901354

Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

PubMed

Nudmamud-Thanoi, Sutisa; Sueudom, Wanvipa; Tangsrisakda, Nareelak; Thanoi, Samur

2016-10-01

Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERβ were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERβ immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERβ were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERβ expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

PubMed

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

Emodin induces neurite outgrowth through PI3K/Akt/GSK-3β-mediated signaling pathways in Neuro2a cells.

PubMed

Park, Shin-Ji; Jin, Mei Ling; An, Hyun-Kyu; Kim, Kyoung-Sook; Ko, Min Jung; Kim, Cheol Min; Choi, Young Whan; Lee, Young-Choon

2015-02-19

In this study, a neurite outgrowth-inducing substance was isolated from the ethylacetate extract of the Polygonum multiflorum roots and identified as emodin by gas-liquid chromatography-mass spectrometry and (1)H NMR and (13)C NMR. Emodin displayed remarkable neurite outgrowth-inducing activity in Neuro2a cells, as demonstrated by morphological changes and immunocytochemistry for class III β-tubulin. Emodin exhibited a stronger neutrophic activity than retinoic acid (RA) known as inducer of neurite outgrowth in Neuro2a cells. Emodin treatment resulted in marked increases in phosphorylation of Akt a direct downstream signaling molecule of phosphatidylinositol 3-kinase (PI3K), but upstream of glycogen synthase kinase-3β (GSK-3β) and cAMP response element-binding protein (CREB). These augmentations and neurite-bearing cells induced by emodin were remarkably reduced by the addition of PI3K inhibitor LY294002. These results demonstrate that emodin induces neuronal differentiation of Neuro2a cells via PI3K/Akt/GSK-3β pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8.

PubMed

Nakamura, Atsushi; Aizawa, Junichi; Sakayama, Kenshi; Kidani, Teruki; Takata, Tomoyo; Norimatsu, Yoshiaki; Miura, Hiromasa; Masuno, Hiroshi

2012-09-26

One of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. In Japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. The purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells. LM8 cells were treated for 3 days with various concentrations of genistein. The effect of genistein on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. The assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. The expression and secretion of MMP-2 were determined by immunohistochemistry and gelatin zymography. The subcellular localization and cellular level of β-catenin were determined by immunofluorescence and Western blot. For examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin (H&E). The expression of osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Genistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 were lower in the genistein-treated cultures than in the untreated cultures. β-Catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. The level of β-catenin was higher in genistein-treated cells than in untreated cells. Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA. Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma

PubMed Central

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-01-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis. PMID:28789364

MazEF toxin-antitoxin proteins alter Escherichia coli cell morphology and infrastructure during persister formation and regrowth.

PubMed

Cho, Junho; Carr, Anita Nicole; Whitworth, Lisa; Johnson, Brent; Wilson, Kevin Scott

2017-03-01

When exposed to antibiotics, many bacteria respond by activating intracellular 'toxin' proteins, which arrest cell growth and induce formation of persister cells that survive antibiotics. After antibiotics are removed, persisters can regrow by synthesizing 'antitoxin' proteins that sequester toxin proteins. In Escherichia coli, MazE antitoxin sequesters the activity of MazF toxin, which extensively cleaves cellular RNAs. Although the functions of MazEF proteins are well characterized, there is surprisingly little known about their effects on cell structure. Here, using a combination of microscopy techniques, we visualized the effects of MazEF and three bactericidal antibiotics on E. coli cell morphology and infrastructure. When ectopically expressed in E. coli, MazF temporarily stalled cell growth and induced persister formation, but only mildly elevated DNA mutagenesis. Viewed by electron microscopy, MazF-expressing persister cells were arrested in cell growth and division. Their chromosomal DNAs were compacted into thread-like structures. Their ribosomes were excluded from their nucleoids. After exposure to ciprofloxacin, persister regrowth was activated by MazE. Cell division remained inhibited while cells became extraordinarily elongated, then divided multiple times during stationary growth phase. This extreme filamentation during persister regrowth was unique to ciprofloxacin-treated persisters, likely caused by inhibition of cell division during regrowth, and was not observed with kanamycin-treated persisters.

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma.

PubMed

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-08-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis.

Modulating effects of epithalamin and epithalon on the functional morphology of the spleen in old pinealectomized rats.

PubMed

Khavinson, V K; Konovalov, S S; Yuzhakov, V V; Popuchiev, V V; Kvetnoi, I M

2001-11-01

Immunohistochemical and morphometric analysis showed that epithalamin and epithalon produced similar effects on the functional morphology of the spleen in pinealectomized rats. Both peptides prevented hyperplasia of lymphoid cells in follicular germinative centers induced by pinealectomy and potentiated the decrease in extramedullary hemopoiesis. These findings confirm the data on functional relationships between the pineal gland and immune system. The effects of epithalamin and epithalon on cell and tissue homeostasis in the spleen of old pinealectomized rats can be regarded as a manifestation of the general regulatory effect of these peptides.

A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line.

PubMed

Collins, Adam; Huett, Alan

2018-05-15

We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.

Carbon and nitrogen depletion-induced nucleophagy and selective autophagic sequestration of a whole nucleus in multinucleate cells of the filamentous fungus Aspergillus oryzae.

PubMed

Kikuma, Takashi; Mitani, Takahiro; Kohara, Takahiro; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2017-05-12

Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed "nucleophagy", has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture. While developing an H2B-EGFP processing assay for the evaluation of nucleophagy in A. oryzae, we found that nucleophagy is efficiently induced by carbon or nitrogen depletion. Microscopic observations in a carbon depletion condition clearly demonstrated that autophagosomes selectively sequester a particular nucleus, despite the presence of multiple nuclei in the same cell. Furthermore, AoNsp1, the A. oryzae homolog of the yeast nucleoporin Nsp1p, mainly localized at the nuclear periphery, but its localization was restricted to the opposite side of the autophagosome being formed around a nucleus. In contrast, the perinuclear ER visualized with the calnexin AoClxA was not morphologically affected by nucleophagy. The findings of nucleophagy-inducing conditions enabled us to characterize the morphological process of autophagic degradation of a whole nucleus in multinucleate cells.

Curcumin induces apoptosis in SGC-7901 gastric adenocarcinoma cells via regulation of mitochondrial signaling pathways.

PubMed

Xue, Xia; Yu, Jin-Long; Sun, De-Qing; Kong, Feng; Qu, Xian-jun; Zou, Wen; Wu, Jing; Wang, Rong-Mei

2014-01-01

Curcumin, a polyphenol compound derived from the rhizome of the plant Curcuma longa L. has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms by which it induces apoptosis is limited. In this study, the anticancer efficacy of curcumin was investigated in human gastric adenocarcinoma SGC-7901 cells. The results demonstrated that curcumin induced morphological changes and decreased cell viability. Apoptosis triggered by curcumin was visualized using Annexin V-FITC/7- AAD staining. Curcumin-induced apoptosis of SGC-7901 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the down-regulation of Bcl-2 and up-regulation of Bax that led to the cleavage of caspase-3 and increased cleaved PARP was observed in SGC-7901 cells treated with curcumin. Therefore, curcumin-induced apoptosis of SGC-7901 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of curcumin as a potential cancer therapeutic compound.

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice.

PubMed

Narahara, Shun; Matsushima, Haruna; Sakai, Eiko; Fukuma, Yutaka; Nishishita, Kazuhisa; Okamoto, Kuniaki; Tsukuba, Takayuki

2012-04-01

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H(2)O(2)) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles.

PubMed

Cetera, Maureen; Leybova, Liliya; Joyce, Bradley; Devenport, Danelle

2018-05-01

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles, which emerges through epidermal-dermal signaling and planar polarized morphogenesis. Here, using live-imaging, we discover that developing hair follicles polarize through dramatic cell rearrangements organized in a counter-rotational pattern of cell flows. Upon hair placode induction, Shh signaling specifies a radial pattern of progenitor fates that, together with planar cell polarity, induce counter-rotational rearrangements through myosin and ROCK-dependent polarized neighbour exchanges. Importantly, these cell rearrangements also establish cell fate asymmetry by repositioning radial progenitors along the anterior-posterior axis. These movements concurrently displace associated mesenchymal cells, which then signal asymmetrically to maintain polarized cell fates. Our results demonstrate how spatial patterning and tissue polarity generate an unexpected collective cell behaviour that in turn, establishes both morphological and cell fate asymmetry.

Anticancer potential of Conium maculatum extract against cancer cells in vitro: Drug-DNA interaction and its ability to induce apoptosis through ROS generation.

PubMed

Mondal, Jesmin; Panigrahi, Ashis Kumar; Khuda-Bukhsh, Anisur Rahman

2014-08-01

Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anti-cancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells. Conium's effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy. Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at sub-G stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase-3 activation led HeLa cells toward apoptosis, down-regulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondria-mediated caspase-3-dependent pathway. CD-spectroscopy revealed that Conium interacted with DNA molecule. Overall results validate anti-cancer potential of Conium and provide support for its use in traditional systems of medicine.

Anticancer potential of Conium maculatum extract against cancer cells in vitro: Drug-DNA interaction and its ability to induce apoptosis through ROS generation

PubMed Central

Mondal, Jesmin; Panigrahi, Ashis Kumar; Khuda-Bukhsh, Anisur Rahman

2014-01-01

Objective: Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anti-cancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells. Materials and Methods: Conium's effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy. Results: Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at sub-G stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase-3 activation led HeLa cells toward apoptosis, down-regulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondria-mediated caspase-3-dependent pathway. CD-spectroscopy revealed that Conium interacted with DNA molecule. Conclusion: Overall results validate anti-cancer potential of Conium and provide support for its use in traditional systems of medicine. PMID:25298670

In vitro anticancer activities of Leonurus heterophyllus sweet (Chinese motherwort herb).

PubMed

Chinwala, Maimoona G; Gao, Min; Dai, Jie; Shao, Jun

2003-08-01

To investigate the anticancer activities of Chinese motherwort herb (Leonurus heterophyllus Sweet; LHS). Dried LHS was extracted and reconstituted in phosphate-buffered saline. The in vitro antiproliferation activities of the extract were tested against seven human cancer cell lines. The DNA ladder assay and cell morphologic studies were performed to verify the drug's apoptotic activities. The possible pathway by which LHS induced apoptosis was also explored by examining mitochondrial depolarization, cytochrome c release, and caspase-3 activation. The LHS extract was effective in inhibiting the growth of all seven cancer cell lines tested. The IC(50) (50% inhibition concentrations, milligrams of raw material per milliliter) were in the range of 8.0-40.0 when the drug exposure time was 48 hours. The inhibitory action of the herbal extract was time- and dose-dependent. A significant decrease in activity was seen when the drug exposure time was shortened. Microscopic examination of the LN CaP and other cancer cell lines after treatment with LHS revealed morphologic changes that are typical of cells undergoing apoptosis. DNA fragmentation was obvious in the DNA latter assay and this confirmed the induction of apoptosis of the cancer cells by LHS. The mitochondria of the LHS-treated cells were found to undergo depolarization. Cytochrome c was released into the cytosol from the LHS-treated cells but not from the control cells. Cells treated with LHS showed cleavage of the full-length poly[ADP(ribose)] polymerase (PARP; 112 kd) to generate the 85-kd cleaved PARP fragment indicating the activation of caspase-3. LHS was able to induce apoptosis of all the tumor cell lines tested. The antiproliferation effect was dose- and time-dependent. The mitochondrion was found to be involved in the apoptosis induced by the LHS extract.

Retention of retinal axon collateral is responsible for induced ipsilateral retinotectal projections in adult goldfish.

PubMed

Sharma, S C; Tsai, C

1991-01-01

In normal goldfish, optic axons innervate only the contralateral optic tectum. When one eye was enucleated and the optic nerve of the other eye crushed, the regenerating optic axons innervated both optic tecta. We studied the presence of bilaterally projecting retinal ganglion cells by double retrograde cell labeling methods using Nuclear Yellow and True Blue dyes. About 10% of the retinal ganglion cells were double labeled and these cells were found throughout the retina. In addition, HRP application to the ipsilateral tectum revealed retrogradely-labeled retinal ganglion cells of all morphological types. These results suggest that induced ipsilateral projections are formed by regenerating axon collaterals and that all cell types are involved in the generation of normal mirror image typography.

Mesenchymal stem cells and their conditioned medium improve integration of purified induced pluripotent stem cell-derived cardiomyocyte clusters into myocardial tissue.

PubMed

Rubach, Martin; Adelmann, Roland; Haustein, Moritz; Drey, Florian; Pfannkuche, Kurt; Xiao, Bing; Koester, Annette; Udink ten Cate, Floris E A; Choi, Yeong-Hoon; Neef, Klaus; Fatima, Azra; Hannes, Tobias; Pillekamp, Frank; Hescheler, Juergen; Šarić, Tomo; Brockmeier, Konrad; Khalil, Markus

2014-03-15

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium.

Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.

PubMed

Ch Ho, Eric; Buckley, Katherine M; Schrankel, Catherine S; Schuh, Nicholas W; Hibino, Taku; Solek, Cynthia M; Bae, Koeun; Wang, Guizhi; Rast, Jonathan P

2016-10-01

The purple sea urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the larval stage. Sea urchin embryos and larvae are morphologically simple and transparent, providing an organism-wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune-associated gene markers for three cell types, including a perforin-like molecule, a scavenger receptor, a complement-like thioester-containing protein and the echinoderm-specific immune response factor 185/333. We elicit larval immune responses by (1) bacterial injection into the blastocoel and (2) seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm-specific immune response as well as those that are shared with other deuterostomes, including vertebrates.

Potential role of voltage-sensing phosphatases in regulation of cell structure through the production of PI(3,4)P2.

PubMed

Yamaguchi, Shinji; Kurokawa, Tatsuki; Taira, Ikuko; Aoki, Naoya; Sakata, Souhei; Okamura, Yasushi; Homma, Koichi J

2014-04-01

Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide-phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg-VSP) induces morphological feature of cell process outgrowths with round cell body in DF-1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg-VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild-type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg-VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1-derived pleckstrin homology (PH) domain suggested that Gg-VSP influenced the distribution of PI(3,4)P2 . These findings raise a possibility that one of the VSP's functions could be to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile. © 2013 Wiley Periodicals, Inc.

Important role of collective cell migration and nerve fiber density in the development of deep nodular endometriosis.

PubMed

Orellana, Renan; García-Solares, Javier; Donnez, Jacques; van Kerk, Olivier; Dolmans, Marie-Madeleine; Donnez, Olivier

2017-04-01

To evaluate deep nodular endometriotic lesions induced in baboons over 12 months and analyze collective cell migration and nerve fiber density. Morphologic and immunohistochemical analysis of endometriotic lesions induced in baboons over the course of 1 year. Academic research unit. Three female baboons (Papio anubis). Recovery of induced deep nodular endometriotic nodules from baboons. Evaluation of the morphology of glands by analysis of the center of lesions and the invasion front; immunohistochemical staining with Ki67, E-cadherin, and β-catenin for investigation of mitotic activity and cell-cell junctions, and with protein gene product 9.5 and nerve growth factor (NGF) for study of nerve fiber density (NFD). All (100%) of the lesions were invasive 1 year after induction, compared with 42.29% after 6 months. Glands from the invasion front showed significantly reduced thickness but significantly higher mitotic activity. E-Cadherin and β-catenin expression were similar between the center and front. NFD was significantly higher in lesions induced after 1 year than after 6 months, and NGF expression was significantly lower in 1-year lesions than in 6-month lesions. Nodular endometriotic lesions induced in the baboon model were found to be significantly more invasive and innervated after 12 months than after 6 months. The invasive phenotype was highly expressed in glands at the invasion front, and our study suggests that nerve fibers play a role in the development of lesions as observed in women. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of hydrocortisone on cell morphology in C6 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berliner, J.A.; Bennett, K.; de Vellis, J.

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases inmore » peripheral microfilaments are shown to be dependent on RNA and protein synthesis. The levels of protein co-electrophorescing with actin are not affected by hydrocortisone.« less

Vigilance or Subversion? Constitutive and Inducible M Cells in Mucosal Tissues.

PubMed

Lo, David D

2018-03-01

Microfold (M) cells are epithelial cells present in mucosal tissues and specialized for the capture of luminal microparticles and their delivery to underlying immune cells; thus, they are crucial participants in mucosal immune surveillance. Multiple phenotypic subsets of M cells have now been described, all sharing a unique apical morphology that provides clues to their ability to capture microbial particles. The existence of diverse M cell phenotypes, especially inflammation-inducible M cells, provides an intriguing puzzle: some variants may augment luminal surveillance to boost mucosal immunity, while others may promote microbial access to tissues. Here, I consider the unique induction requirements of each M cell subset and functional differences, highlighting the potentially distinct consequences in mucosal immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

PubMed Central

Ahmed, Ahmed Ashour; Mills, Anthony D.; Ibrahim, Ashraf E.K.; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E.; Iyer, N. Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D.; Earl, Helena M.; Laskey, Ronald A.; Caldas, Carlos; Brenton, James D.

2007-01-01

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability. PMID:18068629

Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

PubMed Central

MacAuley, A; Pawson, T

1988-01-01

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes. Images PMID:2846881

Altered Calcium Dynamics in Cardiac Cells Grown on Silane-Modified Surfaces

PubMed Central

Ravenscroft-Chang, Melissa S.; Stohlman, Jayna; Molnar, Peter; Natarajan, Anupama; Canavan, Heather E.; Teliska, Maggie; Stancescu, Maria; Krauthamer, Victor; Hickman, J.J.

2013-01-01

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca2+ because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca2+ imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca2+ concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca2+ changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca2+ dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems. PMID:19828193

Investigation of diseases through red blood cells' shape using photoacoustic response technique

NASA Astrophysics Data System (ADS)

Biswas, Deblina; Gorey, Abhijeet; Chen, Goerge C. K.; Sharma, Norman; Vasudevan, Srivathsan

2015-03-01

Photoacoustic (PA) imaging is a non-invasive real-time technique, widely applied to many biomedical imaging studies in the recent years. While most of these studies have been focussed on obtaining an image after reconstruction, various features of time domain signal (e.g. amplitude, width, rise and relaxation time) would provide very high sensitivity in detecting morphological changes in cells during a biological study. Different haematological disorders (e.g., sickle cell anaemia, thalassemia) exhibit significant morphological cellular changes. In this context, this study explores the possibility of utilizing the developed photoacoustic response technique to apply onto blood samples. Results of our preliminary study demonstrate that there is a significant change in signal amplitude due to change in concentration of the blood. Thus it shows the sensitivity of the developed photoacoustic technique towards red blood cell count (related to haematological disease like anaemia). Subsequently, morphological changes in RBC (i.e. swollen and shrunk compared to normal RBC) induced by hypotonic and hypertonic solutions respectively were also experimented. The result shows a distinct change in PA signal amplitude. This would serve as a diagnostic signature for many future studies on cellular morphological disorders.

Artemisinin conferred ERK mediated neuroprotection to PC12 cells and cortical neurons exposed to sodium nitroprusside-induced oxidative insult.

PubMed

Zheng, Wenhua; Chong, Cheong-Meng; Wang, Haitao; Zhou, Xuanhe; Zhang, Lang; Wang, Rikang; Meng, Qian; Lazarovici, Philip; Fang, Jiankang

2016-08-01

The production of nitric oxide (NO) is one of the primary mediators of ischemic damage, glutamate neurotoxicity and neurodegeneration and therefore inhibition of NO-induced neurotoxicity may be considered a therapeutic target for reducing neuronal cell death (neuroprotection). In this study, artemisinin, a well-known anti-malaria drug was found to suppress sodium nitroprusside (SNP, a nitric oxide donor)-induced cell death in the PC12 cells and brain primary cortical neuronal cultures. Pretreatment of PC12 cells with artemisinin significantly suppressed SNP-induced cell death by decreasing the extent of oxidation, preventing the decline of mitochondrial membrane potential, restoring abnormal changes in nuclear morphology and reducing lactate dehydrogenase release and inhibiting caspase 3/7 activities. Western blotting analysis revealed that artemisinin was able to activate extracellular regulated protein kinases (ERK) pathway. Furthermore, the ERK inhibitor PD98059 blocked the neuroprotective effect of artemisinin whereas the PI3K inhibitor LY294002 had no effect. Cumulatively these findings support the notion that artemisinin confers neuroprotection from SNP-induce neuronal cell death insult, a phenomenon coincidentally related to activation of ERK phosphorylation. This SNP-induced oxidative insult in PC12 cell culture model may be useful to investigate molecular mechanisms of NO-induced neurotoxicity and drug-induced neuroprotection, and to generate novel therapeutic concepts for ischemic disease treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

PubMed Central

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee

2013-08-01

Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalizedmore » to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.« less

β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

PubMed

Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

2015-06-01

β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells. © 2014 Wiley Periodicals, Inc.

Effect of hypoxia on the retina and superior colliculus of neonatal pigs

PubMed Central

Ruzafa, Noelia; Rey-Santano, Carmen; Mielgo, Victoria; Pereiro, Xandra; Vecino, Elena

2017-01-01

Purpose To evaluate the effect of hypoxia on the neonatal pig retina and brain, we analysed the retinal ganglion cells (RGCs) and neurons in the superior colliculus, as well as the response of astrocytes in both these central nervous system (CNS) structures. Methods Newborn pigs were exposed to 120 minutes of hypoxia, induced by decreasing the inspiratory oxygen fraction (FiO2: 10–15%), followed by a reoxygenation period of 240 minutes (FiO2: 21–35%). RGCs were quantified using Brn3a, a specific nuclear marker for these cells, and apoptosis was assessed through the appearance of active caspase-3. A morphometric analysis of the cytoskeleton of astrocytes (identified with GFAP) was performed in both the retina and superior colliculus. Results Hypoxia produced no significant change in the RGCs, although, it did induce a 37.63% increase in the number of active caspase-3 positive cells in the superior colliculus. This increase was particularly evident in the superficial layers of the superior colliculus, where 56.93% of the cells were positive for active caspase-3. In addition, hypoxia induced changes in the morphology of the astrocytes in the superior colliculus but not in the retina. Conclusions Hypoxia in the neonatal pig does not affect the retina but it does affect more central structures in the brain, increasing the number of apoptotic cells in the superior colliculus and inducing changes in astrocyte morphology. This distinct sensibility to hypoxia may pave the way to design specific approaches to combat the effects of hypoxia in specific areas of the CNS. PMID:28407001

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

PubMed

Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

2014-09-15

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis. Copyright © 2014 the American Physiological Society.

Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation.

PubMed

Nie, Jing; Mahato, Simpla; Zelhof, Andrew C

2015-02-03

Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure. We found that piperazine-N,N'-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging. We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research.

Streptozotocin produces oxidative stress, inflammation and decreases BDNF concentrations to induce apoptosis of RIN5F cells and type 2 diabetes mellitus in Wistar rats.

PubMed

Bathina, Siresha; Srinivas, Nanduri; Das, Undurti N

2017-04-29

Neurodegenerative disorders, such as deficits in learning, memory and cognition and Alzheimer's disease are associated with diabetes mellitus. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor and is known to possess anti-obesity, anti-diabetic actions and is believed to have a role in memory and Alzheimer's disease. To investigate whether STZ can reduce BDNF production by rat insulinoma (RIN5F) cells in vitro and decrease BDNF levels in the pancreas, liver and brain in vivo. Streptozotocin (STZ)-induced cytotoxicity to RIN5F cells in vitro and type 2 DM in Wistar rats was employed in the present study. Cell viability, activities of various anti-oxidants and secretion of BDNF by RIN5F cells in vitro were measured using MTT assay, biochemical methods and ELISA respectively. In STZ-induced type 2 DM rats: plasma glucose, interleukin-6 and tumor necrosis factor-α levels and BDNF protein expression in the pancreas, liver and brain tissues were measured. In addition, neuronal count and morphology in the hippocampus and hypothalamus areas was assessed. STZ-induced suppression of RIN5F cell viability was abrogated by BDNF. STZ suppressed BDNF secretion by RIN5F cells in vitro. STZ-induced type 2 DM rats showed hyperglycemia, enhanced plasma IL-6 and TNF-αlevels and reduced plasma and pancreas, liver and brain tissues (P < 0.001) and increased oxidative stress compared to untreated control. Hypothalamic and hippocampal neuron in STZ-treated animals showed a decrease in the number of neurons and morphological changes suggesting of STZ cytotoxicity. The results of the present study suggest that STZ is not only cytotoxic to pancreatic beta cells but also to hypothalamic and hippocampal neurons by inducing oxidative stress. STZ ability to suppress BDNF production by pancreas, liver and brain tissues suggests that impaired memory, learning, and cognitive dysfunction seen in diabetes mellitus could be due to BDNF deficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

XB130 translocation to microfilamentous structures mediates NNK-induced migration of human bronchial epithelial cells.

PubMed

Wu, Qifei; Nadesalingam, Jeya; Moodley, Serisha; Bai, Xiaohui; Liu, Mingyao

2015-07-20

Cigarette smoking contributes to the pathogenesis of chronic obstructive pulmonary disease and lung cancer. Nicotine-derived nitrosamine ketone (NNK) is the most potent carcinogen among cigarette smoking components, and is known to enhance migration of cancer cells. However, the effect of NNK on normal human bronchial epithelial cells is not well studied. XB130 is a member of actin filament associated protein family and is involved in cell morphology changes, cytoskeletal rearrangement and outgrowth formation, as well as cell migration. We hypothesized that XB130 mediates NNK-induced migration of normal human bronchial epithelial cells. Our results showed that, after NNK stimulation, XB130 was translocated to the cell periphery and enriched in cell motility-associated structures, such as lamellipodia, in normal human bronchial epithelial BEAS2B cells. Moreover, overexpression of XB130 significantly enhanced NNK-induced migration, which requires both the N- and C-termini of XB130. Overexpression of XB130 enhanced NNK-induced protein tyrosine phosphorylation and promoted matrix metalloproteinase-14 translocation to cell motility-associated cellular structures after NNK stimulation. XB130-mediated NNK-induced cell migration may contribute to airway epithelial repair; however, it may also be involved in cigarette smoking-related chronic obstructive pulmonary disease and lung cancer.

Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells.

PubMed

Dos Santos, Ancély F; Terra, Letícia F; Wailemann, Rosangela A M; Oliveira, Talita C; Gomes, Vinícius de Morais; Mineiro, Marcela Franco; Meotti, Flávia Carla; Bruni-Cardoso, Alexandre; Baptista, Maurício S; Labriola, Leticia

2017-03-15

Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm 2 . We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

[Morphological structure of suprarenal glands in experimental vibration-induced pathology].

PubMed

Kapanadze, N A; Abzianidze, E N; Sumbadze, Ts M; Korkiia, I I; Amiranidze, M V

2009-01-01

Technical progress has caused development of vibration-induced pathology, which is determined by harmful factors or environmental effects. The harmful factors include physical factors--noise, mechanical vibrations, low temperature, high humidity of the air and incorrect lighting. The aim of our study was the investigation of morphological changes in suprarenal glands under condition of vibration-induced pathology. The experiment was conducted on 20 grown-up white male rats weighting 180-200 g. The animals were daily under an hour vibration during 2 months. The vibration frequency was modulated by means of a general vibration. After an experiment, animals were decapitated in condition of general anesthesia. The experiment revealed important changes in the morphological structure of suprarenal glands. The vibration pathology causes following changes: vessels' and sinusoid capillaries' uneven widening, develop the infiltrate cells, bleeding areas, necrosis and other changes. Based on above-stated it is supposed that technical progress and introduction of new technologies is one of the risk factors, which can cause neurohumoral disorders.

A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, So Jung; Park, Young Jun; Shin, Ji Hyun

2011-05-13

Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactivemore » chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.« less

Computerized analysis of cytology and fluorescence in situ hybridization (FISH) in induced sputum for lung cancer detection.

PubMed

Guber, Alexander; Greif, Joel; Rona, Roni; Fireman, Elizabeth; Madi, Lea; Kaplan, Tal; Yemini, Zipi; Gottfried, Maya; Katz, Ruth L; Daniely, Michal

2010-10-25

Lung cancer results from a multistep process, whereby genetic and epigenetic alterations lead to a malignant phenotype. Somatic mutations, deletions, and amplifications can be detected in the tumor itself, but they can also be found in histologically normal bronchial epithelium as a result of field cancerization. The present feasibility study describes a computer-assisted analysis of induced sputum employing morphology and fluorescence in situ hybridization (target-FISH), using 2 biomarkers located at chromosomes 3p22.1 and 10q22.3. Induced sputum samples were collected using a standardized protocol from 12 patients with lung cancer and from 15 healthy, nonsmoking controls. We used an automated scanning system that allows consecutive scans of morphology and FISH of the same slide. Cells derived for the lower airways were analyzed for the presence of genetic alterations in the 3p22.1 and 10q22.3 loci. The cutoff for a positive diagnosis was defined as >4% of cells showing genetic alterations. Eleven of 12 lung cancer patients and 12 of 15 controls were identified correctly, giving an overall sensitivity and specificity of 91.66% and 80%, respectively. This study describes a new technology for detecting lung cancer noninvasively in induced sputum via a combination of morphology and FISH analysis (target-FISH) using computer-assisted technology. This approach may potentially be utilized for mass screening of high-risk populations. © 2010 American Cancer Society.

Physicochemical Constraints on the Distribution of Benthic Foraminiferal Cell Morphology in the Modern Ocean

NASA Astrophysics Data System (ADS)

Keating-Bitonti, C.; Payne, J.

2016-02-01

Patterns in the sizes and shapes of marine organisms often occur across latitude and water depth gradients as a function of metabolic constraints dictated by the physical environment. However, the environmental factors that maintain these gradients in morphology remain incompletely understood because several oceanographic variables of biological importance are intimately correlated, such as temperature, dissolved oxygen concentration, particulate organic carbon (POC) flux, and carbonate saturation. Benthic foraminifera, a diverse group of single-celled protists that occur in nearly all marine environments, provide an ideal opportunity to test statistically among the various hypothesized environmental controls on cell morphology. Here, we use over 7,000 occurrences of 541 species of recent benthic foraminifera that span more than 60 degrees of latitude and 1,600 meters of water depth around the North American continental margin to assess the relative contributions of temperature, oxygen availability, carbonate saturation, and POC flux on their size and volume-to-surface area ratio in the modern ocean. Seawater temperature and dissolved oxygen concentrations best predict both measures of benthic foraminiferal cell morphology from the North American continental margin. These same variables also explain morphological variations from the Pacific continental margin in isolation, but dissolved oxygen is absent from the best model for the Atlantic. Because our results concur with predictions from first principles of cell physiology, we interpret these findings to reflect the physiological selective pressures on cell morphology as determined by the physical environment. Moreover, these findings suggest that warming waters and the expansion of hypoxic zones associated with anthropogenic-induced climate change are more likely to impact benthic foraminiferal communities than changes in primary productivity or ocean acidification.

The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology

PubMed Central

Murk, Kai; Blanco Suarez, Elena M.; Cockbill, Louisa M. R.; Banks, Paul; Hanley, Jonathan G.

2013-01-01

Summary Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult. PMID:23843614

[Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

PubMed

Ji, Ying-Lei; Yan, Jun; Wang, Yan-Sha; Liu, Yi-Chang; Gu, Zhen-Yong

2014-02-01

To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

Aeromonas hydrophila exotoxin induces cytoplasmic vacuolation and cell death in VERO cells.

PubMed

Di Pietro, Angela; Picerno, Isa; Visalli, Giuseppa; Chirico, Cristina; Spataro, Pasquale; Cannavò, Giuseppe; Scoglio, Maria E

2005-07-01

Many organisms are able to cause cell vacuolation, but it is unclear if this can be considered a step of apoptosis or necrosis, or a distinct form of cell death. In this study VERO cells were used to evaluate the relationship between vacuolation and cell death pattern caused by exotoxins produced by environmental strains of A. hydrophila. Cell damage has been evaluated morphologically as well as biochemically. Cytotoxic and vacuolating titres were strictly correlated and the vacuolation has to be considered an early indicator of cytotoxicity that causes cell apoptosis or necrosis in relation to the dose. Signs of apoptosis (chromatin condensation and blebbing) were observed at low concentration and TGase activity, referable to apoptosis induction, confirms morphological observations. In fact, putrescine incorporation was related both to cytotoxin concentration and time of incubation. Moreover, the observed doubling cells with necrotic features permit us to suppose that cell sensitivity and death pattern could change during the different phases of cellular cycle.

From “ES-like” cells to induced pluripotent stem cells: A historical perspective in domestic animals

PubMed Central

Koh, Sehwon; Piedrahita, Jorge A.

2013-01-01

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat, however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as “ES-like” cells due to similar morphological characteristics to mouse ESCs but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of induced pluripotent stem cells. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of induced pluripotent stem cells. PMID:24274415

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

PubMed

Okeyo, Kennedy O; Tanabe, Maiko; Kurosawa, Osamu; Oana, Hidehiro; Washizu, Masao

2018-04-01

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development. © 2018 Japanese Society of Developmental Biologists.

[The anti-tumour effect of Wuxing soup and its mechanism in inducing apoptosis of tumour cells mediated by calcium].

PubMed

Mo, Fei; Hu, Jing-Ying; Gan, Yu; Zhao, Yang-Xing; Zhao, Xin-Tai

2008-09-01

To confirm the anti-cancer effect and mechanism of Wuxing soup. Inhibition of cellular growth under Wuxing soup treatment was observed by MTT; Apoptosis was detected by gel electrophoresis, transmission electron microscopy and FACS; The concentration of calcium was measured by fluorescence probe. After SGC-7901 cell being treated by Wuxing soup, it showed that: 1) Wuxing soup could specifically inhibit cancer cells proliferation in a time and dose dependent manner; 2) Typical apoptotic morphological changes and DNA ladder of SGC-7901 cells were observed; 3) calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. Wuxing soup has an effective inhibition on cancer cells, and can induce SGC-7901 cells to apoptosis by calcium.

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

PubMed Central

Saitakis, Michael; Dogniaux, Stéphanie; Goudot, Christel; Bufi, Nathalie; Asnacios, Sophie; Maurin, Mathieu; Randriamampita, Clotilde; Asnacios, Atef; Hivroz, Claire

2017-01-01

T cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4+ T cell functions are affected by stiffness within the physiological Young’s modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly. DOI: http://dx.doi.org/10.7554/eLife.23190.001 PMID:28594327

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

PubMed

Horibata, Sachi; Rogers, Katherine E; Sadegh, David; Anguish, Lynne J; McElwee, John L; Shah, Pragya; Thompson, Paul R; Coonrod, Scott A

2017-05-26

Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

The effect of Taurolidine on adherent and floating subpopulations of melanoma cells.

PubMed

Shrayer, D P; Lukoff, H; King, T; Calabresi, P

2003-04-01

The annual incidence of malignant melanoma is estimated at 10-12 per 100000 inhabitants in countries of Central Europe and the US, with more recent estimates showing a dramatic upward trend. Taurolidine (Carter/Wallace, Cranberry, NJ) is a novel, potentially effective, antitumor chemotherapeutic agent. We hypothesized that Taurolidine could inhibit the growth, induce apoptosis, affect the cell cycle and change morphology of melanoma cells. We expected this process to be different in adherent and floating subpopulations that may be reflective of solid tumors and their metastases. Analysis of MNT-1 human and B16F10 murine melanoma cells showed that at 72 h the IC(50) of Taurolidine was 25.4+/-3.3 microM for MNT-1 human melanoma cells and 30.9+/-3.6 microM for B16F10 murine melanoma cells. Taurolidine induced DNA fragmentation of melanoma cells in a dose-dependent manner. Taurolidine (75 and 100 microM) induced 52-97% Annexin-V binding (apoptosis), respectively. Evaluation of cell cycle after 72 h exposure to Taurolidine (0-100 microM) revealed that the percentage of melanoma cells in S phase increased from 27 to 40% in the adherent subpopulation and from 33 to 49% in the floating subpopulation. Phase contrast microscopy revealed a marked swelling of melanoma cells and decreasing cell numbers in adherent subpopulation starting at 24 h with 25 microM Taurolidine. Shrinkage of cells dominated at 75-100 microM Taurolidine. Using Cytospin assay in the floating population, we observed swelling of melanoma cells induced by 25-100 micro Taurolidine and appearance of giant (multinuclear) forms resulting from exposure to 75-100 micro Taurolidine. Some floating cells with normal morphology were observed with low concentrations of Taurolidine (0-25 microM). These data show that effects of Taurolidine may be different in adherent and floating subpopulations of melanoma cells. More importantly, floating subpopulations that may contain some viable melanoma cells, may be reflective of potential metastasis after treatment of solid tumors in vivo.

The role of drebrin in glioma migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terakawa, Yuzo; Department of Neurosurgery, Osaka City University Graduate School of Medicine, Osaka; Agnihotri, Sameer

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet beenmore » fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility. - Highlights: ► Drebrin is an actin-binding protein aberrantly expressed in several cancers. ► Role of drebrin in glioma cell morphology and motility is previously unknown. ► We demonstrate that drebrin is expressed in 40% of glioblastoma specimens. ► Drebrin plays a significant role in modulating glioma cell migration and invasion.« less

Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells.

PubMed

Lee, H-Z; Liu, W-Z; Hsieh, W-T; Tang, F-Y; Chung, J-G; Leung, Henry W-C

2009-03-01

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.

Histaminergic regulation of NK-cells: protection against monocyte-induced apoptosis.

PubMed

Hansson, M; Asea, A; Hermodsson, S; Hellstrand, K

1996-08-01

Human natural killer (NK) cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology and degradation of DNA into oligonucleosomal fragments. The monocyte-induced apoptosis in NK-cells was prevented by the biogenic amine histamine at concentrations exceeding 0.1 microM. The protective effect of histamine was blocked by the H2-receptor (H2R) antagonist ranitidine but not by AH202399 A, a chemical control to ranitidine devoid of H2R affinity. It is concluded that histaminergic mechanisms may serve to protect NK cells from damage inflicted by products of the oxidative metabolism of monocytes.

Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma.

PubMed

Higashi, Mayumi; Kolla, Venkatadri; Iyer, Radhika; Naraparaju, Koumudi; Zhuang, Tiangang; Kolla, Sriharsha; Brodeur, Garrett M

2015-08-07

Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

Cytomorphological effects of mitomycin C on urothelial cells: eosinophils may be clue to the drug-induced changes.

PubMed

Guler Simsek, Gulcin; Vargol, Erdem; Simsek, Hulya

2014-01-01

Cytomorphological changes of mitomycin C on urothelial cells may be misinterpreted as a neoplastic process. A 60-year old male patient who was given an eight-week course of intravesical mitomycin C due to non-invasive low grade transitional cell carcinoma. During his follow-up care, the findings of a urine cytology exam were as follows: nuclear enlargement of cells, wrinkled nuclear membranes, little hyperchromasia, pleomorphism, abnormal nuclear morphology and disordered orientation of the urothelium. Furthermore, there were eosinophils nearby the atypical cells. This report aimed at reminding the cytomorphologic changes of mitomycin C may be misinterpreted as carcinoma, so the presence of eosinophils is required to predict the drug-induced changes.

A phosphorescent rhenium(I) histone deacetylase inhibitor: mitochondrial targeting and paraptosis induction.

PubMed

Ye, Rui-Rong; Tan, Cai-Ping; Lin, Yan-Nan; Ji, Liang-Nian; Mao, Zong-Wan

2015-05-14

In this report, we designed a histone deacetylase-targeted phosphorescent Re(I) complex ReLMito. Colocalization studies suggested that ReLMito could specially localize to mitochondria. We also demonstrated that ReLMito could induce paraptosis in cancer cells. These features endowed the complex with potential to induce and monitor mitochondrial morphological changes during the paraptosis simultaneously.

Apoptosis: a basic pathological reaction of injured neonatal muscle.

PubMed

Fidziańska, A; Kamińska, A

1991-01-01

A light and electron microscopic study of immature muscle cell degeneration induced by bupivacaine (BPVC) was performed. The pattern of muscle cell death is related to muscle maturity; in newborn rats, cell death has the morphology of apoptosis, whereas in the older animals muscle cell death resembles cell necrosis and the ultrastructural feature of these changes are essentially the same as those described in adult muscle. The ability to undergo apoptosis in response to a pathological stimulus is a common effector mechanism of immature muscle.

Differential effects of selenite and selenate on human melanocytes, keratinocytes, and melanoma cells.

PubMed

Bandura, Laura; Drukala, Justyna; Wolnicka-Glubisz, Agnieszka; Björnstedt, Mikael; Korohoda, Wlodzimierz

2005-04-01

Among the substances that attracted the attention of oncologists in recent years are selenium-containing compounds, both inorganic and organic. Several epidemiological studies have shown an inverse correlation between selenium intake and cancer incidence. In the experiments reported here, we compared the effects of 2 inorganic selenium-containing salts that differed in the level of selenium oxidation, selenite IV and selenate VI. We tested the effects of these 2 compounds on cell survival and growth, cell cycle processing, cell morphology, cytoskeleton, and lipid peroxidation in 3 human skin cell types: normal keratinocytes, melanocytes, and human melanoma cell line HTB140. The different effects of selenite and selenate on the viability, growth, and morphology of normal cells and tumor cells are reported and provide a base for future research and treatment of some neoplastic diseases. The attention is paid to cell apoptosis induced by selenite and not by selenate, and the effects of tested substances on thioredoxin reductase system are postulated.

Alleviation of hyperglycemia in diabetic rats by intraportal injection of insulin-producing cells generated from surgically resected human pancreatic tissue.

PubMed

Shyu, Jia-Fwu; Wang, Hwai-Shi; Shyr, Yi-Ming; Wang, Shin-E; Chen, Chia-Hsiang; Tan, Joo-Shin; Lin, Meng-Feng; Hsieh, Po-Shiuan; Sytwu, Huey-Kang; Chen, Tien-Hua

2011-03-01

Although islet transplantation holds promise for the treatment of diabetes, the scarcity of donor tissue remains a major drawback. The aim of this study is to generate insulin-producing cells from adult human pancreatic cells isolated from surgically resected pancreatic tissue. To isolate pancreatic endocrine precursor cells from 57 surgically resected pancreases, the cells were cultured and propagated in conditioned medium after which they were differentiated in Matrigel. The resultant cells were characterized using morphology, immunofluorescent studies, expression of differentiated pancreatic islet-specific genes using quantitative reverse transcription-PCR, and glucose-induced insulin secretion through analysis of C-peptide secretion. The relationships between propagation of insulin-producing cells and clinical variables of the donor were also analyzed. Finally, insulin-producing cell function was examined in streptozotocin-induced diabetic rats. Pancreatic endocrine precursor cells were successfully cultured; insulin-producing cells cultured from soft pancreas parenchyma had a significantly higher success rate. Morphological examination revealed islet-like cluster formation upon transfer to Matrigel. The presence of the neural stem cell marker nestin, duct cell marker cytokeratin 19, and endocrine cell markers C-peptide and pancreatic and duodenal homeobox 1, was also observed. In addition, glucose-stimulated C-peptide release was significantly increased in the insulin-producing cells. Furthermore, in diabetic rats, transplantation of insulin-producing cells reduced hyperglycemia. Isolated pancreatic endocrine precursor cells from surgically resected pancreatic tissue differentiated into insulin-producing cells and showed characteristics of functional endocrine cells. Thus, surgically resected pancreatic tissue may represent an alternative source of functional insulin-producing cells.

Epidermal growth factor enhances osteogenic differentiation of dental pulp stem cells in vitro.

PubMed

Del Angel-Mosqueda, Casiano; Gutiérrez-Puente, Yolanda; López-Lozano, Ada Pricila; Romero-Zavaleta, Ricardo Emmanuel; Mendiola-Jiménez, Andrés; Medina-De la Garza, Carlos Eduardo; Márquez-M, Marcela; De la Garza-Ramos, Myriam Angélica

2015-09-03

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) play an important role in extracellular matrix mineralization, a complex process required for proper bone regeneration, one of the biggest challenges in dentistry. The purpose of this study was to evaluate the osteogenic potential of EGF and bFGF on dental pulp stem cells (DPSCs). Human DPSCs were isolated using CD105 magnetic microbeads and characterized by flow cytometry. To induce osteoblast differentiation, the cells were cultured in osteogenic medium supplemented with EGF or bFGF at a low concentration. Cell morphology and expression of CD146 and CD10 surface markers were analyzed using fluorescence microscopy. To measure mineralization, an alizarin red S assay was performed and typical markers of osteoblastic phenotype were evaluated by RT-PCR. EGF treatment induced morphological changes and suppression of CD146 and CD10 markers. Additionally, the cells were capable of producing calcium deposits and increasing the mRNA expression to alkaline phosphatase (ALP) and osteocalcin (OCN) in relation to control groups (p < 0.001). However, bFGF treatment showed an inhibitory effect. These data suggests that DPSCs in combination with EGF could be an effective stem cell-based therapy for bone tissue engineering applications in periodontics and oral implantology.

Oxidative stress induces senescence in human mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandl, Anita; Meyer, Matthias; Bechmann, Volker

Mesenchymal stem cells (MSCs) contribute to tissue repair in vivo and form an attractive cell source for tissue engineering. Their regenerative potential is impaired by cellular senescence. The effects of oxidative stress on MSCs are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of MSCs, subject to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by rtPCR. Sub-lethal doses of oxidative stress reduced proliferation rates and induced senescent-morphological features and senescence-associated {beta}-galactosidase positivity. Prolongedmore » low dose treatment with hydrogen peroxide had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. Following acute oxidant insult p21 was up-regulated prior to returning to initial levels. TRF1 was significantly reduced, TRF2 showed a slight up-regulation. SIRT1 and XRCC5 were up-regulated after oxidant insult and expression levels increased in aging cells. Compared to fibroblasts and chondrocytes, MSCs showed an increased tolerance to oxidative stress regarding proliferation, telomere biology and gene expression with an impaired stress tolerance in aged cells.« less

Model of Oxygen and Glucose Deprivation in PC12 Cells and Detection of HSP70 Protein

NASA Astrophysics Data System (ADS)

He, Jinting; Yang, Le; Shao, Yankun

2018-01-01

Objective: PC12 cell was used to set up a ischemia model by OGD and detected HSP70 protein. Methods: Use of PC12 cells induced by NGF stimulation into nerve cells, oxygen and glucose deprivation to build the nerve cells of oxygen and glucose deprivation model; using Western blot analysis of PC12 cells into neuron-like cells and oxygen-glucose deprivation model established. Results: The application of a final concentration of 50 ng / ml of NGF in DMEM complete mediumPC12 cells showed a typical neuronal morphology with the increase in cell culture time. NGF culture time showed a positive correlation, the establishment of oxygen and glucose deprivation (OGD) training environment, the OGD after nerve element appears different degrees of damage, OGD can effectively induce the expression of HSP70. Conclusion: PC12 cell transformed into cells by NGF; the cell model of OGD was established.

Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.

PubMed

Sun, Zhen; Gou, Wenyu; Kim, Do-Sung; Dong, Xiao; Strange, Charlie; Tan, Yu; Adams, David B; Wang, Hongjun

2017-11-01

The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 10 5 or 1 × 10 6 GFP + ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP + ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP + ASCs migrated to pancreas and differentiated into amylase + cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase + cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.

PubMed

Yutani, Masahiro; Hashimoto, Yukie; Ogita, Akira; Kubo, Isao; Tanaka, Toshio; Fujita, Ken-ichi

2011-11-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625 mM. A hyperosmotic condition (1.2 M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. Copyright © 2011 John Wiley & Sons, Ltd.

Morphometrical and Morphological Alterations of Human Leukocytes Exposed to 1.8 GHz Electromagnetic Radiations: In Vitro Protective Effects Induced by Polyphenols.

PubMed

Biagi, Pier Francesco; Boffola, Sara; Stefanelli, Riccardo; Schiavulli, Luigi; Ligonzo, Teresa; Casamassima, Giuseppe; Pappagallo, Maria Teresa; Kawaguchi, Kiichiro; Takimoto, Hiroaki; Kumazawa, Yoshio; Fontana, Sergio; Laforgia, Flavia; Russo, Matteo Antonio; Magrone, Thea; Jirillo, Emilio

2016-01-01

Our recent findings have demonstrated that electromagnetic radiations (EMR) (1.8 GHz radiofrequency) are able to in vitro induce morphometrical and morphological modifications of human leukocytes from normal donors. In view of the evidence that polyphenols exert many beneficial effects on plants, animals and humans, leukocytes from human peripheral blood were pre-treated for 1 h with two polyphenol preparations from red grape before EMR exposure (1.8 GHz). Our data will show that polyphenol pre-treatment reverts to normality the morphology of irradiated leukocytes in comparison to irradiated cells only. Conversely, leukocyte morphometry seems to be not affected by this treatment. Here, we demonstrate that polyphenols are also able to normalize leukocyte morphology per se altered before as well as after irradiation. Finally, a working hypothesis aimed at clarifying the protective mechanisms exerted by polyphenols on irradiated leukocytes will be illustrated. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

PubMed Central

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field. PMID:26317353

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

PubMed

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

NF-κB inhibitors that prevent foam cell formation and atherosclerotic plaque accumulation.

PubMed

Plotkin, Jesse D; Elias, Michael G; Dellinger, Anthony L; Kepley, Christopher L

2017-08-01

The transformation of monocyte-derived macrophages into lipid-laden foam cells is one inflammatory process underlying atherosclerotic disease. Previous studies have demonstrated that fullerene derivatives (FDs) have inflammation-blunting properties. Thus, it was hypothesized that FD could inhibit the transformation process underlying foam cell formation. Fullerene derivatives inhibited the phorbol myristic acid/oxidized low-density lipoprotein-induced differentiation of macrophages into foam cells as determined by lipid staining and morphology.Lipoprotein-induced generation of TNF-α, C5a-induced MC activation, ICAM-1 driven adhesion, and CD36 expression were significantly inhibited in FD treated cells compared to non-treated cells. Inhibition appeared to be mediated through the NF-κB pathway as FD reduced expression of NF-κB and atherosclerosis-associated genes. Compared to controls, FD dramatically inhibited plaque formation in arteries of apolipoprotein E null mice. Thus, FD may be an unrecognized therapy to prevent atherosclerotic lesions via inhibition of foam cell formation and MC stabilization. Copyright © 2017 Elsevier Inc. All rights reserved.

Normal Fibroblasts Induce E-Cadherin Loss and Increase Lymph Node Metastasis in Gastric Cancer

PubMed Central

Xu, Wen; Hu, Xinlei; Chen, Zhongting; Zheng, Xiaoping; Zhang, Chenjing; Wang, Gang; Chen, Yu; Zhou, Xinglu; Tang, Xiaoxiao; Luo, Laisheng; Xu, Xiang; Pan, Wensheng

2014-01-01

Background A tumor is considered a heterogeneous complex in a three-dimensional environment that is flush with pathophysiological and biomechanical signals. Cell-stroma interactions guide the development and generation of tumors. Here, we evaluate the contributions of normal fibroblasts to gastric cancer. Methodology/Principal Findings By coculturing normal fibroblasts in monolayers of BGC-823 gastric cancer cells, tumor cells sporadically developed short, spindle-like morphological characteristics and demonstrated enhanced proliferation and invasive potential. Furthermore, the transformed tumor cells demonstrated decreased tumor formation and increased lymphomatic and intestinal metastatic potential. Non-transformed BGC-823 cells, in contrast, demonstrated primary tumor formation and delayed intestinal and lymph node invasion. We also observed E-cadherin loss and the upregulation of vimentin expression in the transformed tumor cells, which suggested that the increase in metastasis was induced by epithelial-to-mesenchymal transition. Conclusion Collectively, our data indicated that normal fibroblasts sufficiently induce epithelial-to-mesenchymal transition in cancer cells, thereby leading to metastasis. PMID:24845259

Tocotrienol prevents AAPH-induced neurite degeneration in neuro2a cells.

PubMed

Fukui, Koji; Sekiguchi, Hidekazu; Takatsu, Hirokatsu; Koike, Taisuke; Koike, Tatsuro; Urano, Shiro

2013-01-01

Reactive oxygen species induce neurite degeneration before inducing cell death. However, the degenerative mechanisms have not yet been elucidated. While tocotrienols have a known neuroprotective function, the underlying mechanism remains unclear and may or may not involve antioxidant action. In this study, we hypothesize that free radical-derived membrane injury is one possible mechanism for inducing neurite degeneration. Therefore, we examined the potential neuroprotective effect of tocotrienols mediated through its antioxidant activity. Mouse neuroblastoma neuro2a cells were used to examine the effect of the water-soluble free radical generator 2,2'-azobis(2-methylpropionamide) dihydrochloride (AAPH) on neurite dynamics. After 24 hours of AAPH treatment, cell viability, neurite number, and the number of altered neurites were measured in the presence or absence of α-tocotrienol. Treatment of neuro2a cells with a low concentration of AAPH induces neurite degeneration, but not cell death. Treatment with 5 µM α-tocotrienol significantly inhibited neurite degeneration in AAPH-treated neuro2a cells. Furthermore, morphological changes in AAPH-treated neuro2a cells were similar to those observed with colchicine treatment. α-Tocotrienol may scavenge AAPH-derived free radicals and alkoxyl radicals that are generated from AAPH-derived peroxyl radicals on cell membranes. Therefore, α-tocotrienol may have a neuroprotective effect mediated by its antioxidant activity.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

PubMed Central

2013-01-01

Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

PubMed

Andres, Devon; Keyser, Brian M; Petrali, John; Benton, Betty; Hubbard, Kyle S; McNutt, Patrick M; Ray, Radharaman

2013-04-18

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Imaging the Effects of Prostaglandin Analogues on Cultured Trabecular Meshwork Cells by Coherent Anti-Stokes Raman Scattering

PubMed Central

Lei, Tim C.; Masihzadeh, Omid; Kahook, Malik Y.; Ammar, David A.

2013-01-01

Purpose. The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. Methods. Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. Results. Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. Conclusions. CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo. PMID:23900606

Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wenbin; Chen, Yaomin; Yang, Qun

2010-06-25

Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 {+-} 0.1 {sup o}C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. Withmore » silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.« less

Protective properties of Salvia lavandulifolia Vahl. essential oil against oxidative stress-induced neuronal injury.

PubMed

Porres-Martínez, María; González-Burgos, Elena; Carretero, M Emilia; Gómez-Serranillos, M Pilar

2015-06-01

Salvia lavandulifolia Vahl., known as "Spanish sage", has potential value in dementia for its sedative, antioxidant, anti-inflammatory and anticholinesterase properties. This work aimed to evaluate the in vitro neuroprotective activity of S. lavandulifolia essential oils, obtained from plants at different phenological stages (vegetative and flowering phases) and plants grown at different densities, against H2O2-induced oxidative stress in PC12 cells. The effect on cell viability and morphology, lipid peroxidation, GSH/GSSG ratio, intracellular ROS levels, antioxidant enzymes (CAT, SOD, GR, GPx, HO-1) and apoptotic enzymes was investigated. Comparing with H2O2-treated PC12 cells, pretreatments with essential oil samples attenuated morphological changes and loss of cell viability, decreased MDA levels and intracellular ROS production and increased GSH/GSSG ratio. Moreover, Spanish sage increased antioxidant status as evidenced in an increase of antioxidant enzyme activity and protein expression and inhibited caspase-3 activity. Furthermore, our results suggest that S. lavandulifolia essential oils are able to activate Nrf2 transcription factor. Collectively, the sample of essential oil obtained with the highest densities of planting and at flowering phase exerted the major neuroprotective activity. Our findings demonstrate that S. lavandulifolia essential oils may have therapeutic value for the prevention and treatment of neurodegenerative diseases associated with oxidative stress-induced neuronal injury. Copyright © 2015 Elsevier Ltd. All rights reserved.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D.

PubMed

Tymchenko, Nina; Nilebäck, Erik; Voinova, Marina V; Gold, Julie; Kasemo, Bengt; Svedhem, Sofia

2012-12-01

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.

Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

PubMed

Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

2016-10-01

The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P < 0.05). Spectrophotometric assay showed that gem significantly increased caspase-3 and -9 activity in PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

Simultaneous spin-coating and solvent annealing: Manipulating the active layer morphology to a power conversion efficiency of 9.6% in polymer solar cells

DOE PAGES

He, Zhicai; Liu, Feng; Wang, Cheng; ...

2015-08-20

Here, we developed a simultaneous spin-coating/solvent-annealing process and demonstrated morphology optimization for PTB7 based organic photovoltaics. This novel processing method enhances the edge-on crystalline content in thin films and induces the formation of weak PCBM aggregates. As a result, the efficiency of polymer solar cells increased from 9.2% to a certified high efficiency of 9.61%, owing to an enhanced short-circuit current (J sc, 18.4 mA cm –2vs. 17. 5 mA cm –2) and an improved fill factor.

2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study.

PubMed

Zitta, Karina; Peeters-Scholte, Cacha; Sommer, Lena; Gruenewald, Matthias; Hummitzsch, Lars; Parczany, Kerstin; Steinfath, Markus; Albrecht, Martin

2017-01-01

Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy (HIE), but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB) superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C), and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml). Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml) reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho Rad17. In summary, addition of 2-IB during hypothermia is able to attenuate hypoxia-induced neuronal cell damage in vitro . Combination treatment of hypothermia with 2-IB could be a promising strategy to reduce hypoxia-induced neuronal cell damage and should be considered in further animal and clinical studies.

Rotenone and Paraquat do not Directly Activate Microglia or Induce Inflammatory Cytokine Release

PubMed Central

Klintworth, Heather; Garden, Gwenn; Xia, Zhengui

2009-01-01

Both epidemiological and pathological data suggest an inflammatory response including microglia activation and neuro-inflammation in the Parkinsonian brain. Treatments with lipopolysacchride (LPS), rotenone and paraquat have been used as models for Parkinson’s disease, as they cause dopaminergic neuron degeneration in culture and in animals. Recent studies have suggested that rotenone and paraquat induce neuro-inflammation, however, it is not known if they can directly activate microglia. Here, we use primary cultured microglia to address this question. Microglia activation was analyzed by morphological changes and release of nitric oxide and inflammatory cytokines. Treatment with LPS was used as a positive control. While LPS induced morphological changes characteristic of microglial activation and release of nitric oxide and inflammatory cytokines, rotenone and paraquat did not. Our results suggest that paraquat and rotenone do not act directly on microglia and that neuro-inflammation and microglial activation in animals treated with these agents is likely non-cell autonomous, and may occur as a result of dopaminergic neuron damage or factors released by neurons and other cells. PMID:19559752

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

PubMed

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

PubMed

Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

2004-07-01

The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

Impairment of endoplasmic reticulum is involved in β-cell dysfunction induced by microcystin-LR.

PubMed

Zhao, Yanyan; Cao, Qing; He, Yaojia; Xue, Qingju; Xie, Liqiang; Yan, Yunjun

2017-04-01

Microcystins (MCs) widely distributed in freshwaters have posed a significant risk to human health. Previous studies have demonstrated that exposure to MC-LR impairs pancreatic islet function, however, the underlying mechanisms still remain unclear. In the present study, we explored the role of endoplasmic reticulum (ER) impairment in β-cell dysfunction caused by MC-LR. The result showed that MC-LR modified ER morphology evidenced by increased ER amount and size at low doses (15, 30 or 60 μM) and vacuolar and dilated ER ultrastructure at high doses (100 or 200 μM). Also, insulin content showed increased at 15 or 30 μM but declined at 60, 100, or 200 μM, which was highly accordant with ER morphological alteration. Transcriptomic analysis identified a number of factors and several pathways associated with ER protein processing, ER stress, apoptosis, and diabetes mellitus in the cells treated with MC-LR compared with non-treated cells. Furthermore, MC-LR-induced ER stress significantly promoted the expression of PERK/eIF2α and their downstream targets (ATF4, CHOP, and Gadd34), which indicates that PERK-eIF2α-ATF4 pathway is involved in MC-LR-induced insulin deficiency. These results suggest that ER impairment is an important contributor to MC-LR-caused β-cell failure and provide a new insight into the association between MCs contamination and the occurrence of human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adhesion modification of neural stem cells induced by nanoscale ripple patterns

NASA Astrophysics Data System (ADS)

Pedraz, P.; Casado, S.; Rodriguez, V.; Giordano, M. C.; Buatier de Mongeot, F.; Ayuso-Sacido, A.; Gnecco, E.

2016-03-01

We have studied the influence of anisotropic nanopatterns (ripples) on the adhesion and morphology of mouse neural stem cells (C17.2) on glass substrates using cell viability assay, optical microscopy and atomic force microscopy. The ripples were produced by defocused ion beam sputtering with inert Ar ions, which physically remove atoms from the surface at the energy of 800 eV. The ripple periodicity (∼200 nm) is comparable to the thickness of the cytoplasmatic microspikes (filopodia) which link the stem cells to the substrate. All methods show that the cell adhesion is significantly lowered compared to the same type of cells on flat glass surfaces. Furthermore, the AFM analysis reveals that the filopodia tend to be trapped parallel or perpendicular to the ripples, which limits the spreading of the stem cell on the rippled substrate. This opens the perspective of controlling the micro-adhesion of stem cells and the orientation of their filopodia by tuning the anisotropic substrate morphology without chemical reactions occurring at the surface.

Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

NASA Astrophysics Data System (ADS)

Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

2015-11-01

Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis.

PubMed

Fernández-Arjona, María Del Mar; Grondona, Jesús M; Granados-Durán, Pablo; Fernández-Llebrez, Pedro; López-Ávalos, María D

2017-01-01

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation.Hierarchical cluster and principal components analysis allow morphological classification of microglia.Brain location of microglia is a relevant factor.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis

PubMed Central

Fernández-Arjona, María del Mar; Grondona, Jesús M.; Granados-Durán, Pablo; Fernández-Llebrez, Pedro; López-Ávalos, María D.

2017-01-01

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation.Hierarchical cluster and principal components analysis allow morphological classification of microglia.Brain location of microglia is a relevant factor. PMID:28848398

Magnolol pretreatment prevents sepsis-induced intestinal dysmotility by maintaining functional interstitial cells of Cajal.

PubMed

Miao, Bin; Zhang, Shuwen; Wang, Hong; Yang, Tiecheng; Zhou, Deshan; Wang, Bao-en

2013-08-01

The purpose of this study was to investigate the mechanism by which magnolol treatment prevents lipopolysaccharide (LPS)-induced septic dysmotility in mice. Sepsis was induced by intravenous tail vein injection of LPS (4 mg/kg body weight). Animals were divided into three groups: the magnolol-treated septic group, the placebo-treated septic group, and the control group. Intestinal transit and circular smooth muscle contraction were measured 12 h after LPS injection, and immunocytochemisty was performed to study the morphology of interstitial cells of Cajal (ICCs). Stem cell factor (SCF) expression and c-kit phosphorylation were determined by Western blot analysis, and the mRNA levels of inducible NO synthase (iNOS) were determined by RT-PCR. Nitric oxide (NO) content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using commercial kits. Intestinal transit and muscular contractility were significantly lower in the LPS-treated group than in the control group. Immunocytochemical experiments showed that the total number of ICCs, and the total and average lengths of the ICC processes were significantly decreased in the LPS-treated group compared with those in the control group. In LPS-treated animals, magnolol pretreatment significantly accelerated intestinal transit, increased circular muscle contraction, and prevented ICC morphology changes. Phosphorylation of c-kit and expression of SCF were significantly downregulated in LPS-treated animals compared with control animals. Magnolol pretreatment prevented sepsis-induced decreases in c-kit phosphorylation and SCF expression in LPS-treated animals. Magnolol pretreatment prevented the sepsis-induced increase in NO concentration, iNOS expression, and MDA concentration, and decrease in SOD activity in LPS-treated animals. Our results suggest that magnolol treatment prevents sepsis-induced intestinal dysmotility by regulating SCF/c-kit and NO signaling to maintain functional ICCs.

Flow-enhanced solution printing of all-polymer solar cells

DOE PAGES

Diao, Ying; Zhou, Yan; Kurosawa, Tadanori; ...

2015-08-12

Morphology control of solution coated solar cell materials presents a key challenge limiting their device performance and commercial viability. Here we present a new concept for controlling phase separation during solution printing using an all-polymer bulk heterojunction solar cell as a model system. The key aspect of our method lies in the design of fluid flow using a microstructured printing blade, on the basis of the hypothesis of flow-induced polymer crystallization. Our flow design resulted in a similar to 90% increase in the donor thin film crystallinity and reduced microphase separated donor and acceptor domain sizes. The improved morphology enhancedmore » all metrics of solar cell device performance across various printing conditions, specifically leading to higher short-circuit current, fill factor, open circuit voltage and significantly reduced device-to-device variation. However, we expect our design concept to have broad applications beyond all-polymer solar cells because of its simplicity and versatility.« less

Anticancer activity of Moringa oleifera mediated silver nanoparticles on human cervical carcinoma cells by apoptosis induction.

PubMed

Vasanth, Karunamoorthy; Ilango, Kaliappan; MohanKumar, Ramasamy; Agrawal, Aruna; Dubey, Govind Prasad

2014-05-01

Silver nanomaterial plays a crucial role in the growing field of nanotechnology as there is an increasing commercial demand for silver nanoparticles (AgNPs) owing to their wide biological applications. The present investigation aims at developing anti-cancerous colloidal silver using Moringa olifera stem bark extract. Electron and atomic force microscopic images were taken to analyze the surface morphology of the synthesized AgNPs. The effects of synthesized AgNPs were tested against human cervical carcinoma cells (HeLa) and cell morphology was further evaluated using 4,6-diamidino-2-phenylindole (DAPI) staining. The efficiency of green synthesized AgNPs was studied with the help of fluorescence activated cell sorting (FACS) and was shown to induce apoptosis through reactive oxygen species (ROS) generation in HeLa cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Flow-enhanced solution printing of all-polymer solar cells

PubMed Central

Diao, Ying; Zhou, Yan; Kurosawa, Tadanori; Shaw, Leo; Wang, Cheng; Park, Steve; Guo, Yikun; Reinspach, Julia A.; Gu, Kevin; Gu, Xiaodan; Tee, Benjamin C. K.; Pang, Changhyun; Yan, Hongping; Zhao, Dahui; Toney, Michael F.; Mannsfeld, Stefan C. B.; Bao, Zhenan

2015-01-01

Morphology control of solution coated solar cell materials presents a key challenge limiting their device performance and commercial viability. Here we present a new concept for controlling phase separation during solution printing using an all-polymer bulk heterojunction solar cell as a model system. The key aspect of our method lies in the design of fluid flow using a microstructured printing blade, on the basis of the hypothesis of flow-induced polymer crystallization. Our flow design resulted in a ∼90% increase in the donor thin film crystallinity and reduced microphase separated donor and acceptor domain sizes. The improved morphology enhanced all metrics of solar cell device performance across various printing conditions, specifically leading to higher short-circuit current, fill factor, open circuit voltage and significantly reduced device-to-device variation. We expect our design concept to have broad applications beyond all-polymer solar cells because of its simplicity and versatility. PMID:26264528

Decreasing mitochondrial fission diminishes vascular smooth muscle cell migration and ameliorates intimal hyperplasia

PubMed Central

Wang, Li; Yu, Tianzheng; Lee, Hakjoo; O'Brien, Dawn K.; Sesaki, Hiromi; Yoon, Yisang

2015-01-01

Aims Vascular smooth muscle cell (VSMC) migration in response to arterial wall injury is a critical process in the development of intimal hyperplasia. Cell migration is an energy-demanding process that is predicted to require mitochondrial function. Mitochondria are morphologically dynamic, undergoing continuous shape change through fission and fusion. However, the role of mitochondrial morphology in VSMC migration is not well understood. The aim of the study is to understand how mitochondrial fission contributes to VSMC migration and provides its in vivo relevance in the mouse model of intimal hyperplasia. Methods and results In primary mouse VSMCs, the chemoattractant PDGF induced mitochondrial shortening through the mitochondrial fission protein dynamin-like protein 1 (DLP1)/Drp1. Perturbation of mitochondrial fission by expressing the dominant-negative mutant DLP1-K38A or by DLP1 silencing greatly decreased PDGF-induced lamellipodia formation and VSMC migration, indicating that mitochondrial fission is an important process in VSMC migration. PDGF induced an augmentation of mitochondrial energetics as well as ROS production, both of which were found to be necessary for VSMC migration. Mechanistically, the inhibition of mitochondrial fission induced an increase of mitochondrial inner membrane proton leak in VSMCs, abrogating the PDGF-induced energetic enhancement and an ROS increase. In an in vivo model of intimal hyperplasia, transgenic mice expressing DLP1-K38A displayed markedly reduced ROS levels and neointima formation in response to femoral artery wire injury. Conclusions Mitochondrial fission is an integral process in cell migration, and controlling mitochondrial fission can limit VSMC migration and the pathological intimal hyperplasia by altering mitochondrial energetics and ROS levels. PMID:25587046

FS laser processing of bio-polymer thin films for studying cell-to-substrate specific response

NASA Astrophysics Data System (ADS)

Daskalova, A.; Nathala, Chandra S. R.; Kavatzikidou, P.; Ranella, A.; Szoszkiewicz, R.; Husinsky, W.; Fotakis, C.

2016-09-01

The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell's responses to substrate morphology. Mice fibroblasts migration was monitored after 3 days cultivation period using FESEM. We found that fibroblasts cells tend to migrate and adhere along the laser modified zones. The performed study proved that the immobilized collagen based biofilms suite as a template for successful fibroblasts cell guidance and orientation. Fs laser induced morphological modification of biomimetic materials exhibit direct control over fibroblasts behaviour due to induced change in their wettability state.

Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans.

PubMed

Khan, Amber; Ahmad, Aijaz; Khan, Luqman Ahmad; Manzoor, Nikhat

2014-01-01

Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents - methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: a comparison with the effect of doxorubicin.

PubMed

Rogalska, Aneta; Gajek, Arkadiusz; Szwed, Marzena; Jóźwiak, Zofia; Marczak, Agnieszka

2011-12-01

In the present study, we investigated the anticancer activity of WP 631, a new anthracycline analog, in weakly doxorubicin-resistant SKOV-3 ovarian cancer cells. We studied the time-course of apoptotic and necrotic events: the production of reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in human ovarian cancer cells exposed to WP 631 in the presence and absence of an antioxidant, N-acetylcysteine (NAC). The effect of WP 631 was compared with the activity of doxorubicin (DOX), the best known first-generation anthracycline. Cytotoxic activity was determined by the MTT assay. The morphological changes characteristic of apoptosis and necrosis in drug-treated cells were analyzed by double staining with Hoechst 33258 and propidium iodide (PI) using fluorescence microscopy. The production of reactive oxygen species and changes in mitochondrial membrane potential were studied using specific fluorescence probes: DCFH2-DA and JC-1, respectively. The experiments showed that WP 631 was three times more cytotoxic than DOX in the tested cell line. It was found that the new anthracycline analog induced mainly apoptosis and, marginally, necrosis. Apoptotic cell death was associated with morphological changes and a decrease in mitochondrial membrane potential. In comparison to DOX, the novel bisanthracycline induced a significantly higher level of ROS and a greater drop in the membrane potential. The results provide direct evidence that the novel anthracycline WP 631 is considerably more cytotoxic to human SKOV-3 ovarian cancer cells than doxorubicin. The drug can produce ROS, which are immediately involved in the induction of apoptotic cell death. Copyright © 2011 Elsevier Ltd. All rights reserved.

Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

PubMed Central

Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

2011-01-01

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

PubMed

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme

PubMed Central

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-01-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance. PMID:27584787

Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme.

PubMed

Günzle, Jessica; Osterberg, Nadja; Saavedra, Joseph E; Weyerbrock, Astrid

2016-09-01

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

Canine neoplasia

PubMed Central

Prier, J. E.; Brodey, R. S.

1963-01-01

The authors review current knowledge of spontaneous neoplasms in the dog. The prevalence of certain types of canine tumour has been studied, and comparisons have been made with the occurrence of similar neoplasms in man. Where there are appropriate analogies between the two species, the dog with spontaneous tumours can be used for studies that are not practicable in man. Nutritional and morphological studies have been done on cells cultured from canine tumours. Some consistency has been demonstrated in the morphology of cultures of different tumours of the same type. Nutritional studies with the transmissible venereal sarcoma of the dog have shown the cells to be subject to a growth-repressing effect by SH-containing amino-acids. Attempts to transmit tumours to other dogs or other species have generally been unsuccessful. A transplantable tumour developed in a mouse injected with non-cellular material from a canine thyroid carcinoma, but it is not certain that the tumour was induced. Cell-culture studies have shown that some tumours yield a factor that is cytopathogenic for normal cells, but none has been shown capable of inducing neoplasms in vivo. ImagesFIG. 3FIG. 4FIG. 5FIG. 1FIG. 2FIG. 6 PMID:14058226

Mineralization and Expression of Col1a1-3.6GFP Transgene in Primary Dental Pulp Culture

PubMed Central

Balic, Anamaria; Rodgers, Barbara; Mina, Mina

2008-01-01

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and β-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and β-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization PMID:18781059

[Methuosis: a novel type of cell death].

PubMed

Cai, Hongbing; Liu, Jinkun; Fan, Qin; Li, Xin

2013-12-01

Cell death is a major physiological or pathological phenomenon in life activities. The classic forms of cell death include apoptosis, necrosis, and autophagy. Recently, a novel type of cell death has been observed and termed as methuosis, in which excessive stimuli can induce cytoplasmic uptake and accumulation of small bubbles that gradually merge into giant vacuoles, eventually leading to decreased cellular metabolic activity, cell membrane rupture and cell death. In this article, we describe the nomenclature, morphological characteristics and underlying mechanisms of methuosis, compare methuosis with autophagy, oncosis and paraptosis, and review the related researches.

A morphological study of the changes in the ultrastructure of a bacterial biofilm disrupted by an ac corona discharge in air

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stepanova, Olga, E-mail: o.m.stepanova@spbu.ru; Astafiev, Alexander; Kudryavtsev, Anatoly

The morphology of bacterial cells and biofilms subjected to a low frequency (∼10{sup 5} Hz) ac (∼10{sup −1} A) corona discharge was investigated using electron microscopy. A low-frequency ac corona discharge in air is shown to have a bactericidal and bacteriostatic effect on Escherichia coli M17 culture at both the cellular and population levels. Corona exposure inhibits the formation of a microbial community and results in the destruction of formed biofilms. This paper presents data on changes in the ultrastructure of cells and biofilms after corona treatment. Our results suggest that the E. coli M17 cells inside biofilms are affectedmore » with results similar to sub-lethal and lethal thermal exposure. Some of the biological aspects of colony and biofilm cells death are evaluated. Morphological changes in the ultrastructure of the biofilms under corona treatment are described. Our results indicate that the heating effect is the main factor responsible for the corona-induced inactivation of bacteria.« less

Defining the free-energy landscape of curvature-inducing proteins on membrane bilayers

PubMed Central

Tourdot, Richard W.; Ramakrishnan, N.; Radhakrishnan, Ravi

2015-01-01

Curvature-sensing and curvature-remodeling proteins, such as Amphiphysin, Epsin, and Exo70, are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors; they aggregate to membrane regions matching their intrinsic curvature; as well as induce curvature in cell membranes to stabilize emergent high curvature, nonspherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test particle (or field) insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membrane—in particular, we use this method to track the onset of morphological transitions in the membrane at elevated protein densities. We validate this approach by comparing the results from the Widom method with those of thermodynamic integration and Bennett acceptance ratio methods. Furthermore, the predictions from the Widom method have been tested against analytical calculations of the excess chemical potential at infinite dilution. Our results are useful in precisely quantifying the free-energy landscape, and also in determining the phase boundaries associated with curvature-induction, curvature-sensing, and morphological transitions. This approach can be extended to studies exploring the role of thermal fluctuations and other external (control) variables, such as membrane excess area, in shaping curvature-mediated interactions on bilayer membranes. PMID:25215768

Defining the free-energy landscape of curvature-inducing proteins on membrane bilayers.

PubMed

Tourdot, Richard W; Ramakrishnan, N; Radhakrishnan, Ravi

2014-08-01

Curvature-sensing and curvature-remodeling proteins, such as Amphiphysin, Epsin, and Exo70, are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors; they aggregate to membrane regions matching their intrinsic curvature; as well as induce curvature in cell membranes to stabilize emergent high curvature, nonspherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test particle (or field) insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membrane-in particular, we use this method to track the onset of morphological transitions in the membrane at elevated protein densities. We validate this approach by comparing the results from the Widom method with those of thermodynamic integration and Bennett acceptance ratio methods. Furthermore, the predictions from the Widom method have been tested against analytical calculations of the excess chemical potential at infinite dilution. Our results are useful in precisely quantifying the free-energy landscape, and also in determining the phase boundaries associated with curvature-induction, curvature-sensing, and morphological transitions. This approach can be extended to studies exploring the role of thermal fluctuations and other external (control) variables, such as membrane excess area, in shaping curvature-mediated interactions on bilayer membranes.

Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

PubMed

Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

2016-08-26

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. Published by Elsevier Ireland Ltd.

The Efficacy of Dandelion Root Extract in Inducing Apoptosis in Drug-Resistant Human Melanoma Cells

PubMed Central

Chatterjee, S. J.; Ovadje, P.; Mousa, M.; Hamm, C.; Pandey, S.

2011-01-01

Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible), and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE) specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS) generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells. PMID:21234313

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells

PubMed Central

Lugini, Luana; Valtieri, Mauro; Federici, Cristina; Cecchetti, Serena; Meschini, Stefania; Condello, Maria; Signore, Michele; Fais, Stefano

2016-01-01

Background Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. Results CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Materials and Methods Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. Conclusions CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer. PMID:27418137

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells.

PubMed

Lugini, Luana; Valtieri, Mauro; Federici, Cristina; Cecchetti, Serena; Meschini, Stefania; Condello, Maria; Signore, Michele; Fais, Stefano

2016-08-02

Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer.

The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology.

PubMed

Morissette, G; Germain, L; Marceau, F

2007-03-01

The 'cosmeceutical' agent 2-dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations. We hypothesized that a 337 mmol L(-1) (3%) DMAE reservoir applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. Morphological and functional assays were applied to cultured rabbit dermal fibroblasts treated with tertiary amines in vitro. A morphological verification of the vacuolization caused by topical DMAE was also attempted in vivo using the inner skin of the rabbit ear and in vitro using primary cultures of human cutaneous epithelial cells. Fibroblasts responded to DMAE (2.5-10 mmol L(-1)) by massive vacuolization (0.5-4 h; phase contrast observations). Triethanolamine, another chemical frequently used topically, was also active in this respect (10 mmol L(-1)). The vacuolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- or triethanolamine-induced vacuolization; adding bafilomycin A1 or cell washout slowly reversed the established vacuolization induced by DMAE. Further effects of DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L(-1)) that was abated by bafilomycin A1 cotreatment, a concentration-dependent mitotic arrest (2.5 mmol L(-1)) and transient and mild effects on cell ploidy. The epidermis of the rabbit external ear was significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h; paraffin tissue sections). Cultured human cutaneous epithelial cells responded to DMAE by vacuolization (inhibited by bafilomycin A1 cotreatment). The vacuolar cytopathology induced by concentrated organic amines may be the cellular basis of the antiwrinkle effect of DMAE.

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

Apigenin induced apoptosis in esophageal carcinoma cells by destruction membrane structures.

PubMed

Zhu, Haiyan; Jin, Hua; Pi, Jiang; Bai, Haihua; Yang, Fen; Wu, Chaomin; Jiang, Jinhuan; Cai, Jiye

2016-07-01

Apigenin has shown to have killing effects on some kinds of solid tumor cells. However, the changes in cell membrane induced by apigenin on subcellular- or nanometer-level were still unclear. In this work, human esophageal cancer cells (EC9706 and KYSE150 cells) were employed as cell model to detect the cytotoxicity of apigenin, including cell growth inhibition, apoptosis induction, membrane toxicity, etc. MTT assay showed that apigenin could remarkably inhibit the growth and proliferation in both types of cells. Annexin V/PI-based flow cytometry analysis showed that the cytotoxic effects of apigenin in KYSE150 cells were mainly through early apoptosis induction, while in EC9706 cells, necrosis, and apoptosis were both involved in cell death. The morphological and ultrastructural properties induced by apigenin were investigated at single cellular- or nanometer-level using atomic force microscopy (AFM). Additionally, lactate dehydrogenase (LDH) leakage was measured to assess the changes in membrane permeability. The results indicated that apigenin increased the membrane permeability and caused leakage of LDH, which was consistent with damages on membrane ultrastructure detected by AFM. Therefore, membrane toxicity, including membrane ultrastructure damages and enhanced membrane permeability, played vital roles in apigenin induced human esophageal cancer cell apoptosis. SCANNING 38:322-328, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

PubMed

Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

2014-10-01

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.

Phase imaging microscopy for the diagnostics of plasma-cell interaction

NASA Astrophysics Data System (ADS)

Ohene, Yolanda; Marinov, Ilya; de Laulanié, Lucie; Dupuy, Corinne; Wattelier, Benoit; Starikovskaia, Svetlana

2015-06-01

Phase images of biological specimens were obtained by the method of Quadriwave Lateral Shearing Interferometry (QWLSI). The QWLSI technique produces, at high resolution, phase images of the cells having been exposed to a plasma treatment and enables the quantitative analysis of the changes in the surface area of the cells over time. Morphological changes in the HTori normal thyroid cells were demonstrated using this method. There was a comparison of the cell behaviour between control cells, cells treated by plasma of a nanosecond dielectric barrier discharge, including cells pre-treated by catalase, and cells treated with an equivalent amount of H2O2. The major changes in the cell membrane morphology were observed at only 5 min after the plasma treatment. The primary role of reactive oxygen species (ROS) in this degradation is suggested. Deformation and condensation of the cell nucleus were observed 2-3 h after the treatment and are supposedly related to apoptosis induction. The coupling of the phase QWLSI with immunofluorescence imaging would give a deeper insight into the mechanisms of plasma induced cell death.

Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8

PubMed Central

2012-01-01

Background One of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. In Japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. The purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells. Methods LM8 cells were treated for 3 days with various concentrations of genistein. The effect of genistein on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2’-deoxyuridine (BrdU) incorporation study. The assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. The expression and secretion of MMP-2 were determined by immunohistochemistry and gelatin zymography. The subcellular localization and cellular level of β-catenin were determined by immunofluorescence and Western blot. For examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin (H&E). The expression of osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results Genistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 were lower in the genistein-treated cultures than in the untreated cultures. β-Catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. The level of β-catenin was higher in genistein-treated cells than in untreated cells. Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA. Conclusions Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects. PMID:23013480

The effect of Si nano-columns in 2-D and 3-D on cellular behaviour: nanotopography-induced CaP deposition from differentiating mesenchymal stem cells.

PubMed

Guvendik, S; Trabzon, L; Ramazanoglu, M

2011-10-01

Si nano-columns were deposited in 2-D and 3-D in the form of well-defined geometries by physical vapor deposition. The films were grown by e-beam evaporation with an angle between source and substrate. The Si nano-columns were deposited in the shape of spiral with two different incoming atomic flux angle so that the manipulation of nano-columns in 3-D (out-of-plane) was obtained. The Si nano-columns were also grown as vertical stick with square, triangle and linear cross sections in 2D (in-plane). Rat bone marrow mesenchymal stem cells (MSCs) were cultured on these different Si nanosurfaces. MTS assay was carried out to determine the cell proliferation and viability based on different nanotopographies. For the evaluation of cell distribution and morphology, a SEM (Scanning Electron Microscopy) analysis was performed. Any CaP deposition on Si nanosurfaces was observed using energy dispersive X-Ray spectroscopy in SEM (SEM-EDX). After 4 days of culture, there was a higher value of cell proliferation on square columns and spiral Si nano-columns grown with 85 degrees of incoming atomic flux. The cell attachment and spreading was also affected by the geometry of Si nano-columns. While there were still cells showing round/spherical morphology with minimal spreading on conventional Si surfaces, most of the cells cultured on different Si nanotopographies attached on the surface and displayed flattened morphology, especially on the square columns surface. Moreover, CaP deposition was discovered on square columns and spiral films with 85 degrees substrate angle. So, it can be concluded that there is a clear correlation between cell responses and nano-sized geometry on Si surface and it is possible to induce cellular differentiation and CaP formation in certain geometrical constraints.

Preliminary studies on anti-tumor activity of 2',4'-dihydroxychalcone isolated from Herba Oxytropis in human gastric cancer MGC-803 cells.

PubMed

Lou, Chenghua; Wang, Mingyan; Yang, Guangming; Cai, Hao; Li, Yu; Zhao, Fengming; Yang, Huan; Tong, Li; Cai, Baochang

2009-08-01

2',4'-Dihydroxychalcone (TFC), one of the main components in Herba Oxytropis, belongs to the flavonoid group, which is known to have anti-tumor activity in vitro. In this study, the authors examined the effects of TFC on cell proliferation and apoptosis in human gastric cancer MGC-803 cells. The MTT assay results showed that TFC was able to induce cytotoxicity in MGC-803 cells in a concentration- and time-dependent manner. Acridine orange/ethidium bromide (AO/EB) staining analysis indicated that the cytotoxicity induced by TFC was mediated by apoptosis, and flow cytometry analysis indicated an increase in apoptotic cells after treatment with TFC. Furthermore, typical apoptotic morphology such as condensed chromatin, irregular nuclei, vacuoles, and dispersed granular material in the nuclear compartment were also observed using a transmission electron microscope. These results suggested that TFC can inhibit the growth of MGC-803 cells and induce apoptosis. However, further studies are necessary to investigate the possible mechanism.

3,3′-Diindolylmethane Suppressed Cyprodinil-Induced Epithelial-Mesenchymal Transition and Metastatic-Related Behaviors of Human Endometrial Ishikawa Cells via an Estrogen Receptor-Dependent Pathway

PubMed Central

Kim, Bo-Gyoung; Kim, Jin-Wook; Kim, Soo-Min; Go, Ryeo-Eun; Hwang, Kyung-A

2018-01-01

Cyprodinil (CYP) is a pyrimidine amine fungicide that has been extensively used in agricultural areas. 3,3′-Diindolylmethane (DIM) is a derivative of the dietary phytoestrogen, indole-3-carbinol (I3C), which is derived from cruciferous vegetables and considered to be a cancer-preventive phytonutrient agent. In this study, the effects of CYP and DIM were examined on the cell viability, invasion, and metastasis of human endometrial cancer cells, Ishikawa, via epithelial mesenchymal transition (EMT). CYP increased the level of cell viability of Ishikawa cells compared to DMSO as a control, as did E2. Ishikawa cells lost cell-to-cell contact and obtained a spindle-shaped or fibroblast-like morphology in response to the application of E2 or CYP by the cell morphology assay. In the cell migration and invasion assay, CYP enhanced the ability of migration and invasion of Ishikawa cells, as did E2. E2 and CYP increased the expressions of N-cadherin and Snail proteins, while decreasing the expression of E-cadherin protein as EMT-related markers. In addition, E2 and CYP increased the protein expressions of cathepsin D and MMP-9, metastasis-related markers. Conversely, CYP-induced EMT, cell migration, and invasion were reversed by fulvestrant (ICI 182,780) as an estrogen receptor (ER) antagonist, indicating that CYP exerts estrogenic activity by mediating these processes via an ER-dependent pathway. Similar to ICI 182,780, DIM significantly suppressed E2 and CYP-induced proliferation, EMT, migration, and invasion of Ishikawa cancer cells. Overall, the present study revealed that DIM has an antiestrogenic chemopreventive effect to withdraw the cancer-enhancing effect of E2 and CYP, while CYP has the capacity to enhance the metastatic potential of estrogen-responsive endometrial cancer. PMID:29316692

Effects of 5-fluorouracil in nuclear and cellular morphology, proliferation, cell cycle, apoptosis, cytoskeletal and caveolar distribution in primary cultures of smooth muscle cells.

PubMed

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

PubMed Central

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer. PMID:23646193

Interplay of CREB and ATF2 in Ionizing Radiation-Induced Neuroendocrine Differentiation of Prostate Cancer Cells

DTIC Science & Technology

2011-06-01

h-actin. Analysis of ATF2 and CREB subcellular localization. LNCaP cells were fixed in ice-cold 3.7% formaldehyde for 20 min, followed by... phenol -free RPMI 1640 supplemented with 10% charcoal-dextran–treated FBS (CD-FBS) for 3 wk and similarly assayed for morphologic changes and the... phenol -free RPMI 1640 supplemented with 10% CD-FBS for the indicated times. Cell viability for IR- and docetaxel-treated cells was determined by a 3

Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

PubMed

Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

2010-04-01

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

NASA Astrophysics Data System (ADS)

Khetan, Sudhir; Guvendiren, Murat; Legant, Wesley R.; Cohen, Daniel M.; Chen, Christopher S.; Burdick, Jason A.

2013-05-01

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Evaluation of testicular toxicity and sperm morphology in rats treated with methyl methanesulphonate (MMS).

PubMed

Kuriyama, Kazuya; Kitamura, Tsuyoshi; Yokoi, Ryohei; Hayashi, Morimichi; Kobayashi, Kazuo; Kuroda, Junji; Tsujii, Hirotada

2005-10-01

Methyl methanesulphonate (MMS), a potent alkylating agent and testicular toxicant, was orally administered to rats for 5 days at 40 mg/kg. During the recovery period of up to 5 weeks, males were evaluated for testicular toxicity and sperm morphology. The 5-week recovery period were designated as follows: Day 1 (the day after final treatment); Week 1, Week 2, Week 3, Week 4 and Week 5 (1, 2, 3, 4 and 5 weeks after final treatment). Morphologically abnormal sperm increased beginning in Week 3, peaked in Week 4 and declined slightly in Week 5. Histopathological examinations indicated retention of step 19 spermatids at stage IX from Day 1 through Week 3. Quantitative evaluation of spermatogenic cells indicated a decrease in the number of late pachytene spermatocytes and early spermatids on Day 1. TUNEL examination showed a significantly high frequency of apoptosis in the meiosis cells in Week 1. In the present study, genetic damage induced by treatment with MMS affected spermatogenesis and a wide variety of spermatogenic cells in the testis. Apoptosis in the course of meiosis seemed to be involved in the elimination process of genetically insulted germ cells, and this process seems to play an important role in eliminating and/or decreasing the germ cells with retention of spermatids and the potential to express morphologically abnormal spermatozoa.

Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K., E-mail: swapan.ray@uscmed.sc.edu

Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly inmore » SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-{kappa}B), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression. -- Highlights: Black-Right-Pointing-Pointer Survivin shRNA + EGCG controlled growth of human malignant neuroblastoma cells. Black-Right-Pointing-Pointer Survivin knockdown induced neuronal differentiation in neuroblastoma cells. Black-Right-Pointing-Pointer Survivin shRNA + EGCG induced morphological and biochemical features of apoptosis. Black-Right-Pointing-Pointer Combination therapy inhibited invasion, proliferation, and angiogenesis as well. Black-Right-Pointing-Pointer So, combination therapy showed multiple anti-cancer mechanisms in neuroblastoma.« less

Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Jackson, Desmond N.; Lipke, Peter N.; Dufrêne, Yves F.

2013-01-01

The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33215a

(abstract) Effects of Radiation and Oxidative Stress on Development and Morphology of Intestinal Cells

NASA Technical Reports Server (NTRS)

Honda, Shuji; Nelson, Gregory; Schubert, Wayne

1993-01-01

Intestinal cells when subjected to oxidative stress or radiation exhibit abnormal nuclear divisions observed as: 1) supernumerary cell divisions in anterior intestinal cells or 2) incomplete nuclear division and the persistence of anaphase bridges between daughter nuclei. Two oxygen sensitive mutants, mev-1 and rad-8 were observed to exhibit spontaneous supernumerary nuclear divisions at low frequency. N2 can be induced to undergo these divisions by treatment with the superoxide dismutase (SOD) inhibitor diethyl dithicarbamate or with the free radical generator methyl viologen. By contrast, the free radical generator bleomycin produces anaphase bridges in N2 intestinal nuclei at high frequency. Intestinal anaphase bridges can be induced by ionizing radiation and their formation is dependent on dose and radiation type.

Optimization of surface morphology and scattering properties of TCO/AIT textured glass front electrode for thin film solar cells

NASA Astrophysics Data System (ADS)

Addonizio, M. L.; Fusco, L.; Antonaia, A.; Cominale, F.; Usatii, I.

2015-12-01

Aluminium induced texture (AIT) method has been used for obtaining highly textured glass substrate suitable for silicon based thin film solar cell technology. Wet etch step parameters of AIT process have been varied and effect of different etchants and different etching times on morphological and optical properties has been analyzed. The resulting morphology features (shape, size distribution, inclination angle) have been optimized in order to obtain the best scattering properties. ZnO:Ga (GZO) films have been deposited by sputtering technique on AIT-processed glass. Two different ZnO surface morphologies have been obtained, strongly depending on the underlying glass substrate morphology induced by different etching times. Very rough and porous texture (σrms ∼ 150 nm) was obtained on glass etched 2 min showing cauliflower-like structure, whereas a softer texture (σrms ∼ 78 nm) was obtained on glass etched 7 min giving wider and smoother U-shaped craters. The effect of different glass textures on optical confinement has been tested in amorphous silicon based p-i-n devices. Devices fabricated on GZO/high textured glass showed a quantum efficiency enhancement due to both an effective light trapping phenomenon and an effective anti-reflective optical behaviour. Short etching time produce smaller cavities (<1 μm) with deep U-shape characterized by high roughness, high inclination angle and low autocorrelation length. This surface morphology promoted a large light scattering phenomenon, as evidenced by haze value and by angular resolved scattering (ARS) behaviour, into a large range of diffraction angles, giving high probability of effective light trapping inside a PV device.

Evaluation of PBS Treatment and PEI Coating Effects on Surface Morphology and Cellular Response of 3D-Printed Alginate Scaffolds.

PubMed

Mendoza García, María A; Izadifar, Mohammad; Chen, Xiongbiao

2017-11-01

Three-dimensional (3D) printing is an emerging technology for the fabrication of scaffolds to repair/replace damaged tissue/organs in tissue engineering. This paper presents our study on 3D printed alginate scaffolds treated with phosphate buffered saline (PBS) and polyethyleneimine (PEI) coating and their impacts on the surface morphology and cellular response of the printed scaffolds. In our study, sterile alginate was prepared by means of the freeze-drying method and then, used to prepare the hydrogel for 3D printing into calcium chloride, forming 3D scaffolds. Scaffolds were treated with PBS for a time period of two days and seven days, respectively, and PEI coating; then they were seeded with Schwann cells (RSC96) for the examination of cellular response (proliferation and differentiation). In addition, swelling and stiffness (Young's modulus) of the treated scaffolds was evaluated, while their surface morphology was assessed using scanning electron microscopy (SEM). SEM images revealed significant changes in scaffold surface morphology due to degradation caused by the PBS treatment over time. Our cell proliferation assessment over seven days showed that a two-day PBS treatment could be more effective than seven-day PBS treatment for improving cell attachment and elongation. While PEI coating of alginate scaffolds seemed to contribute to cell growth, Schwann cells stayed round on the surface of alginate over the period of cell culture. In conclusion, PBS-treatment may offer the potential to induce surface physical cues due to degradation of alginate, which could improve cell attachment post cell-seeding of 3D-printed alginate scaffolds.

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

PubMed

Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

2004-07-01

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

Dynamic Reorganization of the Cytoskeleton during Apoptosis: The Two Coffins Hypothesis.

PubMed

Povea-Cabello, Suleva; Oropesa-Ávila, Manuel; de la Cruz-Ojeda, Patricia; Villanueva-Paz, Marina; de la Mata, Mario; Suárez-Rivero, Juan Miguel; Álvarez-Córdoba, Mónica; Villalón-García, Irene; Cotán, David; Ybot-González, Patricia; Sánchez-Alcázar, José A

2017-11-11

During apoptosis, cells undergo characteristic morphological changes in which the cytoskeleton plays an active role. The cytoskeleton rearrangements have been mainly attributed to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent results have shown that microtubules are reorganized during the execution phase of apoptosis forming an apoptotic microtubule network (AMN). Evidence suggests that AMN is required to maintain plasma membrane integrity and cell morphology during the execution phase of apoptosis. The new "two coffins" hypothesis proposes that both AMN and apoptotic cells can adopt two morphological patterns, round or irregular, which result from different cytoskeleton kinetic reorganization during the execution phase of apoptosis induced by genotoxic agents. In addition, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocyte responses. These findings suggest that knowing the type of apoptosis may be important to predict how fast apoptotic cells undergo secondary necrosis and the subsequent immune response. From a pathological point of view, round-shaped apoptosis can be seen as a physiological and controlled type of apoptosis, while irregular-shaped apoptosis can be considered as a pathological type of cell death closer to necrosis.

Dynamic Reorganization of the Cytoskeleton during Apoptosis: The Two Coffins Hypothesis

PubMed Central

Povea-Cabello, Suleva; Oropesa-Ávila, Manuel; de la Cruz-Ojeda, Patricia; Villanueva-Paz, Marina; de la Mata, Mario; Álvarez-Córdoba, Mónica; Villalón-García, Irene; Cotán, David; Ybot-González, Patricia

2017-01-01

During apoptosis, cells undergo characteristic morphological changes in which the cytoskeleton plays an active role. The cytoskeleton rearrangements have been mainly attributed to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent results have shown that microtubules are reorganized during the execution phase of apoptosis forming an apoptotic microtubule network (AMN). Evidence suggests that AMN is required to maintain plasma membrane integrity and cell morphology during the execution phase of apoptosis. The new “two coffins” hypothesis proposes that both AMN and apoptotic cells can adopt two morphological patterns, round or irregular, which result from different cytoskeleton kinetic reorganization during the execution phase of apoptosis induced by genotoxic agents. In addition, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocyte responses. These findings suggest that knowing the type of apoptosis may be important to predict how fast apoptotic cells undergo secondary necrosis and the subsequent immune response. From a pathological point of view, round-shaped apoptosis can be seen as a physiological and controlled type of apoptosis, while irregular-shaped apoptosis can be considered as a pathological type of cell death closer to necrosis. PMID:29137119

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

PubMed

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-11-02

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

PubMed Central

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-01-01

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

Cytotoxic and apoptotic effects of chalcone derivatives of 2-acetyl thiophene on human colon adenocarcinoma cells.

PubMed

de Vasconcelos, Alana; Campos, Vinicius Farias; Nedel, Fernanda; Seixas, Fabiana Kömmling; Dellagostin, Odir A; Smith, Kevin R; de Pereira, Cláudio Martin Pereira; Stefanello, Francieli Moro; Collares, Tiago; Barschak, Alethéa Gatto

2013-06-01

Recent studies report that chalcones exhibit cytotoxicity to human cancer cell lines. Typically, the form of cell death induced by these compounds is apoptosis. In the context of the discovery of new anticancer agents and in light of the antitumour potential of several chalcone derivatives, in the present study, we synthesized and tested the cytotoxicity of six chalcone derivatives on human colon adenocarcinoma cells. Six derivatives of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one were prepared and characterized on the basis of their (1) H and (13) C NMR spectra. HT-29 cells were treated with synthesized chalcones on two concentrations by three different incubation times. Cells were evaluated by cell morphology, Tetrazolium dye (MTT) colorimetric assay, live/dead, flow cytometry (annexin V) and gene expression analyses to determine the cytotoxic way. Chalcones 3-(4-bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C06) and 3-(2-nitrophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C09) demonstrated higher cytotoxicity than other chalcones as shown by cell morphology, live/dead and MTT assays. In addition, C06 induced apoptosis on flow cytometry annexin V assay. These data were confirmed by a decreased expression of anti-apoptotic genes and increased pro-apoptotic genes. Our findings indicate in summary that the cytotoxic activity of chalcone C06 on colorectal carcinoma cells occurs by apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.

Automated Morphological Analysis of Microglia After Stroke.

PubMed

Heindl, Steffanie; Gesierich, Benno; Benakis, Corinne; Llovera, Gemma; Duering, Marco; Liesz, Arthur

2018-01-01

Microglia are the resident immune cells of the brain and react quickly to changes in their environment with transcriptional regulation and morphological changes. Brain tissue injury such as ischemic stroke induces a local inflammatory response encompassing microglial activation. The change in activation status of a microglia is reflected in its gradual morphological transformation from a highly ramified into a less ramified or amoeboid cell shape. For this reason, the morphological changes of microglia are widely utilized to quantify microglial activation and studying their involvement in virtually all brain diseases. However, the currently available methods, which are mainly based on manual rating of immunofluorescent microscopic images, are often inaccurate, rater biased, and highly time consuming. To address these issues, we created a fully automated image analysis tool, which enables the analysis of microglia morphology from a confocal Z-stack and providing up to 59 morphological features. We developed the algorithm on an exploratory dataset of microglial cells from a stroke mouse model and validated the findings on an independent data set. In both datasets, we could demonstrate the ability of the algorithm to sensitively discriminate between the microglia morphology in the peri-infarct and the contralateral, unaffected cortex. Dimensionality reduction by principal component analysis allowed to generate a highly sensitive compound score for microglial shape analysis. Finally, we tested for concordance of results between the novel automated analysis tool and the conventional manual analysis and found a high degree of correlation. In conclusion, our novel method for the fully automatized analysis of microglia morphology shows excellent accuracy and time efficacy compared to traditional analysis methods. This tool, which we make openly available, could find application to study microglia morphology using fluorescence imaging in a wide range of brain disease models.

Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy

PubMed Central

Liu, Hong; Gu, Liu-bao; Tu, Yue; Hu, Hao; Huang, Yan-ru; Sun, Wei

2016-01-01

Aim: A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro. Methods: The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. Results: Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects. Conclusion: Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity. PMID:26775661

Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy.

PubMed

Liu, Hong; Gu, Liu-bao; Tu, Yue; Hu, Hao; Huang, Yan-ru; Sun, Wei

2016-02-01

A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro. The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects. Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity.

BONE MARROW MESENCHYMAL STEM CELLS ARE PROGENITORS IN VITRO FOR INNER EAR HAIR CELLS

PubMed Central

Jeon, Sang-Jun; Oshima, Kazuo; Heller, Stefan; Edge, Albert S.B.

2011-01-01

Stem cells have been demonstrated in the inner ear but they do not spontaneously divide to replace damaged sensory cells. Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages including neurons, and we therefore asked whether MSCs could generate sensory cells. Overexpression of the prosensory transcription factor, Math1, in sensory epithelial precursor cells induced expression of myosin VIIa, espin, Brn3c, p27Kip, and jagged2, indicating differentiation to inner ear sensory cells. Some of the cells displayed F-actin positive protrusions in the morphology characteristic of hair cell stereociliary bundles. Hair cell markers were also induced by culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event, because the chick hair cells could be identified with a chick-specific antibody and chick and mouse antigens were never found in the same cell. PMID:17113786

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

Tetrandrine Induces Apoptosis in Human Nasopharyngeal Carcinoma NPC-TW 039 Cells by Endoplasmic Reticulum Stress and Ca2+/Calpain Pathways.

PubMed

Liu, Kuo-Ching; Lin, Ya-Jing; Hsiao, Yung-Ting; Lin, Meng-Liang; Yang, Jiun-Long; Huang, Yi-Ping; Chu, Yung-Lin; Chung, Jing-Gung

2017-11-01

Tetrandrine is an alkaloid extracted from a traditional China medicine plant, and is considered part of food therapy as well. In addition, it has been widely reported to induce apoptotic cell death in many human cancer cells. However, the mechanism of Tetrandrine on human nasopharyngeal carcinoma cells (NPC) is still questioned. In our study, we examined whether Tetrandrine can induce apoptosis of NPC-TW 039 cells. We found that cell morphology was changed after treatment with different concentrations of Tetrandrine. Further, we indicated that the NPC-TW 039 cells viability decreased in a Tetrandrine dose-dependent manner. We also found that tetrandrine induced cell cycle arrest in G 0 /G 1 phase. Tetrandrine induced DNA condensation by DAPI staining as well. In addition, we found that Tetrandrine induced Ca 2+ release in the cytosol. At the same time, endoplasmic reticulum (ER) stress occurred. Then we used western blotting to examine the protein expression which is associated with mitochondria-mediated apoptotic pathways and caspase-dependent pathways. To further examine whether Ca 2+ was released or not with Tetrandrine induced-apoptosis, we used the chelator of Ca 2+ and showed that cell viability increased. At the same time, caspase-3 expression was decreased. Furthermore, confocal microscopy examination revealed that Tetrandrine induced expression of ER stress-related proteins GADD153 and GRP78. Our results indicate that Tetrandrine induces apoptosis through calcium-mediated ER stress and caspase pathway in NPC-TW 039 cells. In conclusion, Tetrandrine may could be used for treatment of human nasopharyngeal carcinoma in future. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Physangulidine A, a withanolide from Physalis angulata, perturbs the cell cycle and induces cell death by apoptosis in prostate cancer cells.

PubMed

Reyes-Reyes, E Merit; Jin, Zhuang; Vaisberg, Abraham J; Hammond, Gerald B; Bates, Paula J

2013-01-25

Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Sulfated lentinan induced mitochondrial dysfunction leads to programmed cell death of tobacco BY-2 cells.

PubMed

Wang, Jie; Wang, Yaofeng; Shen, Lili; Qian, Yumei; Yang, Jinguang; Wang, Fenglong

2017-04-01

Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H 2 O 2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H 2 DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD. Copyright © 2016. Published by Elsevier Inc.

Ap4A induces apoptosis in human cultured cells.

PubMed

Vartanian, A; Alexandrov, I; Prudowski, I; McLennan, A; Kisselev, L

1999-07-30

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'''-P1,P3-triphosphate to diadenosine 5',5'''-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Ferroptosis is Involved in Acetaminophen Induced Cell Death.

PubMed

Lőrincz, Tamás; Jemnitz, Katalin; Kardon, Tamás; Mandl, József; Szarka, András

2015-09-01

The recently described form of programmed cell death, ferroptosis can be induced by agents causing GSH depletion or the inhibition of GPX4. Ferroptosis clearly shows distinct morphologic, biochemical and genetic features from apoptosis, necrosis and autophagy. Since NAPQI the highly reactive metabolite of the widely applied analgesic and antipyretic, acetaminophen induces a cell death which can be characterized by GSH depletion, GPX inhibition and caspase independency the involvement of ferroptosis in acetaminophen induced cell death has been investigated. The specific ferroptosis inhibitor ferrostatin-1 failed to elevate the viability of acetaminophen treated HepG2 cells. It should be noticed that these cells do not form NAPQI due to the lack of phase I enzyme expression therefore GSH depletion cannot be observed. However in the case of acetaminophen treated primary mouse hepatocytes the significant elevation of cell viability could be observed upon ferrostatin-1 treatment. Similar to ferrostatin-1 treatment, the addition of the RIP1 kinase inhibitor necrostatin-1 could also elevate the viability of acetaminophen treated primary hepatocytes. Ferrostatin-1 has no influence on the expression of CYP2E1 or on the cellular GSH level which suggest that the protective effect of ferrostatin-1 in APAP induced cell death is not based on the reduced metabolism of APAP to NAPQI or on altered NAPQI conjugation by cellular GSH. Our results suggest that beyond necroptosis and apoptosis a third programmed cell death, ferroptosis is also involved in acetaminophen induced cell death in primary hepatocytes.

SIRT1 overexpression protects non-small cell lung cancer cells against osteopontin-induced epithelial-mesenchymal transition by suppressing NF-κB signaling.

PubMed

Li, Xuejiao; Jiang, Zhongxiu; Li, Xiangmin; Zhang, Xiaoye

2018-01-01

Osteopontin (OPN) is a promoter for tumor progression. It has been reported to promote non-small cell lung cancer (NSCLC) progression via the activation of nuclear factor-κB (NF-κB) signaling. As the increased acetylation of NF-κB p65 is linked to NF-κB activation, the regulation of NF-κB p65 acetylation could be a potential treatment target for OPN-induced NSCLC progression. Sirtuin 1 (SIRT1) is a deacetylase, and the role of SIRT1 in tumor progression is still controversial. The effect and mechanism of SIRT1 on OPN-induced tumor progression remains unknown. The results presented in this research demonstrated that OPN inhibited SIRT1 expression and promoted NF-κB p65 acetylation in NSCLC cell lines (A549 and NCI-H358). In this article, overexpression of SIRT1 was induced by infection of SIRT1-overexpressing lentiviral vectors. The overexpression of SIRT1 protected NSCLC cells against OPN-induced NF-κB p65 acetylation and epithelial-mesenchymal transition (EMT), as indicated by the reduction of OPN-induced changes in the expression levels of EMT-related markers and cellular morphology. Furthermore, SIRT1 overexpression significantly attenuated OPN-induced cell proliferation, migration and invasion. Moreover, overexpression of SIRT1 inhibited OPN-induced NF-κB activation. As OPN induced NSCLC cell EMT through activation of NF-κB signaling, OPN-induced SIRT1 downregulation may play an important role in NSCLC cell EMT via NF-κB signaling. The results suggest that SIRT1 could be a tumor suppressor to attenuate OPN-induced NSCLC progression through the regulation of NF-κB signaling.

Differentiation of Induced Pluripotent Stem Cells to Neural Retinal Precursor Cells on Porous Poly-Lactic-co-Glycolic Acid Scaffolds

PubMed Central

Worthington, Kristan S.; Wiley, Luke A.; Guymon, C. Allan; Salem, Aliasger K.

2016-01-01

Abstract Purpose: Cell replacement therapy for the treatment of retinal degeneration is an increasingly feasible approach, but one that still requires optimization of the transplantation strategy. To this end, various polymer substrates can increase cell survival and integration, although the effect of their pore size on cell behavior, particularly differentiation, has yet to be explored. Methods: Salt crystals of varying known size were used to impart structure to poly(lactic-co-glycolic acid) (PLGA) scaffolds by a salt leaching/solvent evaporation process. Mouse induced pluripotent stem cells (miPSCs) were seeded to the polymer scaffolds and supplemented with retinal differentiation media for up to 2 weeks. Proliferation was measured during the course of 2 weeks, while differentiation was evaluated using cell morphology and expression of early retinal development markers. Results: The salt leaching method of porous PLGA fabrication resulted in amorphous smooth pores. Cells attached to these scaffolds and proliferated, reaching a maximum cell number at 10 days postseeding that was 5 times higher on porous PLGA than on nonporous controls. The morphology of many of these cells, including their formation of neurites, was suggestive of neural phenotypes, while their expression of Sox2, Pax6, and Otx2 indicates early retinal development. Conclusions: The use of porous PLGA scaffolds to differentiate iPSCs to retinal phenotypes is a feasible pretransplantation approach. This adds to an important knowledge base; understanding how developing retinal cells interact with polymer substrates with varying structure is a crucial component of optimizing cell therapy strategies. PMID:26692377

Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway.

PubMed

Chen, Wei; Liu, Yongxia; Zhang, Limei; Gu, Xiaolong; Liu, Gang; Shahid, Muhammad; Gao, Jian; Ali, Tariq; Han, Bo

2017-01-01

Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz . 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly ( p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 ( p < 0.01) and casepase-3 ( p < 0.05) levels, significantly ( p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin. The present study is the first comprehensive insight into patho-morphological ultrastructural features of apoptosis/necrosis induced by N. cyriacigeorgica , which concluded that the clinical N. cyriacigeorgica induced apoptotic changes in the bMECs through mitochondrial-caspase dependent apoptotic pathway.

Influence of LOX/COX inhibitors on cell differentiation induced by all-trans retinoic acid in neuroblastoma cell lines.

PubMed

Redova, Martina; Chlapek, Petr; Loja, Tomas; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-02-01

We investigated the possible modulation by LOX/ COX inhibitors of all-trans retinoic acid (ATRA)-induced cell differentiation in two established neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor of cyclooxygenase-2, were chosen for this study. The effects of the combined treatment with ATRA and LOX/COX inhibitors on neuroblastoma cells were studied using cell morphology assessment, detection of differentiation markers by immunoblotting, measurement of proliferation activity, and cell cycle analysis and apoptosis detection by flow cytometry. The results clearly demonstrated the potential of caffeic acid to enhance ATRA-induced cell differentiation, especially in the SK-N-BE(2) cell line, whereas application of celecoxib alone or with ATRA led predominantly to cytotoxic effects in both cell lines. Moreover, the higher sensitivity of the SK-N-BE(2) cell line to combined treatment with ATRA and LOX/COX inhibitors suggests that cancer stem cells are a main target for this therapeutic approach. Nevertheless, further detailed study of the phenomenon of enhanced cell differentiation by expression profiling is needed.

Mitochondria are targets for the antituberculosis drug rifampicin in cultured epithelial cells.

PubMed

Erokhina, M V; Kurynina, A V; Onishchenko, G E

2013-10-01

Rifampicin is a widely used drug for antituberculosis therapy. Its target is the bacterial RNA polymerase. After entry into the human or mammalian organism, rifampicin is accumulated in cells of epithelial origin (kidneys, liver, lungs) where it induces apoptosis, necrosis, and fibrosis. The purpose of this study was to determine the intracellular mechanisms leading to rifampicin-induced pathological changes and cell death. We analyzed the survival and state of the chondriome of cultured epithelial cells of the SPEV line under the influence of rifampicin. Our data show that the drug induces pronounced pathological changes in the network and ultrastructure of mitochondria, and their dysfunction results in excessive production of reactive oxygen species and release of cytochrome c. These data suggest the initiation of the mitochondrial pathway of apoptosis. Simultaneously, we observed inhibition of cell proliferation and changes in morphology of the epithelial cells toward fibroblast-like appearance, which could indicate induction of epithelial-mesenchymal transition. Thus, mitochondria are the main potential target for rifampicin in cells of epithelial origin. We suggest that similar mechanisms of pathological changes can be induced in vivo in organs and tissues accumulating rifampicin during chemotherapy of bacterial infectious diseases.

Effects of macro- versus nanoporous silicon substrates on human aortic endothelial cell behavior

PubMed Central

2014-01-01

Human aortic endothelial cells play a key role in the pathogenesis of atherosclerosis, which is a common, progressive, and multifactorial disease that is the clinical endpoint of an inflammatory process and endothelial dysfunction. Study and development of new therapies against cardiovascular disease must be tested in vitro cell models, prior to be evaluated in vivo. To this aim, new cell culture platforms are developed that allow cells to grow and respond to their environment in a realistic manner. In this work, the cell adhesion and morphology of endothelial cells are investigated on functionalized porous silicon substrates with two different pore size configurations: macroporous and nanoporous silicon. Herein, we modified the surfaces of porous silicon substrates by aminopropyl triethoxysilane, and we studied how different pore geometries induced different cellular response in the cell morphology and adhesion. The cell growth over the surface of porous silicon becomes an attractive field, especially for medical applications. Surface properties of the biomaterial are associated with cell adhesion and as well as, with proliferation, migration and differentiation. PMID:25246859

Overexpression of the erythropoietin receptor in RAMA 37 breast cancer cells alters cell growth and sensitivity to tamoxifen.

PubMed

Ilkovičová, Lenka; Trošt, Nina; Szentpéteriová, Erika; Solár, Peter; Komel, Radovan; Debeljak, Nataša

2017-08-01

Erythropoietin (EPO) is the main regulator of erythropoiesis, and its receptor (EPOR) is expressed in various tissues, including tumors. Expression of EPOR in breast cancer tissue has been shown to correlate with expression of the estrogen receptor (ER). However, EPOR promotes proliferation in an EPO-independent manner. In patients with breast cancer, EPOR is associated with impaired tamoxifen response in ER-positive tumors, but not in ER-negative tumors. Furthermore, a positive correlation between EPOR/ER status and increased local cancer recurrence has been demonstrated, and EPOR expression is associated with G-protein coupled ER (GPER). Herein, we assessed the effects of EPOR on cell physiology and tamoxifen response in the absence of EPO stimulation using two cell lines that differ only in their EPOR expression status: RAMA 37 cells (low EPOR expression) and RAMA 37-28 cells (high EPOR expression). Alterations in cell growth, morphology, response to tamoxifen cytotoxicity, and EPOR-activated signal transduction were observed. RAMA 37 cells showed higher proliferation capacity without tamoxifen treatment, while RAMA 37-28 cells were more resistant to tamoxifen and proliferated more rapidly in the presence of tamoxifen. EPOR overexpression induced cell-morphology changes upon tamoxifen treatment, which resulted in the production of cell protrusions and subsequent cell death. Short-term treatment with tamoxifen (6 h) prompted RAMA 37 cells to acquired longer protrusions than RAMA 37-28 cells, which indicated a pre-apoptotic stage. Furthermore, prolonged treatment with tamoxifen (72 h) caused a greater reduction in RAMA 37 cell numbers, which indicated a higher rate of cell death. RAMA 37-28 cells showed prolonged activation of AKT signaling. We propose sustained AKT phosphorylation in EPOR-overexpressing cells as a mechanism that can lead to EPOR-induced tamoxifen resistance.

[Studies on red orpiment induction of NB4 and HL-60 cell apoptosis].

PubMed

Bai, Y; Huang, S

1998-09-01

To study the possible mechanism of red orpiment, which is main component of composite indigo naturalis tablets, in the treatment of acute promyelocytic leukemia(APL). The effect of red orpiment on induction of APL cell line NB4 and HL-60 apoptosis were studied by cell morphology, DNA gel electrophoresis and flow cytometry assay. Red orpiment induced NB4 and HL-60 cell apoptosis. When treated with different concentration of red orpiment(25-200 micrograms/ml) for 16 hours, both NB4 and HL-60 cells showed typical apoptosis features. If decreased the concentration of red orpiment to 12.5 micrograms/ml, the NB4 cell still showed apoptosis features while the HL-60 cell did not when cultured for 72 hours. Arsenic disulfide(As2S2) had the same effect as red orpiment did under the same experiment condition. It is the main component, As2S2 of the red orpiment that can induces NB4 and HL-60 cell apoptosis. and the red orpiment is responsible for the high CR rate of APL induced by the composite indigo naturalis tablets.

Effect of propofol on hypoxia re-oxygenation induced neuronal cell damage in vitro*.

PubMed

Huang, Y; Zitta, K; Bein, B; Scholz, J; Steinfath, M; Albrecht, M

2013-01-01

Propofol may protect neuronal cells from hypoxia re-oxygenation injury, possibly via an antioxidant actions under hypoxic conditions. This study investigated the molecular effects of propofol on hypoxia-induced cell damage using a neuronal cell line. Cultured human IMR-32 cells were exposed to propofol (30 μm) and biochemical and molecular approaches were used to assess cellular effects. Propofol significantly reduced hypoxia-mediated increases in lactate dehydrogenase, a marker of cell damage (mean (SD) for normoxia: 0.39 (0.07) a.u.; hypoxia: 0.78 (0.21) a.u.; hypoxia+propofol: 0.44 (0.17) a.u.; normoxia vs hypoxia, p<0.05; hypoxia vs hypoxia+propofol, p<0.05), reactive oxygen species and hydrogen peroxide. Propofol also diminished the morphological signs of cell damage. Increased amounts of catalase, which degrades hydrogen peroxide, were detected under hypoxic conditions. Propofol decreased the amount of catalase produced, but increased its enzymatic activity. Propofol protects neuronal cells from hypoxia re-oxygenation injury, possibly via a combined direct antioxidant effect along with induced cellular antioxidant mechanisms. Anaesthesia © 2012 The Association of Anaesthetists of Great Britain and Ireland.

Mitochondrial Dysfunction and Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2.

PubMed

Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Lee, Won-Sup; Kim, Eun-Hee; Kim, Gon Sup

2016-06-01

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells. © 2015 Wiley Periodicals, Inc.

Amarogentin secoiridoid inhibits in vivo cancer cell growth in xenograft mice model and induces apoptosis in human gastric cancer cells (SNU-16) through G2/M cell cycle arrest and PI3K/Akt signalling pathway.

PubMed

Zhao, Jian-Guo; Zhang, Ling; Xiang, Xiao-Jun; Yu, Feng; Ye, Wan-Li; Wu, Dong-Ping; Wang, Jian-Fang; Xiong, Jian-Ping

2016-01-01

To investigate the in vitro and in vivo antitumor effects of amarogentin in SNU-16 human gastric cancer cells as well as in nude mice xenograft model. The effects of this compound on cell apoptosis, cell cycle phase distribution and PI3K/Akt and m-TOR signalling pathways were also studied in detail. MTT assay was used to study the effect of amarogentin on SNU-16 cell viability while clonogenic assay indicated the effect of the compound on colony formation tendency of these cells. Phase contrast microscopy revealed the effect on cellular morphology while flow cytometry was engaged to study the effects on cell apoptosis and cell cycle arrest. SNU-16 cancer cells were subcutaneously inoculated into nude mice to investigate the in vivo antitumor effects of amarogentin. Amarogentin induced potent, dose-dependent as well as time-dependent cytotoxic effects on the growth of SNU-16 human gastric cancer cells. Amarogentin also inhibited the colony forming capability of these tumor cells and its treatment led to morphological alterations in these cells in which the cells became withered and rounded, detached from one another and adopted irregular shapes while floating freely in the culture medium. In comparison to untreated control cells, the amarogentin treated cells with 10, 50 and 75 μM exhibited 32.5, 45.2 and 57.1 % apoptotic cells, respectively. Amarogentin induced potent and dose-dependent G2/M cell cycle arrest in these cells and led to downregulation of m-TOR, p-PI3K, PI3K, p-Akt and Akt and upregulation of cyclin D1 and cyclin E protein expressions. The tumor tissues obtained from the amarogentin-treated mice were much smaller than the tumor tissues derived from the control group. Amarogentin exerts potent in vitro and in vivo antitumor effects in SNU-16 cell model as well as in nude mice xenograft model. These antitumor effects were found to be mediated through apoptosis induction, G2/M cell cycle arrest and downregulation of PI3K/Akt/m-TOR signalling pathways.

The in vitro study of apoptosis in NB4 cell induced by citral.

PubMed

Xia, Hailong; Liang, Wei; Song, Qin; Chen, Xiaowen; Chen, Xin; Hong, Jian

2013-01-01

Citral, 3,7-dimethyl-2,6-octadienal, is a key component of the essential oils extracted from several lemon-scented herbal plants. Besides its antifungal activity, the anticancer effect of citral was studied in recent years. In this study, we investigated the effect of citral on the acute promyelocytic leukemia cell line NB4. Citral treatment had an antiproliferative effect in NB4 cells via the induction of apoptosis assessed by morphology, proliferation assay, DNA electrophoresis, Annexin V-FITC/PI staining and caspase-3 activation. And citral induced apoptosis of NB4 cells in a dose- and time-dependent manner. In addition, citral treatment induced decreased mitochondrial membrane potential, indicating that citral induced apoptosis via the mitochondrial pathway. Bax up-regulation and Bcl-2 down-regulation on mRNA level and NF-κB down-regulation on protein level was found in this study, suggesting that Bcl-2, Bax and NF-κB may be involved in the mechanism of the apoptotic effect of citral on NB4 cells. These data suggest that citral has a potential therapeutic effect on leukemia.

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

PubMed Central

Mondia, Jessica P.; Levin, Michael; Omenetto, Fiorenzo G.; Orendorff, Ryan D.; Branch, Mary Rose; Adams, Dany Spencer

2011-01-01

Background With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration. Methods and Results Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis. Conclusions We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches. PMID:21949803

Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production

PubMed Central

Fortes, Guilherme B.; Alves, Leticia S.; de Oliveira, Rosane; Dutra, Fabianno F.; Rodrigues, Danielle; Fernandez, Patricia L.; Souto-Padron, Thais; De Rosa, María José; Kelliher, Michelle; Golenbock, Douglas; Chan, Francis K. M.

2012-01-01

Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4−/− or to Myd88−/− macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1−/−) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS. PMID:22262768

PDGF-AA-induced filamentous mitochondria benefit dermal papilla cells in cellular migration.

PubMed

Mifude, C; Kaseda, K

2015-06-01

Human dermal papilla cells (HDPCs) play essential roles in hair follicular morphogenesis and postnatal hair growth cycles. Previous reports demonstrated that platelet-derived growth factor-AA (PDGF-AA) enhanced the formation of dermal condensates in hair follicular development. Additionally, PDGF-AA induces/maintains the anagen phase of the hair cycle. It is likely that mitochondrial morphology and functions are tightly coupled with maintenance of these energy-demanding activities. However, little is known about the mitochondrial regulation in HDPCs. Thus, we investigated the PDGF-involved mitochondrial regulation in HDPCs. The mitochondrial morphologies of HDPCs were examined in the presence or absence of PDGF-AA under a fluorescent microscope. ATP production and cellular motility were investigated. The relationship between mitochondrial morphology and the cellular functions was discussed. We observed that primary HDPCs contained mitochondria with filamentous and/or rounded morphologies. Both types of mitochondria showed similar membrane potentials. Interestingly, in the presence of PDGF-AA, but not PDGF-BB, the balance between the two morphologies shifted towards the filamentous form. Concomitantly, both mitochondrial enzymatic activity and total cellular ATP level were augmented by PDGF-AA. These two parameters were closely correlated, suggesting the mitochondrial involvement in the PDGF-augmented ATP production. Moreover, PDGF-AA accelerated the migration of HDPCs in a gap-filling assay, but did not change the rate of cellular proliferation. Notably, filamentous mitochondria dominated migrating HDPCs. PDGF-AA benefits HDPCs in the process of migration, by increasing the number of filamentous mitochondria. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Neurite outgrowth in human iPSC-derived neurons

EPA Pesticide Factsheets

Data on morphology of rat and human neurons in cell cultureThis dataset is associated with the following publication:Druwe, I., T. Freudenrich , K. Wallace , T. Shafer , and W. Mundy. Comparison of Human Induced PluripotentStem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth. Applied In vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, USA, 2(1): 26-36, (2016).

Cholinergic Neurotransmission in the Mammalian Retina

DTIC Science & Technology

1984-11-30

synthesis of (3H)acetylcholine from a (3H) choline precursor and identification of the cells which produce the acetylcholine destructive enzyme...3H) choline , to contain acetylcholinesterase, and to be highly susceptible to morphological changes induced by a presumptive cholinotoxin. These...drawing of starburst-like amacrine cell from cat retina 10 2. Freeze-dry autoradiograph of rabbit retina, incubated with (3H) choline for synthesis of (3H

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

PubMed

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

NASA Astrophysics Data System (ADS)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Effects of Modified Simiao Decoction on IL-1 β and TNF α Secretion in Monocytic THP-1 Cells with Monosodium Urate Crystals-Induced Inflammation.

PubMed

Liu, Ya-Fei; Tu, Sheng-Hao; Chen, Zhe; Wang, Yu; Hu, Yong-Hong; Dong, Hui

2014-01-01

Simiao pill, a Chinese herbal formula containing four herbs, has been used in the treatment of gouty arthritis for many years. The aim of this study was to explore the effects of modified Simiao decoction (MSD) on IL-1 β and TNF α secretion in monocytic THP-1 cells with monosodium urate (MSU) crystals-induced inflammation. The MSU crystals-induced inflammation model in THP-1 cells was successfully established by the stimulation of phorbol 12-myristate 13-acetate (PMA) and MSU crystals. Then, the MSD-derived serum or control serum extracted from rat was administered to different treatment groups. The morphology of MSU crystals and THP-1 cells was observed. IL-1 β and TNF α protein expression in supernatant of THP-1 cells were determined by ELISA. Our data demonstrated that MSU crystals induced time-dependent increase of IL-1 β and TNF α . Moreover, MSD significantly decreased IL-1 β release in THP-1 cells with MSU crystals-induced inflammation. These results suggest that MSD is promising in the treatment of MSU crystals-induced inflammation in THP-1 cells. MSD may act as an anti-IL-1 agent in treating gout. The underlying mechanism may be related to NALP3 inflammasome which needs to be validated in future studies.

Effects of Modified Simiao Decoction on IL-1β and TNFα Secretion in Monocytic THP-1 Cells with Monosodium Urate Crystals-Induced Inflammation

PubMed Central

Liu, Ya-Fei; Tu, Sheng-Hao; Chen, Zhe; Wang, Yu; Hu, Yong-Hong; Dong, Hui

2014-01-01

Simiao pill, a Chinese herbal formula containing four herbs, has been used in the treatment of gouty arthritis for many years. The aim of this study was to explore the effects of modified Simiao decoction (MSD) on IL-1β and TNFα secretion in monocytic THP-1 cells with monosodium urate (MSU) crystals-induced inflammation. The MSU crystals-induced inflammation model in THP-1 cells was successfully established by the stimulation of phorbol 12-myristate 13-acetate (PMA) and MSU crystals. Then, the MSD-derived serum or control serum extracted from rat was administered to different treatment groups. The morphology of MSU crystals and THP-1 cells was observed. IL-1β and TNFα protein expression in supernatant of THP-1 cells were determined by ELISA. Our data demonstrated that MSU crystals induced time-dependent increase of IL-1β and TNFα. Moreover, MSD significantly decreased IL-1β release in THP-1 cells with MSU crystals-induced inflammation. These results suggest that MSD is promising in the treatment of MSU crystals-induced inflammation in THP-1 cells. MSD may act as an anti-IL-1 agent in treating gout. The underlying mechanism may be related to NALP3 inflammasome which needs to be validated in future studies. PMID:24999366

Non-thermal plasma induces mitochondria-mediated apoptotic signaling pathway via ROS generation in HeLa cells.

PubMed

Li, Wei; Yu, K N; Ma, Jie; Shen, Jie; Cheng, Cheng; Zhou, Fangjian; Cai, Zhiming; Han, Wei

2017-11-01

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NTP exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NTP exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP- ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-l-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NTP exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NTP as a novel tool in cervical cancer treatment. Copyright © 2017. Published by Elsevier Inc.